WO2019218876A1 - 一种荧光探针及其制备方法和用途 - Google Patents

一种荧光探针及其制备方法和用途 Download PDF

Info

Publication number
WO2019218876A1
WO2019218876A1 PCT/CN2019/085192 CN2019085192W WO2019218876A1 WO 2019218876 A1 WO2019218876 A1 WO 2019218876A1 CN 2019085192 W CN2019085192 W CN 2019085192W WO 2019218876 A1 WO2019218876 A1 WO 2019218876A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
alkyl
probe
optionally
protein
Prior art date
Application number
PCT/CN2019/085192
Other languages
English (en)
French (fr)
Inventor
朱麟勇
杨弋
鲍丙坤
苏倪
张大生
陈显军
林秋宁
包春燕
Original Assignee
华东理工大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华东理工大学 filed Critical 华东理工大学
Priority to US17/250,061 priority Critical patent/US20210382060A1/en
Priority to EP19803374.8A priority patent/EP3798221A4/en
Publication of WO2019218876A1 publication Critical patent/WO2019218876A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07D403/02—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 two hetero rings
    • C07D403/12—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 two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
    • 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
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C09B23/005—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a COOH and/or a functional derivative thereof
    • C09B23/0058—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a COOH and/or a functional derivative thereof the substituent being CN
    • 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
    • C09B68/00—Organic pigments surface-modified by grafting, e.g. by establishing covalent or complex bonds, in order to improve the pigment properties, e.g. dispersibility or rheology
    • C09B68/40—Organic pigments surface-modified by grafting, e.g. by establishing covalent or complex bonds, in order to improve the pigment properties, e.g. dispersibility or rheology characterised by the chemical nature of the attached groups
    • C09B68/46—Aromatic cyclic groups
    • C09B68/467—Heteroaromatic groups
    • C09B68/4677—6-Membered rings
    • 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
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • 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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
    • 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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • 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
    • C09K2211/1074—Heterocyclic compounds characterised by ligands containing more than three nitrogen atoms as heteroatoms
    • 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
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
    • 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"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention relates to a fluorescent probe, a preparation method thereof and use thereof.
  • Fluorescence-specific labeling is an important means of studying protein function and quantification. Fluorescent labels are an irreplaceable advantage over other research methods in terms of sensitivity, in situ, instant, and visual. At present, the most common method for protein-specific fluorescent labeling is to express the fluorescent protein in situ on the target protein by gene fusion technology, thereby achieving specific illumination of the target protein and enabling it to be in a cell or tissue under a fluorescence microscope. Target proteins were followed for follow-up studies. Fluorescent protein technology has been developed for a long time, and the technology is relatively mature. However, there are still many defects. For example, fluorescent protein matures and folds slowly and easily aggregates; and once expressed, it is difficult to carry out post-modification; in addition, most fluorescent proteins still exist in light. Insufficient stability, etc.; these limit the application of fluorescent proteins to some extent.
  • the molecular structure of the fluorescent protein chromophore is relatively simple, and different types or functionalized fluorescent proteins are constructed, and there are often no rules to follow, and the sea selection can only be carried out by means of random mutation.
  • organic small molecule fluorescent dyes are rich in molecular structure, but small molecule fluorescent probes still have many defects in protein-specific labeling.
  • chemical tag technology has effectively solved the problem of protein-specific labeling of small molecule fluorescent probes.
  • the technology fuses a polypeptide or a protein tag having a specific recognition function with a target protein, and utilizes the label to bind to a substrate with high specificity, thereby realizing a small molecule fluorescent probe-specific protein label, and therefore, chemistry Label technology not only inherits the advantages of gene fusion technology, but also fully inherits the advantages of various aspects of organic dye probes compared to fluorescent proteins.
  • SNAP-tag K. Johnsson et. al. WO 2004031405.
  • CLIP-tag K. Johnsson et. al. WO 2008012296.
  • Halo-tag Wang, Keith V et. al. WO 2004072232
  • Protein labeling technology has been commercialized, especially SNAP-tag and CLIP-tag are the most widely used and have been recognized by the market.
  • a method for fluorescent-activated protein-specific labeling for SNAP-tag and CLIP-tag is designed, it is dark or emits very weak fluorescence before labeling, and once it is labeled on the protein, the fluorescence of the dye is sharp. Enhanced. Undoubtedly, probes of this type will be able to achieve the same specificity as fluorescent proteins, which will not only eliminate free probe washing, but also greatly reduce the background interference of free probes, and will widen SNAP-tag and CLIP-tag.
  • a method for designing a fluorescently activated protein-specific label suitable for this technique must consider a suitable fluorescence loss/activation mechanism. The FRET mechanism is first applied to the design of this aspect by adding an additional fluorescence quenching group to the ligand.
  • the small molecule fluorescence is quenched by the group to which it is attached; once the ligand is combined with the chemical tag, the quenching group is quenched. Leaving, fluorescence activation is achieved (T. Komatsu. et. al. J. Am. Chem. Soc. 2011, 133, 6745-6751.).
  • the introduction of the quenching group greatly increases the molecular volume of the probe, greatly reduces the labeling speed, severely limits the real-time tracking and detection of the probe protein in cells and tissues, and the fluorescent probe and quenching group. There must be a good energy level match between the clusters, which makes the long wavelength, for example, the FRET design of the red light emitting dye very difficult.
  • fluorescence-sensitive dyes have also been used to design activated probes (TKLiu.et.al.ACS Chem.Biol. 2014, 9, 2359-2365.), the dye is in a large polarity such as cell fluid In the solvent, the probe has no fluorescence or has weak fluorescence. When the ligand binds to the protein, the probe is in the protein non-polar pocket, and the probe emits stronger fluorescence.
  • the fluorescence enhancement of the probe is limited; on the other hand, the cell or tissue itself is a very complex system, and the polarity of each organelle changes very much.
  • the inventors have found that by linking the ligand moiety to the electron donor moiety of the viscosity-responsive fluorescent dye, the fluorescence intensity of the ligand-labeled protein-activated fluorescence can be greatly enhanced, thereby obtaining a novel structure of fluorescence detection.
  • the needle has viscosity responsiveness and can be used for specific labeling of proteins. It has high labeling speed, high fluorescence activation brightness, strong bleaching resistance and wide application range, and can be effectively used for labeling, tracking, localization and quantification of target proteins.
  • the present invention provides a fluorescent probe comprising: a ligand moiety A, an optional linker moiety C and a fluorescent dye moiety, the fluorescent dye moiety being a viscosity responsive fluorescent dye group, including an electron donor moiety D.
  • ligand moiety A being a group capable of specifically recognizing and labeling a target protein of a protein tag or a fusion protein tag, optionally, said ligand moiety A is a group capable of specifically recognizing and covalently labeling a target protein of a protein tag or a fusion protein; characterized in that the ligand moiety A is directly covalently attached to the electron donor moiety D of the fluorescent dye moiety, or The linker moiety C is covalently attached to the electron donor moiety D of the fluorescent dye moiety.
  • the fluorescent probe described above has a structure as shown in formula (I),
  • the ligand moiety A is derived from an O 6 -alkylguanine compound, an alkyl 4-chloropyrimidine compound or an alkyl cytosine compound;
  • Linker moiety C is an optionally present group selected from the group consisting of alkylene groups, modified alkylene groups;
  • the fluorescent dye moiety has a structure as shown in the formula (I-R).
  • the conjugated system B has a structure represented by the formulae (I-1-1) to (I-1-7).
  • the electron acceptor moiety has a structure represented by the following formula (I-2),
  • R 1 is selected from the group consisting of hydrogen, a halogen atom, a nitro group, an alkyl group, an aryl group, a heteroaryl group, a hydrophilic group or a modified alkyl group;
  • R 2 is selected from the group consisting of hydrogen, cyano, carboxyl, keto, ester, amide, thioamino, thioester, sulfonate, sulfonate, sulfone, sulfoxide, aryl, hetero An aryl group, an alkyl group or a modified alkyl group;
  • R 3 is a cyano group
  • the electron acceptor moiety optionally forms a cyclic structure of the following formula (I-2-a), (I-2-b), (I-2-c):
  • R a , R b are independently selected from the group consisting of hydrogen, a hydrophilic group, an alkyl group, and a modified alkyl group, and R a and R b are optionally bonded to each other to form an alicyclic or heterocyclic ring;
  • Each R c is independently selected from the group consisting of hydrogen, a halogen atom, a nitro group, an alkyl group, an aryl group, a heteroaryl group, a hydrophilic group or a modified alkyl group; each R d is independently selected from the group consisting of hydrogen, a halogen atom, and a nitro group.
  • R e is selected from the group consisting of cyano, carboxyl, keto, ester, amide, phosphite, phosphate, sulfonate, sulfonate, sulfone, sulfoxide, aryl, heteroaryl, An alkyl group or a modified alkyl group;
  • R 2 or R e is aryl or heteroaryl
  • the hydrogen atom on the ring is optionally independently selected from a halogen atom, a cyano group, a nitro group, a hydrophilic group, an alkyl group or a modified alkyl group.
  • a substituent substituted; optionally, the substituents are linked to each other to form a saturated or unsaturated alicyclic or heteroalicyclic;
  • alkyl group is a C 1 -C 30 linear or branched alkyl group; preferably, a C 1 -C 10 linear or branched alkyl group; preferably, a C 1 -C 7 straight chain Or a branched alkyl group; preferably, a C 1 -C 5 straight or branched alkyl group; preferably, selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, Tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl , 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl,
  • alkylene is a C 1 -C 30 linear or branched alkylene group; preferably, a C 1 -C 7 linear or branched alkylene group; preferably, a C 1 -C 5 linear or branched alkylene;
  • the carbon atom is replaced, meaning that a carbon atom or a carbon atom is replaced by a corresponding group together with a hydrogen atom thereon;
  • the "alicyclic ring” is a saturated or unsaturated 4 to 10 membered monocyclic or polycyclic alicyclic ring;
  • the "aliphatic heterocycle” is a saturated or unsaturated 4 to 10 membered monocyclic or polycyclic heterocyclic ring containing at least one hetero atom selected from N, O, S or Si.
  • the ring contains an S atom, it is -S-, -SO- or -SO 2 -; the heterocyclic ring is optionally modified by a halogen atom, a nitro group, an alkyl group, an aryl group, a hydrophilic group, and Alkyl substitution
  • aryl or aromatic ring is a 5- to 10-membered monocyclic or fused bicyclic or polycyclic aromatic group
  • heteroaryl or aromatic heterocyclic ring is a 5- to 10-membered monocyclic or fused bicyclic or polycyclic heteroaromatic group having at least one hetero atom selected from N, O, S or Si on the ring;
  • halogen atoms are each independently selected from the group consisting of F, Cl, Br, I;
  • the "hydrophilic group” is a hydroxyl group, a sulfonic acid group, a carboxyl group, a phosphite group, a primary amino group, a secondary amino group or a tertiary amino group;
  • the "monocyclic subcyclic hydrocarbon group” is a 4- to 7-membered cycloalkylene group
  • the "bicyclic subcyclic hydrocarbon group” is a 5- to 10-membered bicyclic ring-opened hydrocarbon group
  • bias heterocycle is a group formed by conjugated linking of two or more aromatic heterocycles
  • bridged heterocyclic ring is a 5- to 20-membered bridged heterocyclic ring containing at least one hetero atom selected from N, O, or S on the ring;
  • the "amide group” is a RCONHR' group
  • the "sulfonic acid group” is an RSO 3 H group
  • the "sulfonate group” is an RSO 2 OR'group
  • the "sulfone group” is an RSO 2 R'group
  • the "primary amino group” is an RNH 2 group
  • the "secondary amino group" is an RNHR' group
  • the "tertiary amino group” is an RNR 'R" group
  • the "monosaccharide unit” is a saccharide unit that can no longer be simply hydrolyzed into smaller sugar molecules
  • the "disaccharide unit” is a saccharide unit in which two monosaccharides are dehydrated
  • polysaccharide unit is a saccharide unit in which ten or more monosaccharides are dehydrated
  • the C 1 -C 8 alkyl group is a methyl group, an ethyl group, a propyl group, an isopropyl group
  • the C 1 -C 8 alkoxy group is a methoxy group, an ethoxy group, a propoxy group, Isopropoxy group
  • the C 1 -C 8 acyloxy group is acetoxy group, ethyl group, propyl group, isopropyl group
  • the C 1 -C 8 haloalkyl group is trifluoromethyl group, chloromethyl group, bromine group methyl;
  • the heterocyclic ring is selected from the group consisting of azetidine, pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine;
  • the aromatic heterocycle is selected from,
  • the aromatic ring is selected from
  • the fluorescent probe described above is characterized in that:
  • the protein tag is a purified product, an unpurified product, or an in situ state present in a cell or tissue;
  • the protein tag is O 6 -alkylguanine-DNA alkyltransferase (SNAP-tag) or a mutant thereof, alkyl cytosine transferase (CLIP-tag) or a mutant thereof;
  • the mutant of the O 6 -alkylguanine-DNA alkyltransferase is selected from the group consisting of SNAP F33G or SNAP V164E;
  • the protein tag is O 6 -alkylguanine-DNA alkyltransferase (SNAP-tag) or a mutant thereof;
  • the ligand moiety A suitable for use in the SNAP-tag is derived from an O 6 -alkylguanine compound or an alkyl 4-chloropyrimidine compound; alternatively, the ligand moiety A suitable for the CLIP-tag is derived from Alkyl cytosine compounds;
  • the ligand moiety A- is selected from the group consisting of:
  • X 1 is a C 1-30 straight or branched alkyl group optionally substituted by one or more groups selected from the group consisting of a hydroxyl group, a cyano group, a halogen atom, a carboxyl group, and a quaternary ammonium group, and X 2 is optional.
  • X 1 and X 2 are each independently optionally a C 2-30 ether chain group having 1 to 10 oxygen atoms substituted with one or more groups selected from a sulfonic acid group or a carboxyl group; or, -NX 1 -X 2 is formed from the following formula (Ii-1) Any group of (Ii-2):
  • X 1 is a C 1-10 straight or branched alkyl group optionally substituted by one or more groups selected from the group consisting of a hydroxyl group, a cyano group, a halogen atom, a carboxyl group, and a quaternary ammonium group
  • X 2 is any a C 1-10 linear or branched alkyl or alkylene group substituted by one or more groups selected from the group consisting of a hydroxyl group, a cyano group, a halogen atom, a carboxyl group, and a quaternary ammonium group;
  • the R 2 and R e are independently a group selected from the following structures, or a bicyclic or polycyclic fused aromatic ring or a fused aromatic heterocyclic ring formed by the following structures themselves or fused to each other: preferably a bicyclic or tricyclic fused aromatic ring or a fused aromatic heterocyclic ring;
  • H in the above structure of R 2 or R e is substituted by a halogen atom, a nitro group, a hydrophilic group, an alkyl group or a modified alkyl group; alternatively, R 2 or R e is selected From the NH-containing group in the above structure, optionally, H on the NH is substituted with an alkyl group or a modified alkyl group;
  • the R 2 is selected from the group consisting of hydrogen, cyano, carboxyl, keto, ester, amide, thioamino, thioester, and, when selected from a keto, ester or amide group, a ketone
  • the carbonyl group in the group, ester group or amide group is bonded to the alkenyl carbon of the formula (I-2), the formula (I-2-a), the formula (I-2-b), and the formula (I-2-c)
  • a thioamino acyl group or a thioester group it is bonded to the formula (I-2), the formula (I-2-a), and the formula (I-) through a thiocarbonyl group in a thioamino group or a thioester group.
  • R e is selected from the group consisting of a cyano group, a keto group, an ester group, an amide group, and when selected from a keto group, an ester group, an amide group, a ketone group a carbonyl group in the ester group or the amide group is attached to the alkenyl carbon of the formula (I-2-a) or the formula (I-2-c);
  • the electron acceptor moiety is one selected from the group consisting of the following formulas (I-2-1) to (I-2-22):
  • the fluorescent probe described above, wherein the fluorescent probe is selected from the group consisting of:
  • a method of preparing a fluorescent probe comprising the step of reacting a fluorescent dye of formula (II) with a ligand and, optionally, a linker:
  • a D-group can be formed to bond with a linking group or a ligand.
  • the present invention also provides a fluorescence-activated protein-specific labeling method, comprising the steps of: contacting the fluorescent probe with a target protein of a protein tag or a fusion protein tag, and a ligand portion of the fluorescent probe Labeling with a protein tag, labeling the fluorescent probe onto the protein tag; optionally, labeling the fluorescent probe to the protein tag is a covalent label;
  • the reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or a protein label or an in situ medium in which the target protein of the fusion protein tag is located; alternatively, the in situ medium is intracellular Media, organelle media, living tissue media, blood or body fluids.
  • the present invention also provides the use of the above fluorescent probe in protein fluorescent labeling, protein quantification, detection or kinetics research, and cell, tissue, and in vivo imaging.
  • the present invention also provides a probe kit comprising the above fluorescent probe.
  • the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is selected from at least one of dimethyl sulfoxide, a buffer, and physiological saline;
  • the buffer comprises a phosphate buffer.
