WO2019062876A1 - 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 PDFInfo
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- WO2019062876A1 WO2019062876A1 PCT/CN2018/108519 CN2018108519W WO2019062876A1 WO 2019062876 A1 WO2019062876 A1 WO 2019062876A1 CN 2018108519 W CN2018108519 W CN 2018108519W WO 2019062876 A1 WO2019062876 A1 WO 2019062876A1
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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 requires the participation of oxygen, is easy to aggregate, and once expressed, it is difficult to carry out post-modification. In addition, most of the fluorescent proteins also have shortcomings such as poor photostability, which limits 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.
- protein labels such as SNAP-tag (K. Johnsson et. al. WO 2004031405.), CLIP-tag (K. Johnsson et. al. WO 2008012296.), HaloTag (Wood, Keith V et. al. WO 2004072232) Technology has been commercialized.
- a method for the fluorescent-activated protein-specific labeling of HaloTag is designed, it is dark or emits very weak fluorescence before it is labeled, and once it is labeled on the protein, the fluorescence of the dye is sharply enhanced.
- probes of this type will have 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 the application of HaloTag technology.
- 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 present inventors have found that by attaching a ligand moiety to a viscosity-responsive fluorescent dye moiety, a significant increase in fluorescence brightness after activation of fluorescence by binding of a ligand to a tagged protein can be achieved, thereby obtaining a novel structure of a fluorescent probe. It has viscosity responsiveness and can be used for specific labeling of proteins, and can be effectively used for labeling, tracking, localization and or quantification of target proteins.
- a fluorescent probe comprising a ligand moiety A, an optional linker moiety B, a fluorescent dye moiety C, the fluorescent dye moiety C being a viscosity responsive fluorescent dye, comprising an electron donor moiety D, a conjugated system E And an electron acceptor moiety, the ligand moiety A being a group capable of specifically recognizing and labeling a target protein tagged with a protein tag or a fusion protein, optionally, the ligand moiety A is capable of being tagged or fused to a protein The target protein of the protein tag specifically recognizes and covalently labels the group.
- the above fluorescent probe is a compound having a structure represented by the formula (I), or a salt thereof,
- Linker moiety B is an optionally present group selected from the group consisting of alkylene groups, modified alkylene groups;
- the fluorescent dye moiety C in the formula (I) is a structural moiety represented by the formula (I-R).
- the electron donor moiety -D is -NX 1 -X 2 , X 1 is selected from hydrogen, alkyl, or modified alkyl, and X 2 is selected from hydrogen, alkyl, or modified alkyl, X 1 , X 2 Selectively interconnected to form an aliphatic heterocycle together with the N atom;
- the conjugated system E is a group formed by conjugated at least one selected from the group consisting of a double bond, a hydrazone bond, an aromatic ring, and an aromatic hetero ring, and is a structure represented by the following formula (I-1), which contains Each of the hydrogen atoms is optionally independently substituted with a substituent selected from the group consisting of a halogen atom, a nitro group, a hydrophilic group, an alkyl group, and a modified alkyl group, which are optionally linked to each other to form an alicyclic or a lipocyclic group. ring;
- the structure of formula (I-1) is linked to X 1 and X 2 to form an aliphatic heterocyclic ring;
- 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, phosphite, phosphate, sulfonate, sulfonate, sulfone a sulfoxide group, an aryl group, a heteroaryl 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 heteroalicyclic 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; alternatively, a C 1 -C 10 linear or branched alkyl group; alternatively, C 1 -C 7 a straight or branched alkyl group; optionally, a C 1 -C 5 straight or branched alkyl group; optionally, 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 Base, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3
- alkylene is a C 1 -C 30 linear or branched alkylene group; alternatively, a C 1 -C 7 linear or branched alkylene group; alternatively, C 1 -C 5 straight 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 aromatic group
- heteroaryl or aromatic heterocyclic ring is a 5- to 10-membered monocyclic or fused bicyclic heteroaromatic group having at least one hetero atom selected from N, O, S or Si on the ring;
- heteroaryl or aromatic heterocyclic ring is an 11- to 14-membered fused tricyclic 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 7-membered bicyclic ring-opened hydrocarbon group
- 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 RCONR' 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 heteroaryl ring is selected from the group consisting of thiophene, furan, and pyrrole.