  • the protein tag of the present invention or the portion of the target protein to which the tag is fused can be prepared by existing genetic engineering techniques.
  • the viscosity responsive fluorescent dye means that the fluorescence intensity of the dye molecule responds to the viscosity of the solution, and as the viscosity of the solution increases, the fluorescence intensity increases.
  • the viscosity responsive fluorescent dye is: at 25 ° C, the same concentration and excitation wavelength, the ratio of the maximum fluorescence emission intensity of the dye in glycerol to the fluorescence intensity in methanol is greater than 2, preferably more than 5, more preferably more than 10 organic dye molecules.
  • the concentration of the viscosity responsive dye ranges from 1 x 10 -7 M to 1 x 10 -5 M.
  • the instrument equipment used includes equipment and facilities capable of testing or displaying fluorescence, such as fluorescence spectrometer and fluorescence, as needed. Microscopes, confocal fluorescence microscopes, microplate readers, flow cytometers, and live imagers.
  • the operator can select dyes of different kinds or emission/excitation wavelengths as needed.
  • the fluorescent probe has a wide range of fluorescence emission wavelengths.
  • the fluorescence intensity of the fluorescent probe increases with increasing environmental viscosity, is sensitive to viscosity, and has viscosity responsiveness.
  • the fluorescent probe can be used for specific labeling of a target protein of a protein tag or a fusion protein tag, fluorescence can be activated after fluorescent probe binding protein tag, has good fluorescent molecular switching properties, and fluorescence activation multiple High, fluorescence activation is high.
  • the fluorescent probe labels the protein at a very fast rate.
  • the fluorescence intensity of the fluorescent probe and the protein label concentration have a good linear relationship and can be used for quantitative detection of the target protein.
  • the fluorescent probe can achieve specific labeling of intracellular protein tags and achieve fluorescence-specific illumination while the probe fluorescence is unaffected by the intracellular environment.
  • the fluorescent probe can be used as a powerful tool for cell subcellular organelle markers, such as for nuclei, mitochondria, Golgi, endoplasmic reticulum, whole cells, cytoskeleton, extracellular membrane, lysosome, intracellular membrane Mark of etc.
  • cell subcellular organelle markers such as for nuclei, mitochondria, Golgi, endoplasmic reticulum, whole cells, cytoskeleton, extracellular membrane, lysosome, intracellular membrane Mark of etc.
  • the spectra of different fluorescent probe fluorophores do not interfere with each other.
  • the fluorescent probes of different color systems of the present invention can perform multi-color labeling on samples, and can perform orthogonal marker imaging simultaneously.
  • the fluorescence of the fluorescent probe is not affected by the environment of the animal, and can be applied to a living animal, for example, a SNAP protein tag that specifically expresses at the liver site, and generates a strong fluorescent signal.
  • a fluorescent probe can be used to track and monitor the degradation process of the protein of interest.
  • the fluorescent probe monitors in real time the assembly and degradation processes of biological macromolecules in mammalian cells.
  • the fluorescent probe can perform rapid contrast imaging of a tissue that is not suitable for washing, such as tissue, living body, and the like.
  • the fluorescent probe is excellent in bleaching resistance and light stability.
  • the fluorescent probe does not generate a detection signal when labeling the target protein of the protein tag or the fusion protein, and does not interfere with the detection of the sample, can realize rapid quantitative detection of complex samples, and can track the labeling reaction process. Dynamic process.
  • Figure 1 is a fluorescence emission diagram of fluorescence at different wavelengths after different probes are bound to a protein tag
  • 2 to 6 are standard curves of the fluorescence intensity of probe 1, probe 5, probe 13, probe 16, and probe 28 for different SNAP protein label concentrations;
  • Figure 7 is a fluorescence map of different probe-labeled cells, wherein (1) to (3) correspond to probe 16, probe 17, and probe 18, respectively, group A is a Hela cell expressing a protein tag, and group B is a Hela-WT. Cells (Hela original cells, no protein labeling);
  • Fig. 8 shows different organelles with different probe labels, wherein group A to group C are probe 16, probe 17, and probe 18, respectively, and (1) to (5) are cytoplasmic matrix, Golgi apparatus, mitochondria, and chromosomes, respectively. ,Cytoskeleton;
  • Figure 9 is a two-color labeling of the same cells by different probes, wherein A is probe 16 labeled mitochondria, B is probe 9 labeled nuclei, and C is orthogonal imaging of A and B;
  • Figure 10 is a marker of probe 16 used in the liver of a living mouse, wherein group A is a control group and group B is a sample group;
  • Figure 11 is a graph showing changes in fluorescence of probe 16 in mammalian cells as a function of protein degradation
  • Figure 12 is a comparison of photostability of probe 16, probe 17, probe 18-labeled SNAP protein and fluorescent protein IFP682 in mammalian cells, wherein A to C are probe 16, probe 17, probe 18, respectively. D is IFP682;
  • Figure 13 is a comparison of photostability of probe 16, probe 17, probe 18 and reference probe 33 labeled SNAP protein in mammalian cells, wherein A to C are probe 16, probe 17, probe, respectively. 18, D is a reference probe 33;
  • Figure 14 is a diagram showing the photostability of SNAP protein in mammalian cells by probe 2, probe 5, probe 11, probe 13, probe 16, probe 28, and probe 30, wherein A to G are respectively Needle 16, probe 28, probe 5, probe 11, probe 30, probe 2, probe 13.
  • a fluorescently activated covalently labeled fluorescent probe 4 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 5 suitable for SNAP protein tagging was constructed:
  • N-methyl-N-hydroxyethylaniline (1.88 g, 12.5 mmol) and NaHCO 3 (1.57 g, 18.7 mmol) were dissolved in 48 mL dichloromethane and 36 mL water, cooled to 0 ° C, and slowly added I 2 (3.0 g , 11.8mmol), after adding, the system is gradually raised to room temperature, stirred for 30min, the system is diluted with 300ml of dichloromethane 40ml water, the organic phase is separated, the organic phase is washed with water, sodium thiosulfate solution, brine, anhydrous sulfuric acid The sodium was dried and evaporated to dryness.
  • a fluorescently activated covalently labeled fluorescent probe 6 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 7 suitable for SNAP protein tagging was constructed:
  • a molecular rotor as a viscosity responsive fluorescent dye, construct a fluorescently activated covalently labeled fluorescent probe 8 suitable for CLIP protein labeling:
  • a fluorescently activated covalently labeled fluorescent probe 9 suitable for SNAP protein tagging was constructed:
  • 6-Bromo-1-benzofuran 0.4 g, 2 mmol dissolved in 15 mL of N-methyl-N-hydroxyethylamine, copper powder (6.4 mg, 0.01 mmol), copper iodide (19 mg, 0.01 mmol) ), tripotassium phosphate (0.850g, 4mmol), heated in an oil bath at 80 ° C under Ar protection conditions, the reaction is completed, cooled to room temperature, the system is poured into 50mL water, extracted twice with dichloromethane ⁇ 50mL, combined organic phase, rotating The solvent was evaporated to dryness.
  • a fluorescently activated covalently labeled fluorescent probe 10 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 11 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 12 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 13 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 14 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 15 suitable for the CLIP protein tag was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 16 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 17 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 18 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 19 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 20 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 21 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 22 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 23 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 24 suitable for use in the CLIP protein tag was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 25 suitable for the CLIP protein tag was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 26 suitable for the CLIP protein tag was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 27 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 28 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 29 suitable for the CLIP protein tag was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 30 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 31 suitable for SNAP protein tagging was constructed:
  • a fluorescently activated covalently labeled fluorescent probe 32 suitable for SNAP protein tagging was constructed:
  • Reference probes BG-CCVJ and BG-Gly-CCVJ were prepared by the method reported in the literature (T.Y. Wang et. al. Chem Sci. 2016, 7, 301-307.).
  • the probes 1-32 and the reference probes BG-CCVJ and BG-Gly-CCVJ were respectively dissolved in dimethyl sulfoxide to prepare a mother liquor having a concentration of 1 ⁇ 10 -2 M, respectively, and added to glycerin and methanol. The mixture was uniformly mixed, and a solution having a final concentration of 1 ⁇ 10 -5 M was prepared. According to the difference of the probes, the fluorescence emission spectra of the probes were sequentially measured under the same conditions under the same conditions. The results are shown in Table 1.
  • the probes of the examples have a wide range of fluorescence emission wavelengths, and have a large difference in fluorescence intensity in glycerin and methanol, are sensitive to viscosity changes, and have viscosity responsiveness.
  • the mixed sample is incubated at 37 ° C for 1 hour, and the fluorescence intensity of the sample is detected by a fluorescence spectrophotometer. Changes, the results are shown in Table 1.
  • the fluorescence of the probe bound to the protein of the example can be activated, and has good fluorescent molecular switching properties.
  • the SNAP protein tag was added to a solution of 30 uM of probe 1, probe 5, probe 13, probe 16, and probe 28, respectively, to obtain a final concentration of SNAP protein tags of 0.1 uM, 0.5 uM, 0.7 uM, and 1.2 uM. , 4.5uM, 8.1uM, 13.1uM, 14.8uM mixed sample solution, the mixed sample solution was reacted at 37 ° C for 1 hour, the fluorescence emission spectrophotometer was used to detect the excitation emission spectrum change of the sample, and the SNAP protein label concentration was plotted according to the emission spectrum intensity. The relationship between the fluorescence intensity and the fluorescence intensity is shown in Figures 2-6.
  • the SNAP protein label concentration has a good linear relationship with the fluorescence intensity of the probe in the range of 0.1 uM to 14.8 uM. Therefore, the protein label can be quantitatively detected according to the standard curve.
  • Hela cells are used as an example to examine the labeling effect of compounds in mammalian cells.
  • Hela cells stably expressing protein tags, Hela-WT cells (Hela original cells, unexpressed protein tags) were planted in 14 mm glass bottom 96-well cell culture plates and stabilized for 10 hours.
  • Probe 16, probe 17, and probe 18 were separately added to the medium and diluted to 5 ⁇ M.
  • the cells were incubated for 2 hours in a 37 ° C carbon dioxide incubator, and the labeling cell fluorescence changes were detected using a Leica TPS-8 confocal microscope.
  • probe 17, and probe 18 can be applied to target proteins labeled with different organelles.
  • Hela cells were used as an example to examine the effect of probes labeling different subcellular protein tags.
  • Hela cells 5000 cells were seeded in a 96-well glass-bottomed cell culture plate. After 14 hours, the protein was labeled with different organelle localization plasmids using the lipo2000 kit. The original medium was removed 24 hours after transfection and cultured with phenol-free red DMEM. The cells were washed twice, and the cells were incubated for 2 hours using a phenol red-free medium containing a 0.2 ⁇ M probe, and the cell labeling effect was examined using a leica TCS-8 confocal microscope. The results are shown in Figure 8.
  • the probe can clearly display a variety of subcellular organelle structures in the absence of wash, including but not limited to cytoplasmic matrix, Golgi, mitochondria, chromosomes, cytoskeleton;
  • the probes described above can serve as a powerful tool for cell subcellular organelle markers.
  • Hela cells 5000 cells were seeded per well in 96-well glass bottom cell culture plates, and 14 hours later, pcdna3.1-CLIP-histone (CLIP protein tag chromosomal localization plasmid), pcdna3.1-mito-SNAP were co-transfected with lipo2000 kit. (SNAP protein tag mitochondrial localization plasmid), 0.1 ⁇ g per well; the original medium was removed 24 hours after transfection, and the phenol red-free DMEM medium was used twice, respectively, using 0.2 ⁇ M of probe 16 and probe 9 respectively. The cells were incubated for 2 hours in phenol red medium and examined for cell labeling using a leica TCS-8 confocal microscope. The results are shown in Figure 9.
  • Probe 16 and probe 9 can clearly display mitochondria and chromosomal structures, respectively, while washing-free, and the co-localization coefficient of chromosomal fluorescence labeled by probe 9 and mitochondrial fluorescent channel labeled by probe 16. Below 0.1, the two fluorescent channels do not interfere with each other.
  • the plasmid pcdna3.1-SNAP (sample group) expressing the SNAP protein tag and the control plasmid pcdna3.1-CAT (control group) not expressing the SNAP protein tag were introduced into the mouse.
  • the plasmid is dissolved in a large volume of solution and rapidly injected into the mouse by tail vein injection, and the mouse liver absorbs the plasmid to express the protein of interest.
  • Twenty hours after the plasmid injection 0.4 ⁇ M probe 16 dissolved in 200 ul of PBS was injected into the mouse to mark the SNAP protein tag by tail vein injection method; after 6 hours, the mice were dissected and the small difference was detected by the Kodak multispectral living imaging system.
  • the above experiments show that the fluorescence of the probe is not affected by the environment of the animal, and can be applied to living animals, and can specifically label the SNAP protein tag expressed in the liver and generate a strong fluorescent signal.
  • the SNAP protein of the mammalian cell is taken as an example, and the fluorescence of the probe bound to the SNAP protein after protein degradation is detected by using the AID degradation system in Hela cells as an example. Variety.
  • HeLa cells were seeded at 2000/cm 2 in a 20 mm glass bottom cell culture dish, and 14 hours later, pcdna3.1-TIR1 and pcdna3.1-SNAP-IAA17-H2B plasmid were transfected with invitogen lipofectmain2000 transfection reagent.
  • the original cell culture medium-labeled cells were replaced with phenol red-free DMEM medium containing 1 ⁇ M of probe 16, and the cell samples were incubated in a carbon dioxide incubator at 37 ° C for 1 hour.
  • the Leica SP8 laser confocal microscope was used to detect the fluorescent signal of the cell marker, and indoleacetic acid (IAA) was added to induce the degradation of SNAP-IAA17-H2B protein, and the change of cell fluorescence during protein degradation was detected.
  • IAA indoleacetic acid
  • the fluorescence signal of SNAP-IAA17-H2B protein gradually decreased.
  • the fluorescence signal was almost invisible, and the rate of protein degradation was consistent with the results reported in the literature.
  • the above experiments show that the fluorescent properties of the probe are also dependent on the presence of the protein in mammalian cells.
  • the fluorescence is activated.
  • the fluorescence disappears and can be used to track and monitor the degradation process of the target protein.
  • the mammalian cell SNAP protein was taken as an example, and the light stability and simultaneous expression in the cells of the probe 16, probe 17 and probe 18 labeled proteins were detected in HeLa cells.
  • the fluorescent proteins IFP682 were compared for their photostability under the same conditions.
  • HeLa cells were plated into 96-well glass bottom plates at 5000 cells/well. After 12 hours, the histone-localized SNAP protein or fluorescent protein was transfected, and cultured for 36 hours, and then imaged using a leica SP8 confocal microscope. The output power was A 200 ⁇ W 633 nm laser was taken.
  • Figure 12 is a graph showing the fluorescence intensity of a protein as a function of bleaching time, wherein A to C are probe 16, probe 17, and probe 18, respectively, and D is the fluorescent protein IFP682. As can be seen from the figure, the ability of the probe 16, the probe 17 and the probe 18 to label the protein is significantly better than the fluorescent protein IFP682 reported in the literature.
  • a fluorescently activated covalently labeled reference fluorescent probe 33 suitable for SNAP protein tagging was constructed (prepared by the method of CN107641121A):
  • probe 16 and probe 18 of the present invention were detected in HeLa cells using mammalian cell SNAP protein as an example.
  • HeLa cells were plated into 96-well glass bottom plates at 5000 cells/well. After 12 hours, the histone-localized SNAP protein was transfected, and after 36 hours of culture, it was imaged using a leica SP8 confocal microscope, using a 633 nm output of 200 ⁇ W. The laser is used for shooting.
  • FIG. 13 is a graph showing the change in fluorescence intensity of a protein as a function of bleaching time, wherein A to C are probe 16, probe 17, probe 18, respectively, and D is a reference probe 33. As can be seen from the figure, the bleaching resistance of the probe 16, the probe 17 and the probe 18 after labeling the protein is significantly better than the reference probe 33 reported by the patent.
  • the probe 2 In order to verify that the probes composed of different electron acceptors in the conjugated system B of the formula (I-1-1) to (I-1-7) have excellent photobleaching resistance, the probe 2, the probe 5, The probe 11, the probe 13, the probe 16, the probe 28, and the probe 30 are exemplified, and the mammalian cell SNAP protein is taken as an example, and the light stability in the cell after detecting the labeled protein in HeLa cells.
  • HeLa cells were plated into 96-well glass bottom plates at 5000 cells/well. After 12 hours, the histone-localized SNAP protein or fluorescent protein was transfected, and cultured for 36 hours, and then imaged using a leica SP8 confocal microscope.
  • FIG. 14 is a graph showing the fluorescence intensity of a protein as a function of bleaching time, wherein A to G are probe 16, probe 28, probe 5, probe 11, probe 30, probe 2, and probe 13, respectively. 14 It can be seen that the probe labeled protein has strong bleaching resistance.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Optics & Photonics (AREA)
  • Materials Engineering (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种荧光探针及其制备方法和用途，所述荧光探针对粘度响应敏感并且特异，可用于蛋白的特异性荧光标记，还可用于蛋白的定量、检测或动力学研究以及细胞、组织和活体影像。

Description

一种荧光探针及其制备方法和用途 技术领域
本发明涉及一种荧光探针及其制备方法和用途。
背景技术
荧光特异性标记，是研究蛋白功能并定量的一种重要手段。和其它研究方法相比，荧光标记具有灵敏、原位、即时、可视等不可替代的优势。当前，蛋白的特异性荧光标记最常用的方法就是通过基因融合技术将荧光蛋白原位表达于目标蛋白上，从而实现对目标蛋白的特异性点亮，并能够在荧光显微镜下对细胞或组织内靶蛋白进行跟踪研究。荧光蛋白技术经过较长时间发展，技术相对成熟，然而依然存在不少缺陷，例如，荧光蛋白成熟折叠较慢、容易聚集；并且一旦表达，很难进行后期修饰；另外，大多荧光蛋白还存在光稳定性差等不足；这些在一定程度上限制了荧光蛋白的应用。
事实上，荧光蛋白生色团的分子结构相对单一，构建不同种类或者功能化的荧光蛋白，往往没有多少规律可循，只能借助于无规突变的方式进行海选。相比，有机小分子荧光染料的分子结构丰富的多，但是小分子荧光探针在蛋白特异性标记方面仍存在很多缺陷。最近，化学标签(chemical tag)技术的出现，有效地解决了小分子荧光探针蛋白特异性标记的问题。该技术将具有特异性识别功能的多肽或蛋白标签(tag)与靶蛋白融合，并利用该标签与底物高度特异性结合的特点，实现小分子荧光探针特异性的蛋白标记，因此，化学标签技术不仅继承了基因融合技术的优势，而且与荧光蛋白相比，它充分继承了有机染料探针的各方面的优势。目前，SNAP-tag(K.Johnsson et.al.WO 2004031405.)、CLIP-tag(K.Johnsson et.al.WO 2008012296.)、Halo-tag(Wood，Keith V et.al.WO 2004072232)等蛋白标签技术已经商业化，特别是SNAP-tag和CLIP-tag应用最为广泛，并得到了市场的一致认可。
虽然SNAP-tag和CLIP-tag等化学标签技术对所属蛋白的标记具有特异性。但事实上，标记过程中，无论游离的探针还是已标记的探针都存在同样的荧光发射。也就是说，不管标记上的还是没标记上的探针都在该体系内发射荧光。这种非特征性的荧光发射，显然是当前化学标签技术的严重缺陷。因此，从严格意义上，这种方法依然无法达到与荧光蛋白同等的特异性。将没有标记上的探针通过洗涤的方法加以去除，是当前解决以上问题的唯一有效方法。显然，在一些需要快速，或者无法洗涤的场合，该技术的应用将受到严重限制。
假若设计一种适用于SNAP-tag和CLIP-tag的荧光激活型蛋白特异标记的方法，它在未标记之前是暗的或发射非常弱的荧光，一旦它标记在蛋白上后，染料 的荧光急剧增强。毫无疑问，这类设计的探针将有可能实现与荧光蛋白具有等同的特异性，不仅可以免除游离探针洗涤，大幅降低游离探针的背景干扰，而且势必拓宽SNAP-tag和CLIP-tag技术的应用。设计适用于该技术的荧光激活型蛋白特异标记的方法，必须考虑一个合适的荧光失/激活机制。FRET机制首先应用于这方面的设计，在配体上额外加入荧光淬灭基团，正常情况下小分子荧光被与之相连的基团淬灭；一旦配体与化学标签结合后淬灭基团离去，实现荧光激活(T.Komatsu.et.al.J.Am.Chem.Soc.2011,133,6745—6751.)。但是，淬灭基团的引入，大大增加了探针的分子体积，使标记速度大大下降，严重限制了探针在细胞、组织中蛋白的实时跟踪与检测，并且，荧光探针与淬灭基团之间必须具有较好的能级匹配，这样使得长波长，例如，红光发射染料的FRET设计非常困难。另外，一些荧光对极性敏感的染料也被用来设计激活型探针(T.K.Liu.et.al.ACS Chem.Biol.2014，9，2359—2365.)，染料处于细胞液等大极性溶剂中，探针没有荧光或者具有较弱荧光，当配体与蛋白结合，探针处于蛋白非极性口袋中，探针发射较强荧光。然而，一方面由于蛋白表面本身就存在一个极性较大的水化层，使探针的荧光增强幅度有限；另一方面，细胞或组织本身是个非常复杂的体系，各个细胞器的极性变化很大，这些都会导致极性敏感的探针在细胞或组织造影中具有很高的背景。最近，文献(T.Y.Wang et.al.Chem Sci.2016,7,301-307.)报导了具有粘度响应性的分子转子荧光探针，在蛋白配体与蛋白共价结合后，蛋白位阻的作用降低分子转子自由度，从而使得探针荧光激活。然而，该文献中探针分子荧光激活后的荧光亮度并不高，荧光量子产率非常低。因此，该文献报导的方法不能作为合格的荧光蛋白标签，用于靶蛋白的标记、跟踪、定位和定量。
发明内容
本发明人发现，通过将配体部分与粘度响应性荧光染料的电子供体部分相连，能够极大地提高配体与标签蛋白结合激活荧光后的荧光亮度，从而获得了一种全新结构的荧光探针，其具有粘度响应性，可用于蛋白的特异性标记，标记速度快、荧光激活亮度高、耐漂白能力强、适用范围广，能够有效用于靶蛋白的标记、跟踪、定位和定量。
鉴于此，本发明提供一种荧光探针，包括：配体部分A，任选的连接体部分C和荧光染料部分，所述荧光染料部分为粘度响应性荧光染料基团，包括电子供体部分D、共轭体系B和电子受体部分，所述配体部分A为能够与蛋白标签或融合蛋白标签的靶蛋白特异性识别并标记的基团，可选地，所述配体部分A为能够与蛋白标签或融合蛋白标签的靶蛋白特异性识别并共价标记的基团；其特征在于，所述配体部分A直接共价连接于荧光染料部分的电子供体部分D，或者，通过连接体部分C共价连接于荧光染料部分的电子供体部分D。
可选地，上述的荧光探针，其具有如式(I)所示的结构，
Figure PCTCN2019085192-appb-000001
其中，
所述配体部分A来自于O 6-烷基鸟嘌呤类化合物、烷基4-氯嘧啶类化合物或烷基胞嘧啶类化合物；
连接体部分C为任选存在的基团，选自亚烷基、改性亚烷基；
荧光染料部分具有如式(I-R)所示的结构，
Figure PCTCN2019085192-appb-000002
其中，
电子供体部分-D-为-NX 1-X 2-，X 1选自氢、烷基或改性烷基，X 2选自亚烷基或改性亚烷基，X 1，X 2任选相互连接，与N原子一起形成脂杂环；
共轭体系B具有如式(I-1-1)～(I-1-7)所示的结构，
Figure PCTCN2019085192-appb-000003
任选地，所述式(I-1-1)～(I-1-7)结构与X 1、X 2相互连接形成脂杂环；
电子受体部分具有如下式(I-2)所示的结构，
Figure PCTCN2019085192-appb-000004
其中，
R 1选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基；
R 2选自氢、氰基、羧基、酮基、酯基、酰胺基、硫代胺酰基、硫代酯基、磺酸基、磺酸酯基、砜基、亚砜基、芳基、杂芳基、烷基或改性烷基；
R 3为氰基；
电子受体部分任选形成下式(I-2-a)、(I-2-b)、(I-2-c)环状结构：
Figure PCTCN2019085192-appb-000005
其中，
R a、R b独立地选自氢、亲水性基团、烷基和改性烷基，R a和R b任选地相互连接形成脂环或脂杂环；
各个R c独立地选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基；各个R d独立地选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基或者为双键与芳香环、芳香杂环中的至少一种共轭连接而形成的基团；
各个Y 1独立地选自-O-、-S-、-(S＝O)-和-(NR i)-，其中R i选自氢、烷基或改性烷基；
各个Y 2独立地选自＝O、＝S、＝S＝O和＝NR i，其中R i选自氢、烷基或改性烷基；
各个Y 3独立地选自＝O、＝S、＝S＝O和＝NR i，其中R i选自氢、烷基或改性烷基；
或者，各个Y 3独立地为＝C(R e)(CN)；
R e选自氰基、羧基、酮基、酯基、酰胺基、亚磷酸酯基、磷酸酯基、磺酸基、磺酸酯基、砜基、亚砜基、芳基、杂芳基、烷基或改性烷基；
当R 2或R e为芳基或杂芳基时，环上的氢原子任选独立地被选自卤原子、氰基、硝基、亲水性基团、烷基或改性烷基中的取代基取代；任选地，所述取代基相互连接构成饱和或不饱和的脂环或脂杂环；
其中，
所述“烷基”为C 1-C 30的直链或支链的烷基；优选地，为C 1-C 10直链或支链烷基；优选地，为C 1-C 7直链或支链烷基；优选地，为C 1-C 5直链或支链烷基；优选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
所述“亚烷基”为C 1-C 30的直链或支链的亚烷基；优选地，为C 1-C 7直链或支链亚烷基；优选地，为C 1-C 5直链或支链亚烷基；
所述“改性烷基”为烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-CS-、-NO 2、-CN、-S-、-SO 2-、-(S＝O)-、
Figure PCTCN2019085192-appb-000006
苯基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、饱和或不饱和的单环或双环亚环烃基、联芳杂环、桥联脂杂环中的至少一种基团置换所得的基团，所述改性烷基具有1～30个碳原子，其碳碳单键任选独立地被碳碳双键或碳碳叁键置换；
所述“改性亚烷基”为亚烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-NO 2、-CN、-S-、-CS-、-SO 2-、-(S＝O)-、
Figure PCTCN2019085192-appb-000007
苯基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、饱和或不饱和的单环或双环亚环烃基、联芳杂环、桥联脂杂环中的至少一种基团置换所得的基团，所述改性亚烷基具有1～30个碳原子，其碳碳单键任选独立地被碳碳双键或碳碳叁键置换；
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“脂环”为饱和或不饱和的4～10元单环或多环脂环；
所述“脂杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的饱和或不饱和的4～10元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被卤原子、硝基、烷基、芳基、亲水性基团和改性烷基取代；
所述“芳基或芳香环”为5～10元单环或稠合双环或多环芳香基团；
所述“杂芳基或芳香杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的5～10元单环或稠合双环或多环杂芳香基团；
所述“卤原子”各自独立地选自F、Cl、Br、I；
所述“亲水性基团”为羟基、磺酸基、羧基、亚磷酸酯基、伯氨基、仲氨基或叔氨基；
所述“单环亚环烃基”为4～7元亚环烃基；
所述“双环亚环烃基”为5～10元双环亚环烃基；
所述“联芳杂环”为两个或两个以上芳香杂环共轭连接形成的基团；
所述“桥联脂杂环”为环上含有选自N、O、或S中的至少一种杂原子的5～20元桥联脂杂环；
所述“酮基”为R-(C＝O)R'基团；
所述“酯基”为R(C＝O)OR'基团；
所述“酰胺基”为RCONHR'基团；
所述“硫代胺酰基”为R(C＝S)NHR'基团；
所述“硫代酯基”为R(C＝S)OR'基团；
所述“亚磷酸酯基”为RP(＝O)(OH) 2基团；
所述“磷酸酯基”为ROP(＝O)(OH) 2基团；
所述“磺酸基”为RSO 3H基团；
所述“磺酸酯基”为RSO 2OR'基团；
所述“砜基”为RSO 2R'基团；
所述“亚砜基”RSOR'基团；
所述“伯胺基”为RNH 2基团；
所述“仲胺基”为RNHR'基团；
所述“叔胺基”为RNR'R”基团；
所述“季铵盐基”R R'R”R”'N +基团；
各个R、R'、R”、R”'各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基，所述改性烷基或改性亚烷基为C 1-C 10(优选为C 1-C 6)烷基或亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中一种基团置换所得的基团；
可选地，所述改性烷基或改性亚烷基各自独立地为含有选自-OH、-O-、乙二醇单元(-(CH 2CH 2O) n-)、C 1～C 8烷基、C 1～C 8烷氧基、C 1～C 8酰基氧基、C 1～C 8卤代烷基、单糖基团、二糖基团、多糖基团、-O-CO-、-NH-CO-、-(-NH-CHR””-CO-) n-、-SO 2-O-、-SO-、-SO 2-NH-、-S-S-、-CH＝CH-、- C≡ C-、卤原子、氰基、硝基、邻硝基苯基、苯甲酰甲基、磷酸酯基中至少一种基团的基团，其中，n为1～100，优选为1～50，更优选为1～30，更优选为1～10；R””为H或α氨基酸的残基；所述“磷酸酯基”具有如上所述定义；
所述“单糖单元”为不能再被简单地水解为更小的糖分子的糖类单元；
所述“二糖单元”为两个单糖失水而成的糖类单元；
所述“多糖单元”为十个以上单糖失水而成的糖类单元；
可选地，所述C 1～C 8烷基为甲基、乙基、丙基、异丙基，所述C 1～C 8烷氧基为甲氧基、乙氧基、丙氧基、异丙氧基，所述C 1～C 8酰基氧基为乙酰氧基、乙基、丙基、异丙基，所述C 1～C 8卤代烷基为三氟甲基、氯甲基、溴甲基；
可选地，所述脂杂环选自氮杂环丁烷、吡咯烷、哌啶、四氢呋喃、四氢吡喃、吗啉、硫代吗啉；
可选地，所述芳香杂环选自、
Figure PCTCN2019085192-appb-000008
可选地，所述芳香环选自
Figure PCTCN2019085192-appb-000009
可选地，上述的荧光探针，其特征在于：
所述蛋白标签为提纯品、未提纯品或存在于细胞或组织的原位状态；
可选地，所述蛋白标签为O 6-烷基鸟嘌呤-DNA烷基转移酶(SNAP-tag)或 其突变体、烷基胞嘧啶转移酶(CLIP-tag)或其突变体；
可选地，所述O 6-烷基鸟嘌呤-DNA烷基转移酶的突变体选自SNAP F33G或SNAP V164E；
可选地，所述蛋白标签为O 6-烷基鸟嘌呤-DNA烷基转移酶(SNAP-tag)或其突变体；
可选地，适用于SNAP-tag的配体部分A来自于O 6-烷基鸟嘌呤类化合物或烷基4-氯嘧啶类化合物；可选地，适用于CLIP-tag的配体部分A来自于烷基胞嘧啶类化合物；
可选地，所述配体部分A-选自下式结构：
Figure PCTCN2019085192-appb-000010
可选地，所述连接体部分C选自具有1～30个碳原子的饱和直链或支链的烷基，该烷基链上的一个或多个碳原子被-O-或-(C＝O)-置换；所述的碳原子被-O-或-(C＝O)-置换，是指碳原子或碳原子与其上的氢原子一起被-O-或-(C＝O)-置换；
可选地，X 1为任选被一个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-30直链或支链烷基，X 2为任选被一个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-30直链或支链亚烷基；或者X 1、X 2各自独立地为任选被一个或多个选自磺酸基、羧基的基团取代的含1-10个氧原子的C 2-30醚链基团；或者，-NX 1-X 2形成选自下式(I-i-1)、(I-i-2)的任一基团：
Figure PCTCN2019085192-appb-000011
可选地，X 1为任选被1个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-10直链或支链烷基，X 2为任选被1个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-10直链或支链烷基或亚烷基；
可选地，所述R 2和R e独立地为选自以下结构的基团，或者，由以下结构自身或相互之间稠合形成的双环或多环稠芳香环或稠芳香杂环：优选为双环或三环稠芳香环或稠芳香杂环；
Figure PCTCN2019085192-appb-000012
可选的，R 2或R e的上述结构中CH上的H被卤原子、硝基、亲水性基团、烷基或改性烷基取代；可选地，R 2或R e为选自上述结构中的含NH的基团，可选地，所述NH上的H被烷基或改性烷基取代；
或者，所述R 2选自氢、氰基、羧基、酮基、酯基、酰胺基、硫代胺酰基、硫代酯基，并且，当选自酮基、酯基或酰胺基时，通过酮基、酯基或酰胺基中的羰基连接到式(I-2)、式(I-2-a)、式(I-2-b)、式(I-2-c)的烯基碳上，当选自硫代胺酰基、硫代酯基时，通过硫代胺酰基、硫代酯基中的硫羰基连接到式(I-2)、式(I-2-a)、式(I-2-b)、式(I-2-c)的烯基碳上；R e选自氰基、酮基、酯基、酰胺基，当选自酮基、酯基、酰胺基时，通过酮基、酯基、酰胺基中的羰基连接到式(I-2-a)或式(I-2-c)的烯基碳上；
可选地，所述电子受体部分为选自下式(I-2-1)～(I-2-22)中的一种：
Figure PCTCN2019085192-appb-000013
可选地，上述的荧光探针，其特征在于，所述荧光探针选自下式化合物：
Figure PCTCN2019085192-appb-000014
Figure PCTCN2019085192-appb-000015
Figure PCTCN2019085192-appb-000016
另一方面，还提供制备上述的荧光探针的方法，其特征在于，包括式(II)所示荧光染料与配体以及任选的连接体发生反应的步骤：
Figure PCTCN2019085192-appb-000017
其中，D’反应后能够形成D-基团与连接基团或配体键合。
本发明还提供一种荧光激活型蛋白特异性标记方法，其特征在于，包括以下步骤：将上述的荧光探针与蛋白标签或者融合蛋白标签的靶蛋白接触，所述荧光探针的配体部分与蛋白标签发生标记反应，将荧光探针标记到蛋白标签上；可选地，所述将荧光探针标记到蛋白标签上为共价标记；
可选地，所述标记反应的反应介质选自纯蛋白溶液、细胞裂解液或蛋白标签或融合蛋白标签的靶蛋白所处在的原位介质；可选地，所述原位介质为细胞内介质、细胞器内介质、活体组织介质、血液或体液。
本发明还提供上述的荧光探针在蛋白荧光标记，蛋白的定量、检测或动力学研究，以及细胞、组织、活体影像中的用途。
本发明还提供一种探针试剂盒，其特征在于，包括上述的荧光探针。
可选地，所述探针试剂盒还包含生物相容性介质；可选地，所述生物相容性介质选自二甲基亚砜、缓冲剂、生理盐水中的至少一种；可选地，所述缓冲剂包括磷酸盐缓冲液。
本发明所述蛋白标签(tag)或者是融合该标签的靶蛋白部分可通过现有基因工程技术制备而来。
本发明所述的具有粘度响应性荧光染料是指染料分子的荧光强度对溶液粘 度响应，随溶液的粘度增大，荧光强度增强。可选地，所述的具有粘度响应性荧光染料为：在25℃下，同等浓度和激发波长的条件下，染料最大荧光发射强度在甘油中的荧光强度与在甲醇中的荧光强度之比大于2，优选大于5，更优选大于10的有机染料分子。所述粘度响应性染料的浓度的范围在1×10 -7M～1×10 -5M。
根据具体情况，本领域工作人员可根据需要选择对应的标签与配体。
本领域技术人员可以利用具有相应配制的仪器设备对蛋白标签或者是融合蛋白标签的靶蛋白进行跟踪监测，根据需要，所用的仪器设备包括能测试或显示荧光的设备与设施，如荧光光谱仪、荧光显微镜、共聚焦荧光显微镜、酶标仪、流式细胞仪和活体成像仪等设备。
根据需要，操作者可以选择不同种类或发射/激发波长的染料。
根据一方面的实施方案，荧光探针荧光发射波长范围广。
根据一方面的实施方案，荧光探针的荧光强度随环境粘度的增大而增强，对粘度响应灵敏，具有粘度响应性。
根据一方面的实施方案，荧光探针可用于蛋白标签或融合蛋白标签的靶蛋白的特异性标记，荧光探针结合蛋白标签后荧光可以被激活，具有良好的荧光分子开关性质，而且荧光激活倍数高，荧光激活亮度高。
根据一方面的实施方案，荧光探针标记蛋白的速度非常快。
根据一方面的实施方案，荧光探针的荧光强度和蛋白标签浓度具有很好的线性关系，可用于目标蛋白的定量检测。
根据一方面的实施方案，荧光探针可以实现特异性标记细胞内蛋白标签，并且实现荧光特异性点亮，同时，探针荧光不受细胞内环境影响。
根据一方面的实施方案，荧光探针可以作为细胞亚细胞器标记的有力工具，例如对细胞核、线粒体、高尔基体、内质网、全细胞、细胞骨架、细胞外膜、溶酶体、细胞内膜等的标记。
根据一方面的实施方案，不同的荧光探针荧光基团的光谱不会相互干扰，本发明不同色系的荧光探针，可以对样本进行多色标记，可以同时进行正交标记成像。
根据一方面的实施方案，荧光探针的荧光不受动物内环境影响，可以应用于活体动物体内，例如特异性标记表达在肝脏部位的SNAP蛋白标签，并产生较强荧光信号。
根据一方面的实施方案，荧光探针可以用于跟踪、监测目标蛋白的降解过程。
根据一方面的实施方案，荧光探针实时监测哺乳动物细胞内生物大分子的组装与降解过程。
根据一方面的实施方案，荧光探针可以对组织、活体等不适宜清洗的样本进 行快速造影成像。
根据一方面的实施方案，荧光探针的耐漂白性质优异，光稳定性更好。
根据一方面的实施方案，荧光探针未标记蛋白标签或融合蛋白标签的靶蛋白时几乎不产生检测信号，不干扰对样本的检测，可以实现复杂样本的快速定量检测，还可以跟踪标记反应过程的动力学过程。
附图说明
图1为不同探针与蛋白标签结合后不同波长的荧光被激活的荧光发射图；
图2～图6分别为探针1、探针5、探针13、探针16、探针28的荧光强度对不同SNAP蛋白标签浓度做的标准曲线；