- the above fluorescent probe is a compound having a structure represented by (I-a) or (I-b), or a salt thereof:
- the electron acceptor moiety has a structure represented by the following formulas (I-2) and (I-2-i),
- 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 selected from the group consisting of a dehalogenase
- the protein tag is a haloTag or a mutant thereof
- the ligand moiety A is derived from a halogenated hydrocarbon compound
- the ligand moiety A suitable for HaloTag is derived from a dehalogenase substrate
- the ligand moiety A is a haloalkyl group; optionally a haloethyl group, optionally of the formula:
- X is a halogen, optionally chlorine
- the linker moiety B is selected from a saturated linear or branched alkyl group having from 1 to 30 carbon atoms, one or more carbon atoms on the alkyl chain being one or more -O- Displacement; the carbon atom is replaced by -O-, meaning that a carbon atom or a carbon atom is replaced by -O- together with a hydrogen atom thereon;
- 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
- X 2 is optional.
- a C 1-30 straight 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;
- each of X 1 and X 2 is independently a C 2-30 ether chain group having 1 to 10 oxygen atoms, optionally substituted by one or more groups selected from a sulfonic acid group or a carboxyl group;
- -NX 1 -X 2 forms any group selected from the following formulas (Ii-1), (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;
- two adjacent substituents in the conjugated system E are linked to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring;
- H on the CH in the conjugated system E is substituted with a halogen atom, a nitro group, a hydrophilic group, an alkyl group or a modified alkyl group;
- the conjugated system E contains NH; alternatively, H on the NH is substituted with an alkyl group or a modified alkyl group;
- conjugated system E is selected from the structures of the following formulas (I-1-1) to (I-1-38):
- conjugated system E and -NX 1 -X 2 form an aliphatic heterocyclic ring represented by the following (Ii-3) (Ii-7):
- conjugated system E and -NX 1 -X 2 form a structure as shown in (Ii-8) below:
- 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: Selected as 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 cyano group, a nitro group, a hydrophilic group, an alkyl group or a modified alkyl group; alternatively, R 2 or R e is an NH-containing group selected from the above structures, and 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), formula (I-2-i), and when selected from a thioamino group, a thioester group, a thioamine a thiocarbonyl group in the acyl or thioester group is bonded to the alkenyl carbon of the formula (I-2), formula (I-2-i);
- R e is selected from the group consisting of a cyano group, a keto group, an ester group, an amide group, and is elected From a keto group, an ester group, or an amide group, it is attached to the formula (I-2-a), the formula (I-2-b)
- the fluorescent probe described above wherein the fluorescent probe is selected from the group consisting of a compound of the formula: or a salt thereof:
- the salt of the above compound may be an alkali metal salt, an alkaline earth metal salt or an anionic salt, and optionally a sodium salt, a potassium salt, a calcium salt, a hydrochloride, a sulfate, a sulfonate, a carboxylate or the like.
- 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:
- the D' group can form a D-group to bond with a linking group or a ligand; or R 2 ' can form an R 2 - group to bond with a linking group or a ligand.
- a fluorescence-activated protein-specific labeling method comprising the steps of: contacting the fluorescent probe with a protein label or a fusion protein-tagged target protein, the fluorescent probe The body moiety is labeled with a protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is labeled on the protein tag as 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.
- 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 above protein tag or part of the target protein fused to the tag can be prepared by existing genetic engineering techniques.
- the above-mentioned 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.
- An organic dye molecule optionally greater than 5, optionally greater than 10.