图7为不同探针标记细胞荧光图谱，其中，(1)～(3)分别对应探针16、探针17、探针18，A组是表达蛋白标签的Hela细胞，B组是Hela-WT细胞(Hela原始细胞，未表达蛋白标签)；
图8为不同探针标记不同的细胞器，其中，A组～C组分别为探针16、探针17、探针18，(1)～(5)分别为细胞质基质、高尔基体、线粒体、染色体、细胞骨架；
图9为不同探针对相同细胞进行双色标记，其中，A为探针16标记的线粒体，B为探针9标记的细胞核，C为A和B的正交成像；
图10为探针16用于活体小鼠肝脏的标记，其中，A组为对照组，B组为样品组；
图11为探针16在哺乳动物细胞中的荧光随蛋白降解的变化；
图12为探针16、探针17、探针18标记SNAP蛋白和荧光蛋白IFP682在哺乳动物细胞中的光稳定性对比，其中A～C分别为探针16、探针17、探针18，D为IFP682；
图13为探针16、探针17、探针18和参比探针33标记SNAP蛋白在哺乳动物细胞中的光稳定性对比，其中A～C分别为探针16、探针17、探针18，D为参比探针33；
图14为探针2、探针5、探针11、探针13、探针16、探针28、探针30标记SNAP蛋白在哺乳动物细胞中的光稳定性，其中A～G分别为探针16、探针28、探针5、探针11、探针30、探针2、探针13。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于对本发明进行示例性说明，并不用于限制本发明。
实施例1
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激 活型共价标记的荧光探针1：
Figure PCTCN2019085192-appb-000018
化合物1：
参照文献(Hwan Myung Kim et al.ANAL CHEM.2014，86，308-311.)公开的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝10.13(s，1H)，7.83-7.89(m，2H)，7.25-7.34(m，1H)，7.13(d，1H)，6.73(d，1H)，3.68(t，2H，J＝5.6Hz)，3.53(t，2H，J＝5.6Hz)，3.08(s，3H)。
化合物2：
化合物1(0.461g，2mmol)和氰基乙酸叔丁酯(0.338g，2.4mmol)溶于50ml无水乙醇中，加入催化量无水氯化锌，Ar保护条件下油浴加热5h，反应结束，冷却至室温，旋转蒸发除掉部分溶剂，体系有大量固体析出，过滤，滤饼用冷乙醇洗两次，真空烘干，即得纯的黄色化合物2(0.41g，88％)。产率82％。 1H-NMR(400MHz，CDCl 3)：δ＝8.07(s，1H)，7.83-7.89(m，2H)，7.25-7.34(m，1H)，7.13(d，1H)，6.73(d，1H)，3.68(t，2H，J＝5.6Hz)，3.53(t，2H，J＝5.6Hz)，3.08(s，3H)，1.52(s，9H)。
化合物3：
参照文献(Antje Keppler et.al.Nat Biotechnology.2002,21,86-89.)公开的方法进行合成。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.82(s，1H)，7.39(m，4H)，6.27(s，2H)，5.45(s，2H)，3.71(s，2H)。
探针1:
化合物2(0.353g，1.0mmol)和4-二甲氨基吡啶(0.146g，1.2mmol)于20ml的无水二氯甲烷中溶解，氩气保护条件下缓慢滴加入对硝基氯甲酸苯酯(0.242g，1.2mmol)的10ml无水二氯甲烷溶液，滴加完毕，室温搅拌1h，反应完毕，旋转蒸干溶剂，残余物溶于10ml无水N，N-二甲基甲酰胺中，加入化合物3(0.324g，1.2mmol)，加入无水三乙胺(0.16ml，1.2mmol)，Ar保护条件下室温搅拌30min，反应完毕，旋转蒸干溶剂，残余物过柱分离，即得纯的探1 0.32g，产率90％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.03(s,1H),8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.83-7.89(m，2H)，7.79(s,1H),7.44(d,J＝7.9Hz,2H),7.25-7.34(m，3H)，7.13(d，1H)，6.73(d，1H)，6.27(s,2H),5.44(s,2H), 4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H),1.49(s,9H)。
实施例2
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针2
Figure PCTCN2019085192-appb-000019
化合物4：
参照化合物2的合成方法，产率86％。1H-NMR(400MHz，CDCl 3)：8.08(s，1H)，7.83-7.89(m，2H)，7.49(d，1H，J＝8.4Hz)，7.36-7.42(m，3H)，7.25-7.34(m，1H)，7.13(d，1H)，6.73(d，1H)，3.61(t，2H，J＝8.0Hz)，3.34(t，2H，J＝8.0Hz)，3.11(s，3H)。
探针2：
参照探针1的合成方法，产率82％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.31(s,1H),8.52(t,J＝5.8Hz,1H)，8.31(s,1H),8.04(d,J＝7.8Hz,1H),7.89(d,J＝8.1Hz,1H),7.83-7.89(m，2H),7.79(s,1H),7.48(t,J＝7.6Hz,1H),7.44(d,J＝7.9Hz,2H),7.37(t,J＝7.5Hz,1H),7.25-7.34(m，3H)，7.13(d，1H)，6.73(d，1H)，6.27(s,2H),5.76(s,1H),5.44(s,2H),4.88(d,J＝4.8Hz,2H),4,37(d,J＝5.8Hz,2H),3.65(d,J＝4.7Hz,2H),3.45(s,2H),3.08(s,3H).
实施例3
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针3：
Figure PCTCN2019085192-appb-000020
化合物5：
按照文献公开的方法合成。1H-NMR(400MHz，CD3OD)：δ＝7.84(d，1H， J＝6.0Hz)，7.40(d，2H，J＝8.0Hz)，7.31(d，2H，J＝8.0Hz)，6.14(d，1H，J＝6.0)，5.29(s，2H)，3.78(s，2H)。
探针3：
参照探针1的合成方法，产率62％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.03(s，1H)，7.86(d，1H)，7.79(dd,2H),7.74(t，1H)，7.62(d,1H),7.36(d，2H，J＝6.0Hz)，7.26(d，2H，J＝6.0Hz)，7.22(dd,1H),6.92(d,1H),6.85(bs，2H)，6.08(d，1H)，5.20(s，2H)，4.24(t，2H)，4.15(d，2H)，3.66(t，2H)，3.14(s，3H),1.54(s，9H)。
实施例4
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针4：
Figure PCTCN2019085192-appb-000021
化合物6：
按照文献(Srikun D K et.al.JACS 2010，132，4455-4465.)公开的方法合成。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.33(d，2H，J＝8.0Hz)，7.31(d，2H，J＝8.0Hz)，7.10(brs，2H)，6.10(s，1H)，5.25(s，2H)，3.68(s，2H)。
探针4：
参照探针1的合成方法，产率62％。 1H-NMR(400MHz，DMSO-d 6)：δ＝9.99(brs，1H)，8.01(s，1H)，7.83-7.89(m，2H)，7.72(t，1H)，7.39(d，2H)，7.25-7.34(m，3H)，7.13(d，1H)，6.73(d，1H)，5.26(s，2H)，4.36(d，2H)，3.55-3.59(m，4H)，3.08(s，3H)，1.50(s，9H)。
实施例5
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针5：
Figure PCTCN2019085192-appb-000022
化合物7：
N-甲基-N-羟乙基苯胺(1.88g，12.5mmol)和NaHCO 3(1.57g，18.7mmol)溶于48ml二氯甲烷和36ml水中，冷却至0℃，缓慢加入I 2(3.0g，11.8mmol)，加毕，体系逐渐升至室温，搅拌30min，体系加300ml二氯甲烷40ml水稀释，分出有机相，有机相分别用水、硫代硫酸钠溶液、食盐水洗涤，无水硫酸钠干燥后旋转蒸干，残余物柱色谱分离，得纯化合物7(2.46g，92％)。 1H-NMR(400MHz，CDCl 3)：δ＝7.46(d，1H，J＝7.60Hz)，6.56(d，1H，J＝7.60Hz)，3.78(t，2H，J＝4.80Hz)，3.44(t，2H，J＝4.80Hz)，2.94(s，3H)。
化合物8：
化合物7(0.554g，2mmol)和5-醛基-2-噻吩硼酸(0.374g，2.4mmol)溶于10ml甲苯、10ml乙醇中，加入2ml 2 N K 2CO 3溶液，氩气保护条件下85℃油浴加热5h，反应完毕，冷却至室温，加水10ml淬灭，分出有机相，水相用二氯甲烷萃取，合并有机相，食盐水洗涤，无水硫酸钠干燥，旋转蒸干过柱，得纯化合物0.339g，产率65％。 1H-NMR(400MHz，CDCl 3)：δ＝9.81(s，1H)，7.68(s，1H)，7.55(d，1H，J＝8.00Hz)，7.25(d，2H，J＝8.00Hz)，6.78(d，2H，J＝8.00Hz)，3.86(t，2H，J＝4.80Hz)，3.56(t，2H，J＝4.80Hz)，3.06(s，3H)。
化合物9：
参照化合物2的合成方法，产率98％。 1H-NMR(400MHz，CDCl 3)：δ＝8.01(s，1H)，7.68(d，1H)，7.55(d，1H)，7.25(d，2H，J＝8.00Hz)，6.78(d，2H，J＝8.00Hz)，3.86(t，2H，J＝4.80Hz)，3.56(t，2H，J＝4.80Hz)，3.06(s，3H),1.50(s，9H)。
探针5：
参照探针1的合成方法，产率54％。 1H-NMR(400MHz，CDCl 3)：δ＝12.42(s，1H)，10.01(s，1H)，8.01(s，1H)，7.81(s，1H)，7.68(s，1H)，7.55(d，1H，J＝8.00Hz)，7.40(m，4H)，7.25(d，2H，J＝8.00Hz)，6.78(d，2H，J＝8.00Hz)，6.29(s，2H)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.86(t，2H，J＝4.80Hz)，3.56(t，2H，J＝4.80Hz)，3.06(s，3H)，1.50(s，9H)。
实施例6
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针6：
Figure PCTCN2019085192-appb-000023
化合物10：
氰乙酸(1.0g，10mmol)加入25ml圆底烧瓶中，加入2-甲氧基乙胺，Ar保护条件下室温搅拌，反应完毕，加入10ml无水乙醚，超声分散，过滤，真空干燥得白色固体。 1H-NMR(400MHz，CDCl 3)：δ＝6.5(s，1H)，3.48-3.52(m，4H)，3.38(s，3H)。
化合物11：
参照化合物2的合成方法，产率91％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.31(s，1H)，8.22(bt，1H)，7.82(d，1H，J＝4.00Hz)，7.58(d，2H，J＝8.80Hz)，7.50(d，2H，J＝4.00Hz)，6.77(d，2H，J＝8.80Hz)，4.74(bt，1H)，3.57(t，2H，J＝5.20Hz)，3.41—3.48(m，4H)，3.38(t，2H，J＝5.20Hz)，3.27(s，3H)，3.01(s，3H)。
探针6：
参照探针1的合成方法，产率45％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.42(s，1H)，10.01(s，1H)，8.31(s，1H)，8.22(bt，1H)，7.82(m，2H)，7.58(d，2H，J＝8.80Hz)，7.50(d，2H，J＝4.00Hz)，7.40(m，4H)，6.77(d，2H，J＝8.80Hz)，6.29(s，2H)，5.46(s，2H)，4.74(bt，1H)，4.40(d，2H，J＝4.8Hz)，3.57(t，2H，J＝5.20Hz)，3.41—3.48(m，4H)，3.38(t，2H，J＝5.20Hz)，3.27(s，3H)，3.01(s，3H)。
实施例7
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针7：
Figure PCTCN2019085192-appb-000024
化合物12：
参照化合物2的合成方法，产率98％。1H-NMR(400MHz，CDCl3)：δ＝7.81(s,1H),7.64-7.71(m,3H),7.55(d，1H)，7.30-7.38(m,2H),7.25(d，2H，J＝8.00Hz)，6.78(d，2H，J＝8.00Hz)，4.90(t,J＝5.2Hz,1H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H)。
探针7：
参照探针1的合成方法，产率55％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.55(t,J＝5.8Hz,1H),8.37(s,1H),7.81(s,1H),7.64-7.71(m,3H),7.55(d，1H)，7.44(d,J＝7.9Hz,2H),7.30-7.38(m,4H),7.25(d，2H，J＝8.00Hz)，6.78(d，2H，J＝8.00Hz)，6.27(s,2H),5.44(s,2H),4.90(t,J＝5.2Hz,1H),4,37(d,J＝5.8Hz,2H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H)。
实施例8
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针8：
Figure PCTCN2019085192-appb-000025
探针8：
参照探针1的合成方法，产率73％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.03(s，1H)，7.86(d，1H)，7.74(t，3H)，7.68(d，1H)，7.55(d，1H)，7.36(d，2H，J＝6.0Hz)，7.26(d，2H，J＝6.0Hz)，7.25(d，2H，J＝8.00Hz)，6.85(bs，2H)，6.78(d，2H，J＝8.00Hz)，6.08(d，1H)，5.20(s，2H)，4.24(t，2H)，4.15(d，2H)，3.66(t，2H)，3.14(s，3H),1.54(s，9H)。
实施例9
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针9：
Figure PCTCN2019085192-appb-000026
化合物13：
6-溴-1-苯并呋喃(0.4g，2mmol)溶于15mL N-甲基-N-羟乙基胺，加入铜粉(6.4mg，0.01mmol)，碘化亚铜(19mg，0.01mmol)，磷酸三钾(0.850g，4mmol)，Ar保护条件下80℃油浴加热过夜，反应完毕，冷却至室温，体系倒入50mL水中，二氯甲烷萃取3次×50mL，合并有机相，旋转蒸干溶剂，柱色谱分离得黄色产物0.362g，产率87％。 1H-NMR(400MHz，CDCl 3)：δ＝8.02(s，1H)，7.66(d，1H，J＝8.4Hz)，7.44-7.48(m，1H)，7.41(m，1H)，7.29(m，1H)，3.60(t，2H，J＝5.6Hz)，3.34(t，J＝8.0Hz，3H)，3.10(s，3H)。
化合物14：
化合物13(0.382g，2mmol)溶于50ml无水二氯甲烷中，加入1ml三乙胺，冰浴条件下缓慢滴加醋酸酐(0.3ml，3mmol)，滴加完毕，体系缓慢升至室温，搅拌3h，反应完毕，加水100ml，分出有机相，水相用二氯甲烷50ml二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，旋转蒸干溶剂，残余物无需进一步纯化，直接用于下一步。
上述残余物溶于50ml二氯甲烷中，加入二甲基甲酰胺5ml，冰浴条件下加入三氯氧磷2ml，Ar保护条件下搅拌0.5h，体系缓慢升至室温，继续搅拌5h，反应完毕，加入饱和碳酸钠溶液调pH＝10.0，室温条件下搅拌过夜，次日分出有机相，水相用二氯甲烷50ml萃取三次，合并有机相，饱和食盐水洗涤两次，有机相用无水硫酸钠干燥，旋转蒸干溶剂，残余物柱色谱分离得黄色固体0.235g，产率56％。
1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.10(s，3H)。
化合物15:
参照化合物2的合成方法，产率91％。 1H-NMR(400MHz，CDCl 3)：δ＝8.22(s，1H)，8.02(s，1H)，6.43(s，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.11(s，3H)，1.48(s，9H)。
探针9：
参照探针1的合成方法，产率66％。 1H-NMR(400MHz，CDCl 3)：δ＝12.42(s，1H)，10.01(s，1H)，8.20(s，1H)，7.81(s，2H)，7.68(d，J＝9.0Hz，1H)，7.40(m，4H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，6.29(s，2H)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.11(s，3H)，1.51(s，9H)。
实施例10
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针10：
Figure PCTCN2019085192-appb-000027
化合物16:
参照化合物2的合成方法，产率93％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.45(s，1H)，8.09(d，J＝8.00Hz，2H)，8.07(s，1H)，7.94(d，J＝8.00Hz，2H)，7.51(m，1H)，7.41(m，1H)，6.45(s，1H)，3.61(t，3H，J＝8.0Hz)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H)。
探针10:
参照探针1的合成方法，产率71％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.42(s，1H)，10.01(s，1H)，8.45(s，1H)，8.09(d，J＝8.00Hz，2H)，8.07(s，1H)，7.94(d，J＝8.00Hz，2H)，7.81(s，1H)，7.51(m，1H)，7.41(m，5H)，6.45(s，1H)，6.29(s，2H)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.61(t，3H，J＝8.0Hz)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H)。
实施例11
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针11：
Figure PCTCN2019085192-appb-000028
化合物17：
参照化合物2的合成方法，产率89％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.09(d，1H，J＝8.00Hz)，7.94(d，1H，J＝8.00Hz)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，7.51(m，1H)，7.41(m，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz,1H)，6.45(s，1H)，3.61(t，2H，J＝8.0Hz)，3.34(t，2H，J＝8.0Hz)， 3.21(s，3H)。
探针11：
参照探针1的合成方法，产率66％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.33(s，1H)，10.12(s，1H)，8.03(d，1H，J＝8.00Hz)，7.94(d，1H，J＝8.00Hz)，7.81(s，2H)，7.68(d，J＝9.0Hz，1H)，7.51(m，1H)，7.41(m，5H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz,1H)，6.45(s，1H)，6.29(s，2H)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.62(t，2H，J＝8.0Hz)，3.36(t，J＝8.0Hz，2H)，3.21(s，3H)。
实施例12
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针12：
Figure PCTCN2019085192-appb-000029
探针12：
参照探针1的合成方法，产率61％。 1H-NMR(400MHz，CDCl 3)：δ＝8.22(s，1H)，8.02(s，1H)，7.93(d，1H，J＝5.6Hz)，7.75(s，1H)，7.33(d，2H，J＝8.0Hz)，7.19(d，2H，J＝8.0Hz)，6.43(s，1H)，6.06(d，1H，J＝5.6Hz)，5.27(s，2H)，5.16(s，2H)，4.45(d，2H，J＝5.6Hz)，3.62(t，J＝8.0Hz，3H)，3.35(t，J＝8.0Hz，3H)，3.21(s，3H)，1.48(s，9H)。
实施例13
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针13：
Figure PCTCN2019085192-appb-000030
化合物18：
参照文献(Martinez M.et al.Org.Biomol.Chem.2012.10.3892-3898.)公开的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝7.24(dd，J 1＝5.2Hz，J 2＝1.2Hz，1H)，7.13(dd，1H，J1＝3.6Hz，J2＝1.2Hz)，7.03(dd，1H，J1＝5.2Hz，J2＝1.2Hz)，6.99(d，1H，J＝3.8Hz)，6.93(d，1H，J＝3.6Hz)。
化合物19：
参照化合物13的合成方法，产率78％。 1H-NMR(400MHz，CDCl3)：δ＝7.25(dd，J 1＝5.2Hz，J 2＝1.2Hz，1H)，7.13(dd，1H，J1＝3.6Hz，J 2＝1.2Hz)，7.03(dd，1H，J 1＝5.2Hz，J 2＝1.2Hz)，6.99(d，1H，J＝3.8Hz)，6.93(d，1H，J＝3.6Hz)，3.85(t，2H，J＝4.80Hz)，3.46(t，2H，J＝4.80Hz)，3.10(s，3H)。
化合物20：
参照化合物14的合成方法，产率65％。 1H-NMR(400MHz，DMSO-d 6)：δ＝9.75(s，1H)，7.57(d，1H，J＝4.00Hz)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，5.81(d，1H，J＝4.00Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.27(s，3H)，3.13(s，3H)。
化合物21：
参照化合物2的合成方法，产率98％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.00(s，1H)，7.57(d，1H，J＝4.00Hz)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，5.81(d，1H，J＝4.00Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.13(s，3H)，1.50(s，9H)。
探针13：
参照探针1的合成方法，产率45％。 1H-NMR(400MHz，DMSO-d 6)：δ＝11.52(s，1H)，10.01(s，1H)，8.00(s，1H)，7.57(d，1H，J＝4.00Hz)，7.81(s，1H)，7.40(m，4H)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，6.29(s，2H)，5.81(d，1H，J＝4.00Hz)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.27(s，3H)，3.13(s，3H)，1.50(s，9H)。
实施例14
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针14：
Figure PCTCN2019085192-appb-000031
化合物22：
参照化合物2的合成方法，产率97％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.04(d，1H，J＝8.0Hz)，7.94(d，1H，J＝8.0Hz)，7.89(s，1H)，7.57(d，1H，J＝4.00Hz)，7.53(t，1H，J＝8.0Hz)，7.45(t，1H，J＝8.0Hz)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，5.81(d，1H，J＝4.00Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.13(s，3H)。
探针14：
参照探针1的合成方法，产率48％。 1H-NMR(400MHz，DMSO-d 6)：δ＝11.82(s，1H)，10.21(s，1H)，8.04(d，1H，J＝8.0Hz)，7.94(d，1H，J＝8.0Hz)，7.89(s，1H)，7.81(s，1H)，7.57(d，1H，J＝4.00Hz)，7.53(t，1H，J＝8.0Hz)，7.45(t，1H，J＝8.0Hz)，7.40(m，4H)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，6.29(s，2H)，5.81(d，1H，J＝4.00Hz)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.13(s，3H)。
实施例15
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针15：
Figure PCTCN2019085192-appb-000032
探针15：
参照探针1的合成方法，产率56％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.00(s，1H)，7.93(d，1H，J＝5.6)，7.75(s，1H)，7.57(d，1H，J＝4.00Hz)，7.33(d，2H，J＝8.0Hz)，7.19(d，2H，J＝8.0Hz)，7.13(d，1H，J＝4.00Hz)，6.95(d，1H，J＝4.00Hz)，6.06(d，1H，J＝5.6Hz)，5.81(d，1H，J＝4.00Hz)，5.27(s， 2H)，5.16(s，2H)，4.45(d，2H，J＝5.6Hz)，3.67(t，2H，J＝5.60Hz)，3.35(t，2H，J＝5.60Hz)，3.13(s，3H)，1.50(s，9H)。
实施例16
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针16：
Figure PCTCN2019085192-appb-000033
化合物23：
参照文献(Kimin Lim et al.J.Phys.Chem.C.201,115,22640-22646.)公开的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝7.48(s,1H),7.41(d,1H,J＝8.1Hz),7.32(d,1H,J＝5.1Hz),7.30(d,1H,J＝7.8Hz),7.11(d,1H,J＝4.5Hz),1.46(s,6H)。
化合物24：
参照化合物13的合成方法，产率66％。 1H-NMR(400MHz，CDCl 3)：δ＝7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(d,1H),7.11(d,1H)，3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.46(s,6H)。
化合物25：
参照化合物14的合成方法，产率75％。 1H-NMR(400MHz，CDCl 3)：δ＝9.84(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.46(s,6H)。
化合物26：
参照化合物2的合成方法，产率95％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.50(s，9H)，1.46(s,6H)。
探针16：
参照探针1的合成方法，产率45％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.03(s,1H),8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.48(s,1H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.32(d,1H),7.31(s,1H),7.30(d,J＝7.9Hz,2H),6.27(s,2H),5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H),1.49(s,9H)。
实施例17
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激 活型共价标记的荧光探针17：
Figure PCTCN2019085192-appb-000034
化合物27：
参照化合物2的合成方法，产率89％。 1H NMR(400MHz,DMSO-d 6):δ＝8.37(s,1H),7.81(s,1H),7.64-7.71(m,2H),7.41(d,1H),7.35-7.38(m,2H),7.32(d,1H),6.24(s,1H),4.90(t,J＝5.2Hz,1H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.42(s,6H).
探针17：
参照探针1的合成方法，产率56％。 1H NMR(400MHz,DMSO-d 6):δ＝8.55(t,J＝5.8Hz,1H),8.37(s,1H),7.79(s,1H),7.81(s,1H),7.64-7.71(m,2H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.35-7.38(m,4H),7.32(d,1H),6.27(s,2H),6.24(s,1H),5.44(s,2H),4.90(t,J＝5.2Hz,1H),4,37(d,J＝5.8Hz,2H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.42(s,6H).
实施例18
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针18：
Figure PCTCN2019085192-appb-000035
化合物28：
参照化合物2的合成方法，产率95％。 1H NMR(400MHz,DMSO-d 6):δ＝8.33(s,1H),7.74(s,1H),7.64-7.71(m,2H),7.41(d,1H),7.35-7.38(m,2H),7.22(d,1H),6.24(s,1H),4.90(t,J＝5.2Hz,1H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.41(s,6H).
探针18：
参照探针1的合成方法，产率54％。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(t, J＝5.8Hz,1H),8.20(s,1H),7.79(s,1H),7.73(s,1H),7.64-7.71(m,2H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.35-7.38(m,4H),7.32(d,1H),6.27(s,2H),6.24(s,1H),5.44(s,2H),4.90(t,J＝5.2Hz,1H),4,37(d,J＝5.8Hz,2H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.41(s,6H).
实施例19
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针19：
Figure PCTCN2019085192-appb-000036
化合物29：
参照化合物11的合成方法，产率87％。 1H-NMR(400MHz，CDCl3)：δ＝8.03(s，1H)，7.48(s,1H),7.45(t,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.48-3.52(m，4H)，3.46(t，2H)，3.27(s，3H)，3.10(s，3H)，1.46(s,6H)。
探针19：
参照探针1的合成方法，产率67％。 1H-NMR(400MHz,DMSO-d 6):δ＝8.57(t,1H),8.55(s,1H),8.42(t,1H),8.03(s,1H),7.53(m,2H),7.16(t,2H),7.11(t,2H),6.99(s,2H),6.81(s,1H),6.64(d,1H),5.16(s,2H),4.48(t,2H),4.29(m,2H),4.23(d,2H),3.76(t,2H),3.30(s,3H),3.04(t,2H),2.75(s,3H),1.72(s,6H)。
实施例20
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针20：
Figure PCTCN2019085192-appb-000037
化合物31：
参照文献(Gamba-Sánchez.et.al.Tetrahedron Lett.2015，56，4308-4311)的方法合成。1H-NMR(400MHz，CDCl3)：δ＝6.52(s，2H)，3.48-3.52(m，4H)， 3.38(s，3H)。
化合物31：
参照化合物11的合成方法，产率73％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),7.01(t,1H),3.85(t，2H)，3.48-3.52(m，4H)，3.46(t，2H)，3.27(s，3H)，3.10(s，3H)，1.46(s,6H)。
探针20：
参照探针1的合成方法，产率70％。 1H-NMR(400MHz,DMSO-d 6):δ＝8.57(s,1H),8.03(s,1H),7.63(s,1H),7.53(d,2H),7.25(s,1H),7.16(t,2H),7.11(t,2H),6.99(s,2H),6.83(d,1H),6.81(d,1H),6.64(d,1H),5.16(s,2H),4.48(t,2H),4.29(m,2H),4.23(d,2H),3.76(t,2H),3.30(s,3H),2.75(s,1H),2.73(t,2H),2.0(s,1H),1.72(s,6H)。
实施例21
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针21：
Figure PCTCN2019085192-appb-000038
化合物32：
参照文献(Gamba-Sánchez.et.al.Tetrahedron Lett.2015.56.4308-4311)的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝6.56(s，2H)，3.42(s，3H)。
化合物33：
参照化合物2的合成方法，产率65％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.22(s，3H)，3.10(s，3H)，1.46(s,6H)。
探针21：
参照探针1的合成方法，产率33％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.03(s,1H),8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.48(s,1H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.32(d,1H),7.31(s,1H),7.30(d,J＝7.9Hz,2H),6.27(s,2H),5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.21(s,3H)，3.09(s,3H)。
实施例22
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针22：
Figure PCTCN2019085192-appb-000039
化合物34：
参照化合物2的合成方法，产率67％。 1H-NMR(400MHz，CDCl 3)：δ＝11.03(s，1H)，8.03(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.46(s,6H)。
探针22：
参照探针1的合成方法，产率85％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.03(s,1H),11.22(s,1H)，8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.48(s,1H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.32(d,1H),7.31(s,1H),7.30(d,J＝7.9Hz,2H),6.27(s,2H),5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H)。
实施例23
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针23：
Figure PCTCN2019085192-appb-000040
化合物35：
参照化合物2的合成方法，产率55％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s，1H)，7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.46(s,6H)。
探针23：
参照探针1的合成方法，产率58％。 1H-NMR(400MHz，DMSO-d 6)：δ＝13.58(s,1H),12.22(s,1H)，8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.48(s,1H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.32(d,1H),7.31(s,1H),7.30(d,J＝7.9Hz,2H),6.27(s,2H),5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t, J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H)。
实施例24
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针24：
Figure PCTCN2019085192-appb-000041
探针24：
参照探针1的合成方法，产率85％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.03(s，1H)，7.86(d，1H)，7.74(t，3H)，7.48(s,1H),7.41(d,1H),7.36(d，2H，J＝6.0Hz)，7.32(d,1H),7.30(s,1H),7.26(d，2H，J＝6.0Hz)，6.85(bs，2H)，6.08(d，1H)，5.20(s，2H)，4.24(t，2H)，4.15(d，2H)，3.66(t，2H)，3.14(s，3H),1.54(s，9H)，1.42(s，6H)。
实施例25
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针25：
Figure PCTCN2019085192-appb-000042
探针25：