- 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 the fluorescent groups of different fluorescent probes do not interfere with each other, and the fluorescent probes of different color systems can perform multi-color labeling on the samples, and can perform orthogonal mark 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 HaloTag protein label specifically expressed in 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 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 11 are fluorescence intensity pairs of probe 57, probe 59, probe 60, probe 61, probe 62, probe 63, probe 69, probe 75, probe 76, and probe 82, respectively. a standard curve made from different HaloTag protein label concentrations;
- Figure 12 is a fluorescence map of different probe labeled cells, wherein (1) to (8) are probe 57, probe 59, probe 60, probe 61, probe 69, probe 75, probe 76, Probe 82, group A is a Hela cell expressing a protein tag, and group B is a Hela-WT cell (Hela original cell, no protein tag is expressed);
- Figure 13 shows different organelles with different probe labels, wherein groups A to F are probe 57, probe 59, probe 60, probe 61, probe 62, and probe 63, (1) to (6), respectively. They are cytoskeleton, mitochondria, nucleus, Golgi, whole cells, lysosomes;
- Figure 14 is a two-color labeling of the same cells by different probes, wherein A is the reference probe 85 labeled mitochondria, B is the probe 57 labeled nuclei, and C is the orthogonal imaging of A and B;
- Figure 15 is a marker of probe 77 for living mice, wherein group A is a blank group, group B is a control group, and group C is a sample group;
- Figure 16 is a graph showing changes in fluorescence of probe 61 in mammalian cells as a function of protein degradation
- A represents the fluorescent channel of the probe 61
- B represents the fluorescent channel of the probe 57
- C represents the superimposed fluorescent channel of the probe 61 and the probe 57;
- Figure 18 is a comparison of labeling speed and fluorescence intensity of probe 48 labeled HaloTag protein tag and reference probe 85 labeled SNAP protein tag under the same conditions.
- N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde (0.358 g, 2 mmol) and tert-butyl cyanoacetate (0.338 g, 2.4 mmol) were dissolved in 50 ml of absolute ethanol and catalyzed Anhydrous zinc chloride was heated in an oil bath for 5 h under Ar protection. The reaction was completed, cooled to room temperature, and some of the solvent was removed by rotary evaporation. The system precipitated a large amount of solid, filtered, and the filter cake was washed twice with cold ethanol and dried in vacuo. That is, yellow compound 1 (0.49 g, 81%).
- a fluorescently activated covalently labeled fluorescent probe 3 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 4 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 5 suitable for the HaloTag protein tag was constructed:
- Probe 4 (0.581 g, 1.0 mmol) and 4-dimethylaminopyridine (0.146 g, 1.2 mmol) were dissolved in 20 ml of anhydrous dimethylformamide, and p-nitrochloroformic acid was slowly added dropwise under argon atmosphere.
- a fluorescently activated covalently labeled fluorescent probe 6 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 7 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 8 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 9 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 10 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 11 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 12 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 13 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 14 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 15 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 16 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 17 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 18 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 19 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 20 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 21 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 22 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 23 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 24 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 25 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 26 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 27 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 28 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 29 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 30 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 31 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 32 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 33 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 34 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 35 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 36 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 37 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 38 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 39 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 40 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 41 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 42 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 43 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 44 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 45 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 46 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 47 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 48 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 49 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 50 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 51 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 52 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 53 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 54 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 55 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 56 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 57 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 58 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 59 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 60 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 61 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 62 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 63 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 64 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 65 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 66 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 67 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 68 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 69 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 70 suitable for use in the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 71 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 72 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 73 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 74 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 75 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 76 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 77 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 78 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 79 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 80 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 81 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 82 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 83 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled fluorescent probe 84 suitable for the HaloTag protein tag was constructed:
- a fluorescently activated covalently labeled reference fluorescent probe 85 suitable for SNAP protein tagging was constructed:
- a fluorescently activated covalently labeled fluorescent probe 86 suitable for the HaloTag protein tag was constructed:
- the probe was mixed with the corresponding HaloTag protein label to obtain a mixed sample.
- the final concentration of the probe was 5 ⁇ M in the mixed sample, and the final concentration of the protein label was 10 ⁇ M.
- the mixed sample was incubated at 37 ° C for 1 hour, and the fluorescence of the sample was detected using a fluorescence spectrophotometer. The intensity changes and 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 HaloTag protein tag was separately added to the solution of 30 uM of probe 57, probe 59, probe 60, probe 61, probe 62, probe 63, probe 69, probe 75, probe 76, probe 82.