参照探针1的合成方法，产率88％。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.93(d，1H，J＝7.2Hz)，7.89(s，1H)，7.79(s，1H)，7.74(d，1H，J＝4.0Hz)，7.55(d，1H，J＝4.0Hz),7.42(m，2H)，7.41(d,1H),7.32(d,1H),7.31(d，2H，J＝8.0Hz)，7.18(m，3H)，6.96(d，2H，J＝5.6Hz)，6.06(d，1H，J＝5.6Hz)，5.27(s，2H)，5.15(s，2H)，4.45(d，2H，J＝5.6Hz)，3.85(t，2H，J＝5.6Hz)，4.12(s，2H)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H)。
实施例26
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针26：
Figure PCTCN2019085192-appb-000043
探针26：
参照探针1的合成方法，产率88％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.18(m,1H),8.03(t,1H),8.01(m,1H),7.94(d,1H),7.74(s,2H),7.54(m,1H),7.53(m,3H),7.16(t,2H),7.11(t,2H),6.83(d,1H),6.81(d,1H),6.64(d,1H),6.19(d,1H)5.16(s,2H),4.48(t,2H),4.29(t,2H),4.23(d,2H),2.75(s,3H),1.72(s,6H)。
实施例27
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针27：
Figure PCTCN2019085192-appb-000044
探针27：
参照探针1的合成方法，产率86％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.18(m,1H),8.03(t,1H),8.01(m,1H),7.94(d,1H),7.74(s,2H),7.54(m,1H),7.53(m,3H),7.48(s,1H),7.41(d,1H),7.32(d,1H),7.30(s,1H),7.16(t,2H),7.11(t,2H),6.83(d,1H),6.81(d,1H),6.64(d,1H),6.19(d,1H)5.16(s,2H),4.48(t,2H),4.29(t,2H),4.23(d,2H),2.75(s,3H),1.72(s,6H)。
实施例28
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针28：
Figure PCTCN2019085192-appb-000045
化合物36：
参照文献(Kimin Lim et al.J.Phys.Chem.C.201,115,22640-22646.)公开的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝7.51(s,1H),7.43(d,1H,J＝8.1Hz),7.31(d,1H,J＝5.1Hz),7.27(d,1H,J＝7.8Hz),7.18(d,1H,J＝4.5Hz),1.44(s,6H)。
化合物37：
参照化合物13的合成方法，产率56％。 1H-NMR(400MHz，CDCl 3)：δ＝7.52(s,1H),7.41(d,1H),7.32(d,1H),7.22(d,1H),7.11(d,1H)，3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.45(s,6H)。
化合物38：
参照化合物14的合成方法，产率70％。 1H-NMR(400MHz，CDCl 3)：δ＝9.88(s，1H)，7.53(s,1H),7.40(d,1H),7.32(d,1H),7.30(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.46(s,6H)。
化合物39：
参照化合物2的合成方法，产率95％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s，1H)，7.51(s,1H),7.44(d,1H),7.32(d,1H),7.21(s,1H),3.85(t，2H)，3.46(t，2H)，3.10(s，3H)，1.50(s，9H)，1.45(s,6H)。
探针28：
参照探针1的合成方法，产率75％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.05(s,1H),8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.48(s,1H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.32(d,1H),7.31(s,1H),7.30(d,J＝7.9Hz,2H),6.27(s,2H),5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H),1.49(s,9H)。
实施例29
以分子转子作为粘度响应性荧光染料，构建适用于CLIP蛋白标签的荧光激活型共价标记的荧光探针29：
Figure PCTCN2019085192-appb-000046
探针29：
参照探针1的合成方法，产率74％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.05(s，1H)，7.68(d，1H)，7.58(t，3H)，7.45(s,1H),7.43(d,1H),7.29(d，2H，J＝6.0Hz)，7.27(d,1H),7.24(s,1H),7.11(d，2H，J＝6.0Hz)，6.85(bs，2H)，6.08 (d，1H)，5.20(s，2H)，4.24(t，2H)，4.15(d，2H)，3.66(t，2H)，3.15(s，3H),1.46(s，9H)。
实施例30
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针30：
Figure PCTCN2019085192-appb-000047
化合物40：
参照文献(Eric A.Owens et.al.Dyes and Pigments,2015,113,27-37)的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝7.76(d，1H)，7.60(s，1H)，7.03(d，1H)，2.34(s，3H)，1.42(s，6H)。
化合物41：
化合物40(0.474g，2mmol)溶于50ml二氧六环中，加入0.4g二氧化锡，80度油浴条件下搅拌3h，反应完毕，过滤，加水100ml，二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，旋转蒸干溶剂，柱色谱分离的产物0.45g，产率89％。NMR(400MHz，CDCl 3)：δ＝9.74(s，1H)，7.76(d，1H)，7.60(s，1H)，7.03(d，1H)，1.42(s，6H)。
化合物42：
参照化合物13的合成方法，产率58％。 1H-NMR(400MHz，CDCl 3)：δ＝9.74(s，1H)，7.76(d，1H)，7.60(s，1H)，7.03(d，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.42(s，6H)。
化合物43：
参照化合物2的合成方法，产率98％。 1H-NMR(400MHz，CDCl 3)：δ＝8.05(s，1H)，7.76(d，1H)，7.60(s，1H)，7.03(d，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.45(s，9H)，1.42(s，6H)。
探针30：
参照探针1的合成方法，产率75％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.10(s,1H),8.55(t,J＝5.8Hz,1H),8.12(s,1H),7.79(s,1H),7.76(d，1H)，7.60(s，1H)，7.44(d,J＝7.9Hz,2H),7.30(d,J＝7.9Hz,2H),7.03(d，1H)，6.27(s,2H), 5.44(s,2H),4.89(t,J＝5.6Hz,1H),4.37(d,J＝5.8Hz,2H),3.65(t,J＝5.6Hz,2H),3.48(t,J＝5.6Hz,2H),3.09(s,3H),1.49(s,9H)，1.42(s，6H)。
实施例31
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针31：
Figure PCTCN2019085192-appb-000048
化合物44：
参照化合物2的合成方法，产率93％。 1H NMR(400MHz,DMSO-d 6):δ＝8.33(s,1H),7.74(s,1H),7.64-7.71(m,2H),7.41(d,1H),7.35-7.38(m,2H),7.22(d,1H),4.90(t,J＝5.2Hz,1H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.41(s,6H).
探针31：
参照探针1的合成方法，产率67％。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(t,J＝5.8Hz,1H),8.20(s,1H),7.79(s,1H),7.73(s,1H),7.64-7.71(m,2H),7.44(d,J＝7.9Hz,2H),7.41(d,1H),7.35-7.38(m,4H),7.32(d,1H),6.27(s,2H),5.44(s,2H),4.90(t,J＝5.2Hz,1H),4,37(d,J＝5.8Hz,2H),3.66(t,J＝6.0Hz,2H),3.47(t,J＝6.0Hz,2H),3.10(s,3H),1.41(s,6H).
实施例32
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的荧光探针32：
Figure PCTCN2019085192-appb-000049
探针32：
参照探针1的合成方法，产率87％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.03(s，1H)，7.86(d，1H)，7.74(t，3H)，7.48(s,1H),7.41(d,1H),7.36(d，2H，J＝6.0Hz)，7.32(d,1H),7.26(d，2H，J＝6.0Hz)，6.85(bs，2H)，6.08(d，1H)，5.20(s，2H)，4.24(t，2H)，4.15(d，2H)，3.66(t，2H)，3.14(s，3H),1.54 (s，9H)，1.42(s，6H)。
实施例33
采用文献(T.Y.Wang et.al.Chem Sci.2016,7,301-307.)报道的方法制备得到参比探针BG-CCVJ和BG-Gly-CCVJ。
将探针1-32以及参比探针BG-CCVJ和BG-Gly-CCVJ分别溶于二甲基亚砜中，制得浓度均为1×10 -2M的母液，分别加入甘油和甲醇中，混合均匀，配制终浓度为1×10 -5M的溶液，根据探针不同，依次用各探针的最大激发波长在相同条件下检测其荧光发射图谱，结果如表1所示。
由表1可知，实施例探针荧光发射波长范围广，并且在甘油和甲醇中的荧光强度区别很大，对粘度变化响应灵敏，具有粘度响应性。
实施例34
将探针与对应的蛋白标签混合得混合样品，混合样品中探针终浓度为5μM，蛋白标签终浓度为10μM，将混合样品置于37℃孵育1小时，使用荧光分光光度计检测样品荧光强度变化，结果如表1所示。
由表1中游离探针量子产率可知：实施例的探针以及参比探针未与蛋白标签反应时荧光极低，接近PBS缓冲液本底荧光水平，说明在未与蛋白标签反应时，粘度响应性荧光探针的荧光未被激活，而由结合蛋白标签量子产率可知，实施例的探针同蛋白标签反应后可以在相应的激发发射通道检测到明显的荧光信号增强，荧光激活倍数达到几百倍到一千倍以上，并且具有较高的亮度，说明实施例的探针结合蛋白标签后荧光可以被激活，具有良好的荧光分子开关性质；而参比探针虽然也有荧光激活的性质，但是，激活后荧光量子产率很低，亮度非常低。
综上可知：实施例的探针结合蛋白标签后荧光可以被激活，具有良好的荧光分子开关性质。
表1 不同探针荧光发射图谱检测结果
Figure PCTCN2019085192-appb-000050
Figure PCTCN2019085192-appb-000051
实施例35
将SNAP蛋白标签分别加入30uM的探针1、探针5、探针13、探针16、探针28的溶液中，制得SNAP蛋白标签终浓度为0.1uM、0.5uM、0.7uM、1.2uM、4.5uM、8.1uM、13.1uM、14.8uM的混合样品溶液，将混合样品溶液置于37℃反应1小时，使用荧光分光光度计检测样品激发发射光谱变化，根据发射光谱强度绘制SNAP蛋白标签浓度与荧光强度关系图，结果分别如图2～6所示。
从图2～6中可以看出，SNAP蛋白标签浓度在0.1uM～14.8uM的范围内与探针的荧光强度都具有较好的线性关系，因此，可以根据标准曲线对蛋白标签进行定量检测。
实施例36
Hela细胞为例检测化合物在哺乳动物细胞中的标记效果。将稳定表达蛋白标签的Hela细胞、Hela-WT细胞(Hela原始细胞，未表达蛋白标签)种植于于14mm玻璃底96孔细胞培养板中，稳定10小时。将探针16、探针17、探针18分别加入至培养基中并稀释至5μM。细胞置于37℃二氧化碳培养箱孵育2小时，使用Leica TPS-8共聚焦显微镜成像检测标记细胞荧光变化。图7B组结果显示加入上述探针后在Hela-WT细胞中未能检测到相应的荧光信号，说明探针荧光不受细胞内环境影响；而图7A组中表达蛋白标签的Hela细胞可以检测到强烈的荧光信号，荧光信号增强近700倍。
以上实验说明探针可以实现特异性标记细胞内蛋白标签，并且实现荧光特异性点亮，同时，探针荧光不受细胞内环境影响。
实施例37
为验证探针16、探针17、探针18可以应用于标记不同细胞器定位的目标蛋白，以Hela细胞为例检测了探针标记不同亚细胞蛋白标签的效果。Hela细胞5000细胞每孔种植于96孔玻璃底细胞培养板中，14小时后使用lipo2000试剂盒转染蛋白标签不同细胞器定位质粒；转染后24小时去除原有培养基，使用无酚红DMEM培养基清洗2次，使用含有0.2μM探针的无酚红培养基孵育细胞2小时，使用leica TCS-8共聚焦显微镜成像检测细胞标记效果。结果如图8显示，探针可以在免洗的情况下清晰展示多种亚细胞器结构，其中包括但不限于细胞质基质、高尔基体、线粒体、染色体、细胞骨架；
以上结果说明的探针可以作为细胞亚细胞器标记的有力工具。
实施例38
Hela细胞5000细胞每孔种植于96孔玻璃底细胞培养板中，14小时后使用lipo2000试剂盒共转染pcdna3.1-CLIP-histone(CLIP蛋白标签染色体定位质粒)、pcdna3.1-mito-SNAP(SNAP蛋白标签线粒体定位质粒)，每孔0.1μg；转染后24 小时去除原有培养基，使用无酚红DMEM培养基2次，分别使用含有0.2μM的探针16和探针9的无酚红培养基孵育细胞2小时，使用leica TCS-8共聚焦显微镜成像检测细胞标记效果。结果如图9显示，探针16与探针9可以在同时在免洗的情况下分别清晰展示线粒体和染色体结构，且探针9标记的染色体荧光与探针16标记的线粒体荧光通道共定位系数低于0.1，说明两个荧光通道之间不会相互干扰。
以上实验说明不同探针荧光基团的光谱不会相互干扰，可以同时进行正交标记成像。
实施例39
首先，将表达SNAP蛋白标签的质粒pcdna3.1-SNAP(样品组)和未表达SNAP蛋白标签的对照质粒pcdna3.1-CAT(对照组)导入小鼠体内。此方法是将质粒溶解在很大体积的溶液中通过尾静脉注射迅速注射入小鼠体内，小鼠肝脏吸收质粒，进而表达目的蛋白。质粒注射后20小时，将溶于200ul PBS中的0.4μM探针16通过尾静脉注射方法注射到小鼠体内标记SNAP蛋白标签；6小时后解剖小鼠，通过柯达多光谱活体成像系统检测不同小鼠样品肝脏部位荧光差异。结果如图10显示，注射探针16的对照质粒pcdna3.1-CAT的小鼠肝脏荧光很低，而注射探针16的SNAP质粒pcdna3.1-SNAP的小鼠肝脏具有较强的荧光，信号强度是对照组荧光的20倍以上。
以上实验说明探针的荧光不受动物内环境影响，可以应用于活体动物体内，且可以特异性标记表达在肝脏部位的SNAP蛋白标签，并产生较强荧光信号。
实施例40：
为验证本发明探针的荧光激活与蛋白的存在具有相关性，以哺乳动物细胞SNAP蛋白为例，在Hela细胞中以AID降解系统为例检测了结合SNAP蛋白的探针在蛋白降解后的荧光变化。首先，Hela细胞20000/cm 2种植于20mm玻璃底细胞培养皿中，14小时后通过invirtogen公司的lipofectmain2000转染试剂转染pcdna3.1-TIR1与pcdna3.1-SNAP-IAA17-H2B质粒。细胞转染后24小时，使用含有1μM探针16的无酚红DMEM培养基替换原有细胞培养基标记细胞，细胞样品置于37℃二氧化碳培养箱孵育1小时。标记完成后，使用Leica SP8激光共聚焦显微镜成像检测细胞标记荧光信号，并加入吲哚乙酸(IAA)，诱导SNAP-IAA17-H2B蛋白降解，检测蛋白降解过程中细胞荧光变化情况。结果如图11所示，SNAP-IAA17-H2B蛋白定位于细胞核中(0min)，加入吲哚乙酸诱导蛋白降解，随着时间的增加，SNAP-IAA17-H2B蛋白荧光信号逐渐降低，加入吲哚乙酸140min时，荧光信号基本不可见，蛋白降解速度与文献报道结果一致。以上实验说明探针的荧光性质在哺乳动物细胞中的同样依赖于蛋白的存在，当蛋白存在时荧光被激活，当蛋白降解以后，荧光消失，可用于跟踪、监测目标 蛋白的降解过程。
实施例41：
为验证本发明探针优异的耐光漂白性能，以哺乳动物细胞SNAP蛋白为例，在Hela细胞中检测探针16、探针17和探针18标记蛋白后在细胞内的光稳定性，同时表达荧光蛋白IFP682，并在同样条件下对比它们的光稳定性。将HeLa细胞按5000个/孔铺入96孔玻璃底培养板中，12小时后转染表达组蛋白定位的SNAP蛋白或荧光蛋白，培养36小时后使用leica SP8共聚焦显微镜成像，使用输出功率为200μW的633nm激光进行拍摄。缩放设为2x，拍摄区域为93μm*93μm，扫描电压为600V，每秒拍摄一帧(0.833s/帧)。图12为蛋白的荧光强度随漂白时间的变化图，其中A～C分别为探针16、探针17、探针18，D为荧光蛋白IFP682。由图可知，探针16、探针17和探针18标记蛋白后的耐漂白能力均要明显优于文献报道的荧光蛋白IFP682。
对比实施例42：
以分子转子作为粘度响应性荧光染料，构建适用于SNAP蛋白标签的荧光激活型共价标记的参比荧光探针33(参照CN107641121A中的方法制备)：
Figure PCTCN2019085192-appb-000052
参照专利(CN107641121A)公开的方法合成，产率45％。1H-NMR(400MHz，DMSO-d6)：δ＝12.42(s，1H)，10.01(s，1H)，7.89(s，1H)，7.18(s，1H)，7.81(s，1H)，7.4(m，4H)，6.96(d，2H，J＝5.6Hz)，6.29(s，2H)，5.46(s，2H)，4.40(d，2H，J＝4.8Hz)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(m，15H)。
为验证本发明探针探针16、探针17和探针18优异的耐光漂白性能，以哺乳动物细胞SNAP蛋白为例，在Hela细胞中检测探针16、探针17和探针18和参比探针33标记蛋白后在细胞内的光稳定性，并在同样条件下对比它们的光稳定性。将HeLa细胞按5000个/孔铺入96孔玻璃底培养板中，12小时后转染表达组蛋白定位的SNAP蛋白，培养36小时后使用leica SP8共聚焦显微镜成像，使用输出功率为200μW的633nm激光进行拍摄。缩放设为2x，拍摄区域为93μm*93μm，扫描电压为600V，每秒拍摄一帧(0.833s/帧)。图13为蛋白的荧光强度随漂白时间的变化图，其中A～C分别为探针16、探针17、探针18，D为参比探针33。由图可知，探针16、探针17和探针18标记蛋白后的耐漂白能力均要明显优于专利报道的参比探针33。
以上实验说明本发明探针荧光的耐漂白性质优异，其光稳定性明显优于已公开的参比探针33的光稳定性。
实施例43：
为验证式(I-1-1)～(I-1-7)中共轭体系B连接不同的电子受体所构成的探针均具有优异的耐光漂白性能，以探针2、探针5、探针11、探针13、探针16、探针28、探针30为例，以哺乳动物细胞SNAP蛋白为例，在Hela细胞中检测标记蛋白后在细胞内的光稳定性。将HeLa细胞按5000个/孔铺入96孔玻璃底培养板中，12小时后转染表达组蛋白定位的SNAP蛋白或荧光蛋白，培养36小时后使用leica SP8共聚焦显微镜成像，使用输出功率为200μW的633nm激光进行拍摄。缩放设为2x，拍摄区域为93μm*93μm，扫描电压为600V，每秒拍摄一帧(0.833s/帧)。图14为蛋白的荧光强度随漂白时间的变化图，其中A～G分别为探针16、探针28、探针5、探针11、探针30、探针2、探针13，由图14可知，探针标记蛋白后均拥有很强的耐漂白能力。
以上实验说明本发明探针荧光的耐漂白性质优异。

Claims (8)

  1. 一种荧光探针，包括配体部分A，任选的连接体部分C和荧光染料部分，所述荧光染料部分为粘度响应性荧光染料基团，包括电子供体部分D、共轭体系B和电子受体部分，所述配体部分A为能够与蛋白标签或融合蛋白标签的靶蛋白特异性识别并标记的基团，可选地，所述配体部分A为能够与蛋白标签或融合蛋白标签的靶蛋白特异性识别并共价标记的基团；其特征在于，所述配体部分A直接共价连接于荧光染料部分的电子供体部分D，或者，通过连接体部分C共价连接于荧光染料部分的电子供体部分D。
  2. 根据权利要求1所述的荧光探针，其具有如式(I)所示的结构，
    Figure PCTCN2019085192-appb-100001
    其中，
    所述配体部分A来自于O 6-烷基鸟嘌呤类化合物、烷基4-氯嘧啶类化合物或烷基胞嘧啶类化合物；
    连接体部分C为任选存在的基团，选自亚烷基、改性亚烷基；
    荧光染料部分具有如式(I-R)所示的结构，
    Figure PCTCN2019085192-appb-100002
    其中，
    电子供体部分-D-为-NX 1-X 2-，X 1选自氢、烷基或改性烷基，X 2选自亚烷基或改性亚烷基，X 1，X 2任选相互连接，与N原子一起形成脂杂环；
    共轭体系B具有如式(I-1-1)～(I-1-7)所示的结构，
    Figure PCTCN2019085192-appb-100003
    任选地，所述式(I-1-1)～(I-1-7)结构与X 1、X 2相互连接形成脂杂环；
    电子受体部分具有如下式(I-2)所示的结构，
    Figure PCTCN2019085192-appb-100004
    其中，
    R 1选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基；
    R 2选自氢、氰基、羧基、酮基、酯基、酰胺基、硫代胺酰基、硫代酯基、磺酸基、磺酸酯基、砜基、亚砜基、芳基、杂芳基、烷基或改性烷基；
    R 3为氰基；
    电子受体部分任选形成下式(I-2-a)、(I-2-b)、(I-2-c)环状结构：
    Figure PCTCN2019085192-appb-100005
    其中，
    R a、R b独立地选自氢、亲水性基团、烷基和改性烷基，R a和R b任选地相互连接形成脂环或脂杂环；
    各个R c独立地选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基；各个R d独立地选自氢、卤原子、硝基、烷基、芳基、杂芳基、亲水性基团或改性烷基或者为双键与芳香环、芳香杂环中的至少一种共轭连接而形成的基团；
    各个Y 1独立地选自-O-、-S-、-(S＝O)-和-(NR i)-，其中R i选自氢、烷基或改性烷基；
    各个Y 2独立地选自＝O、＝S、＝S＝O和＝NR i，其中R i选自氢、烷基或改性烷基；
    各个Y 3独立地选自＝O、＝S、＝S＝O和＝NR i，其中R i选自氢、烷基或改性烷基；
    或者，各个Y 3独立地为＝C(R e)(CN)；
    R e选自氰基、羧基、酮基、酯基、酰胺基、亚磷酸酯基、磷酸酯基、磺酸基、磺酸酯基、砜基、亚砜基、芳基、杂芳基、烷基或改性烷基；
    当R 2或R e为芳基或杂芳基时，环上的氢原子任选独立地被选自卤原子、氰基、硝基、亲水性基团、烷基或改性烷基中的取代基取代；任选地，所述取代基相互连接构成饱和或不饱和的脂环或脂杂环；
    其中，
    所述“烷基”为C 1-C 30的直链或支链的烷基；优选地，为C 1-C 10直链或支链烷基；优选地，为C 1-C 7直链或支链烷基；优选地，为C 1-C 5直链或支链烷基；优选地，选自甲基、 乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
    所述“亚烷基”为C 1-C 30的直链或支链的亚烷基；优选地，为C 1-C 7直链或支链亚烷基；优选地，为C 1-C 5直链或支链亚烷基；
    所述“改性烷基”为烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-CS-、-NO 2、-CN、-S-、-SO 2-、-(S＝O)-、
    Figure PCTCN2019085192-appb-100006
    苯基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、饱和或不饱和的单环或双环亚环烃基、联芳杂环、桥联脂杂环中的至少一种基团置换所得的基团，所述改性烷基具有1～30个碳原子，其碳碳单键任选独立地被碳碳双键或碳碳叁键置换；
    所述“改性亚烷基”为亚烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-NO 2、-CN、-S-、-CS-、-SO 2-、-(S＝O)-、
    Figure PCTCN2019085192-appb-100007
    苯基、亚苯基、伯氨基、仲氨基、叔氨基、季铵盐基、饱和或不饱和的单环或双环亚环烃基、联芳杂环、桥联脂杂环中的至少一种基团置换所得的基团，所述改性亚烷基具有1～30个碳原子，其碳碳单键任选独立地被碳碳双键或碳碳叁键置换；
    所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
    所述“脂环”为饱和或不饱和的4～10元单环或多环脂环；
    所述“脂杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的饱和或不饱和的4～10元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被卤原子、硝基、烷基、芳基、亲水性基团和改性烷基取代；
    所述“芳基或芳香环”为5～10元单环或稠合双环或多环芳香基团；
    所述“杂芳基或芳香杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的5～10元单环或稠合双环或多环杂芳香基团；
    所述“卤原子”各自独立地选自F、Cl、Br、I；
    所述“亲水性基团”为羟基、磺酸基、羧基、亚磷酸酯基、伯氨基、仲氨基或叔氨基；
    所述“单环亚环烃基”为4～7元亚环烃基；
    所述“双环亚环烃基”为5～10元双环亚环烃基；
    所述“联芳杂环”为两个或两个以上芳香杂环共轭连接形成的基团；
    所述“桥联脂杂环”为环上含有选自N、O、或S中的至少一种杂原子的5～20元桥联脂杂环；
    所述“酮基”为R-(C＝O)R'基团；
    所述“酯基”为R(C＝O)OR'基团；
    所述“酰胺基”为RCONHR'基团；
    所述“硫代胺酰基”为R(C＝S)NHR'基团；
    所述“硫代酯基”为R(C＝S)OR'基团；
    所述“亚磷酸酯基”为RP(＝O)(OH) 2基团；
    所述“磷酸酯基”为ROP(＝O)(OH) 2基团；
    所述“磺酸基”为RSO 3H基团；
    所述“磺酸酯基”为RSO 2OR'基团；
    所述“砜基”为RSO 2R'基团；
    所述“亚砜基”RSOR'基团；
    所述“伯胺基”为RNH 2基团；
    所述“仲胺基”为RNHR'基团；
    所述“叔胺基”为RNR'R”基团；
    所述“季铵盐基”R R'R”R”'N +基团；
    各个R、R'、R”、R”'各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基，所述改性烷基或改性亚烷基为C 1-C 10(优选为C 1-C 6)烷基或亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中一种基团置换所得的基团；
    可选地，所述改性烷基或改性亚烷基各自独立地为含有选自-OH、-O-、乙二醇单元(-(CH 2CH 2O) n-)、C 1～C 8烷基、C 1～C 8烷氧基、C 1～C 8酰基氧基、C 1～C 8卤代烷基、单糖基团、二糖基团、多糖基团、-O-CO-、-NH-CO-、-(-NH-CHR””-CO-) n-、-SO 2-O-、-SO-、-SO 2-NH-、-S-S-、-CH＝CH-、
    Figure PCTCN2019085192-appb-100008
    卤原子、氰基、硝基、邻硝基苯基、苯甲酰甲基、磷酸酯基中至少一种基团的基团，其中，n为1～100，优选为1～50，更优选为1～30，更优选为1～10；R””为H或α氨基酸的残基；所述“磷酸酯基”具有如上所述定义；
    所述“单糖单元”为不能再被简单地水解为更小的糖分子的糖类单元；
    所述“二糖单元”为两个单糖失水而成的糖类单元；
    所述“多糖单元”为十个以上单糖失水而成的糖类单元；
    可选地，所述C 1～C 8烷基为甲基、乙基、丙基、异丙基，所述C 1～C 8烷氧基为甲氧基、乙氧基、丙氧基、异丙氧基，所述C 1～C 8酰基氧基为乙酰氧基、乙基、丙基、异丙基，所述C 1～C 8卤代烷基为三氟甲基、氯甲基、溴甲基；
    可选地，所述脂杂环选自氮杂环丁烷、吡咯烷、哌啶、四氢呋喃、四氢吡喃、吗啉、硫代吗啉；
    可选地，所述芳香杂环选自、
    Figure PCTCN2019085192-appb-100009
    可选地，所述芳香环选自
    Figure PCTCN2019085192-appb-100010
  3. 根据权利要求1或2所述的荧光探针，其特征在于：
    所述蛋白标签为提纯品、未提纯品或存在于细胞或组织的原位状态；
    可选地，所述蛋白标签为O 6-烷基鸟嘌呤-DNA烷基转移酶(SNAP-tag)或其突变体、烷基胞嘧啶转移酶(CLIP-tag)或其突变体；
    可选地，所述O 6-烷基鸟嘌呤-DNA烷基转移酶的突变体选自SNAP F33G或SNAP V164E；
    可选地，所述蛋白标签为O 6-烷基鸟嘌呤-DNA烷基转移酶(SNAP-tag)或其突变体；
    可选地，适用于SNAP-tag的配体部分A来自于O 6-烷基鸟嘌呤类化合物或烷基4-氯嘧啶类化合物；可选地，适用于CLIP-tag的配体部分A来自于烷基胞嘧啶类化合物；
    可选地，所述配体部分A-选自下式结构：
    Figure PCTCN2019085192-appb-100011
    可选地，所述连接体部分C选自具有1～30个碳原子的饱和直链或支链的烷基，该烷基链上的一个或多个碳原子被-O-或-(C＝O)-置换；所述的碳原子被-O-或-(C＝O)-置换，是指碳原子或碳原子与其上的氢原子一起被-O-或-(C＝O)-置换；
    可选地，X 1为任选被一个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-30直链或支链烷基，X 2为任选被一个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-30直链或支链亚烷基；或者X 1、X 2各自独立地为任选被一个或多个选自磺酸基、羧基的基团取代的含1-10个氧原子的C 2-30醚链基团；或者，-NX 1-X 2形成选自下式(I-i-1)、(I-i-2)的任一基团：
    Figure PCTCN2019085192-appb-100012
    可选地，X 1为任选被1个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-10直链或支链烷基，X 2为任选被1个或多个选自羟基、氰基、卤原子、羧基、季铵基团的基团取代的C 1-10直链或支链烷基或亚烷基；
    可选地，所述R 2和R e独立地为选自以下结构的基团，或者，由以下结构自身或相互之间稠合形成的双环或多环稠芳香环或稠芳香杂环：优选为双环或三环稠芳香环或稠芳香杂环；
    Figure PCTCN2019085192-appb-100013
    可选的，R 2或R e的上述结构中CH上的H被卤原子、硝基、亲水性基团、烷基或改性烷基取代；可选地，R 2或R e为选自上述结构中的含NH的基团，可选地，所述NH上的H被烷基或改性烷基取代；
    或者，所述R 2选自氢、氰基、羧基、酮基、酯基、酰胺基、硫代胺酰基、硫代酯基，并且，当选自酮基、酯基或酰胺基时，通过酮基、酯基或酰胺基中的羰基连接到式(I-2)、式(I-2-a)、式(I-2-b)、式(I-2-c)的烯基碳上，当选自硫代胺酰基、硫代酯基时，通过硫代胺酰基、硫代酯基中的硫羰基连接到式(I-2)、式(I-2-a)、式(I-2-b)、式(I-2-c)的烯基碳上；R e选自氰基、酮基、酯基、酰胺基，当选自酮基、酯基、酰胺基时，通过酮基、酯基、酰胺基中的羰基连接到式(I-2-a)或式(I-2-c)的烯基碳上；
    可选地，所述电子受体部分为选自下式(I-2-1)～(I-2-22)中的一种：
    Figure PCTCN2019085192-appb-100014
  4. 根据权利要求1-3任一项所述的荧光探针，其特征在于，所述荧光探针选自下式化合物：
    Figure PCTCN2019085192-appb-100015
    Figure PCTCN2019085192-appb-100016
  5. 制备权利要求1-4中任一项所述的荧光探针的方法，其特征在于，包括式(II)所示荧光染料与配体以及任选的连接体发生反应的步骤：
    Figure PCTCN2019085192-appb-100017
    其中，D’反应后能够形成D-基团与连接基团或配体键合。
  6. 一种荧光激活型蛋白特异性标记方法，其特征在于，包括以下步骤：将权利要求1-4任一项所述的荧光探针与蛋白标签或者融合蛋白标签的靶蛋白接触，所述荧光探针的配体部分与蛋白标签发生标记反应，将荧光探针标记到蛋白标签上；可选地，所述将荧光探针标记到蛋白标签上为共价标记；
    可选地，所述标记反应的反应介质选自纯蛋白溶液、细胞裂解液或蛋白标签或融合蛋白标签的靶蛋白所处在的原位介质；可选地，所述原位介质为细胞内介质、细胞器内介质、活体组织介质、血液或体液。
  7. 权利要求1-4中任一项所述荧光探针在蛋白荧光标记、蛋白的定量、检测或动力学研究，以及细胞、组织、活体影像中的用途。
  8. 一种探针试剂盒，其特征在于，包括权利要求1-4中任一项所述荧光探针；
    可选地，所述探针试剂盒还包含生物相容性介质；可选地，所述生物相容性介质选自二甲基亚砜、缓冲剂、生理盐水中的至少一种；可选地，所述缓冲剂包括磷酸盐缓冲液。
PCT/CN2019/085192 2018-05-18 2019-04-30 一种荧光探针及其制备方法和用途 WO2019218876A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/250,061 US20210382060A1 (en) 2018-05-18 2019-04-30 Fluorescent probe and preparation method and use thereof
EP19803374.8A EP3798221A4 (en) 2018-05-18 2019-04-30 FLUORESCENT PROBE AND METHOD OF MANUFACTURE AND USE THEREOF

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810485224.4A CN110498799B (zh) 2018-05-18 2018-05-18 一种荧光探针及其制备方法和用途
CN201810485224.4 2018-05-18

Publications (1)

Publication Number Publication Date
WO2019218876A1 true WO2019218876A1 (zh) 2019-11-21

Family

ID=68539423

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/085192 WO2019218876A1 (zh) 2018-05-18 2019-04-30 一种荧光探针及其制备方法和用途

Country Status (4)

Country Link
US (1) US20210382060A1 (zh)
EP (1) EP3798221A4 (zh)
CN (1) CN110498799B (zh)
WO (1) WO2019218876A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023011656A1 (zh) 2021-08-06 2023-02-09 纳莹(上海)生物科技有限公司 一种荧光染料及其制备方法和用途

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349070B (zh) * 2020-02-12 2022-09-20 曲阜师范大学 一种用于检测生物细胞粘度的近红外荧光分子探针及其制备方法和应用
CN113501790A (zh) * 2020-03-23 2021-10-15 纳莹(上海)生物科技有限公司 一种荧光染料及其制备方法和用途
CN112592283B (zh) * 2021-01-25 2022-06-07 井冈山大学 一种用于酒类饮品粘度检测的荧光化合物及其制备和应用
CN114181204B (zh) * 2021-12-13 2023-07-11 洛兮医疗科技(杭州)有限公司 一种检测粘度的近红外荧光探针及其制备和应用
CN115650963B (zh) * 2022-09-09 2024-04-23 安徽大学 一种测定线粒体粘度的近红外双光子荧光探针及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031405A1 (en) 2002-10-03 2004-04-15 Ecole Polytechnique Federale De Lausanne (Epfl) Substrates for o6-alkylguanine-dna alkyltransferase
WO2004072232A2 (en) 2003-01-31 2004-08-26 Promega Corporation Covalent tethering of functional groups to proteins
WO2008012296A1 (en) 2006-07-25 2008-01-31 Epfl - Ecole Polytechnique Federale De Lausanne Labelling of fusion proteins with synthetic probes
CN104745177A (zh) * 2015-04-07 2015-07-01 华东理工大学 一种具有蛋白标签定位的光激活荧光探针及其制备方法和应用
CN107641121A (zh) 2016-07-20 2018-01-30 华东理工大学 一种荧光探针及其制备方法和用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11140083A (ja) * 1997-08-06 1999-05-25 Kumiai Chem Ind Co Ltd ベンゾフラン誘導体及び除草剤
WO2007041241A2 (en) * 2005-09-30 2007-04-12 The Trustees Of Columbia University In The City Of New York Compositions and methods for detecting ligand-receptor interactions
US9863937B2 (en) * 2012-09-26 2018-01-09 Agency For Science, Technology And Research Fluorescent molecular rotors
CN107663384B (zh) * 2016-07-20 2020-05-12 上海高驰资产管理有限公司 一种荧光染料及其制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004031405A1 (en) 2002-10-03 2004-04-15 Ecole Polytechnique Federale De Lausanne (Epfl) Substrates for o6-alkylguanine-dna alkyltransferase
WO2004072232A2 (en) 2003-01-31 2004-08-26 Promega Corporation Covalent tethering of functional groups to proteins
WO2008012296A1 (en) 2006-07-25 2008-01-31 Epfl - Ecole Polytechnique Federale De Lausanne Labelling of fusion proteins with synthetic probes
CN104745177A (zh) * 2015-04-07 2015-07-01 华东理工大学 一种具有蛋白标签定位的光激活荧光探针及其制备方法和应用
CN107641121A (zh) 2016-07-20 2018-01-30 华东理工大学 一种荧光探针及其制备方法和用途

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANTJE KEPPLER, NAT BIOTECHNOLOGY, vol. 21, 2002, pages 86 - 89
ERIC A.OWENS, DYES AND PIGMENTS, vol. 113, 2015, pages 27 - 37
GAMBA-SANCHEZ, TETRAHEDRON LETT, vol. 56, 2015, pages 4308 - 4311
HWAN MYUNG KIM ET AL., ANAL CHEM, vol. 86, 2014, pages 308 - 311
KIMIN LIM ET AL., J. PHYS. CHEM. C., vol. 201, no. 115, pages 22640 - 22646
MARTINEZ M. ET AL., ORG. BIOMOL. CHEM., vol. 10, 2012, pages 3892 - 3898
See also references of EP3798221A4
SRIKUN D K, JACS, vol. 132, 2010, pages 4455 - 4465
T. KOMATSU., J. AM. CHEM. SOC., vol. 133, 2011, pages 6745 - 6751
T. Y. WANG, CHEM SCI, vol. 7, 2016, pages 301 - 307
T.K. LIU., ACS CHEM. BIOL., vol. 9, 2014, pages 2359 - 2365

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023011656A1 (zh) 2021-08-06 2023-02-09 纳莹(上海)生物科技有限公司 一种荧光染料及其制备方法和用途

Also Published As

Publication number Publication date
US20210382060A1 (en) 2021-12-09
CN110498799B (zh) 2022-07-05
EP3798221A1 (en) 2021-03-31
EP3798221A4 (en) 2022-07-06
CN110498799A (zh) 2019-11-26

Similar Documents

Publication Publication Date Title
WO2018014821A1 (zh) 一种荧光探针及其制备方法和用途
WO2019218876A1 (zh) 一种荧光探针及其制备方法和用途
WO2019062876A1 (zh) 一种荧光探针及其制备方法和用途
JP7227741B2 (ja) カルボキシｘローダミン類似体
CN107089937B (zh) 线粒体靶向测定粘度的荧光探针及其制备方法和应用
WO2018223876A1 (zh) 一种荧光探针及其制备方法和用途
US20070134737A1 (en) Fluorophore compounds and their use in biological systems
CN108069967B (zh) 一种用于细胞内蛋白标记的荧光探针及其合成方法和应用
WO2018014820A1 (zh) 一种荧光染料及其制备方法和用途
JPWO2010126077A1 (ja) 近赤外蛍光化合物
WO2020221217A1 (zh) 一种荧光染料及其制备方法和用途
Xiao et al. An aggregation-induced emission platform for efficient Golgi apparatus and endoplasmic reticulum specific imaging
CN109369636A (zh) 一种区分不同粘度的双光子荧光探针及其制备方法和应用
CN113087703A (zh) 一种能特异性标记脂滴的光敏剂及其制备方法
CN116836565B (zh) 一类水溶性方酸菁染料及其合成方法和应用
CN111333640B (zh) 一种快速特异性标记SNAP-tag的荧光探针及其制备和生物应用
CN116535378A (zh) 罗丹明类荧光染料及其应用
CN115873011A (zh) 一种癌细胞靶向的响应线粒体内硝基还原酶的荧光探针及其制备方法和用途
CN116925097A (zh) 一种有机荧光小分子材料、合成方法和应用
CN115124555A (zh) 基于苝酰亚胺衍生物的乏氧酶荧光材料及其制备方法
CN117003759A (zh) 具有黏度响应的tb-吡啶氰基乙烯基衍生物及其合成和应用
CN118146223A (zh) 一种SNAP-tag探针及制备与应用
CN112341411A (zh) 一种类罗非昔布衍生物、制备的有机荧光染料骨架及用途
Wang Synthesis of Fluorescent Molecules and their Applications as Viscosity Sensors, Metal Ion Indicators, and Near-Infrared Probes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19803374

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2019803374

Country of ref document: EP