- a mixed sample solution having a final concentration of HaloTag protein of 0.1 uM, 0.5 uM, 0.7 uM, 1.2 uM, 4.5 uM, 8.1 uM, 13.1 uM, and 14.8 uM was prepared, and the mixed sample solution was subjected to a reaction at 37 ° C for 1 hour, using Fluorescence spectrophotometer was used to detect the change of excitation spectrum of the sample, and the relationship between the label concentration of HaloTag protein and the fluorescence intensity was plotted according to the intensity of the emission spectrum. The results are shown in Figure 2 to Figure 11, respectively.
- the HaloTag 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 HaloTag protein, Hela-WT cells (Hela original cells, unexpressed protein tags) were planted in a 14 mm glass bottom 96-well cell culture plate and stabilized for 10 hours.
- Probe 57, probe 59, probe 60, probe 61, probe 69, probe 75, probe 76, and probe 82 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.
- Hela cells are used as an example to detect different subcellular organelles HaloTag.
- the effect of protein labels Hela cells 5000 cells were seeded in 96-well glass bottom cell culture plates per well. After 14 hours, the HaloTag protein tag was used to transfect the plasmids on different organelles using the lipo2000 kit. The original medium was removed 24 hours after transfection.
- the cells were washed twice with phenol red DMEM medium, and the cells were incubated with a phenol red-free medium containing 0.2 ⁇ M probe for 2 hours, and the cell labeling effect was examined using a leica TCS-8 confocal microscope.
- the probe can clearly display the subcellular structure of the cytoskeleton, mitochondria, nucleus, Golgi apparatus, whole cells, lysosomes, and the like without washing.
- the probes described above can serve as a powerful tool for a variety of 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-SNAP-NLS, pcdna3.1-mito-HaloTag (HaloTag protein tag mitochondrial localization plasmid) was co-transfected with lipo2000 kit. 0.1 ⁇ g per well; the original medium was removed 24 hours after transfection, and the cells were incubated with phenol red-free medium containing 0.2 ⁇ M of reference probe 85 and probe 57, respectively, using phenol red-free DMEM medium twice. The cell marker effect was measured using a leica TCS-8 confocal microscope image for 2 hours. As a result, as shown in Fig.
- the reference probe 85 and the probe 57 can clearly display the mitochondrial and nuclear structures, respectively, while being left-free, and the reference probe 85-labeled nuclear fluorescence and the probe 57-labeled mitochondrial fluorescence.
- the channel co-localization coefficient is less than 0.1, indicating that the two fluorescent channels do not interfere with each other.
- a plasmid pcdna3.1-HaloTag (sample group) expressing a HaloTag protein tag and a control plasmid pcdna3.1-CAT (control group) not expressing a HaloTag protein tag were introduced into mice.
- the plasmid is dissolved in a large volume of solution and rapidly injected into the mouse by tail vein injection, and the liver of the mouse absorbs DNA to express the target protein.
- Twenty hours after the plasmid injection 0.4 ⁇ M probe 77 dissolved in 200 ul of PBS was injected into the mouse to label the HaloTag protein by tail vein injection method; after 6 hours, the mice were dissected and the small difference was detected by a Kodak multispectral living imaging system.
- the mammalian cell HaloTag protein was taken as an example.
- the fluorescence change of the probe bound to the HaloTag protein after protein degradation was detected by using the AID degradation system in Hela cells.
- 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-HaloTag-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 probe 61, 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 the indole acetic acid (IAA) was added to induce the degradation of HaloTag-IAA17-H2B protein, and the change of cell fluorescence during protein degradation was detected.
- the results are shown in Figure 16.
- the HaloTag-IAA17-H2B protein is localized in the nucleus (0 min). Indoleacetic acid is added to induce protein degradation.
- the fluorescence signal of HaloTag-IAA17-H2B protein gradually decreases.
- the fluorescence signal was almost invisible, and the degradation rate of the protein 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. When the protein is present, the fluorescence is activated. When the protein is degraded, the fluorescence disappears and can be used to track and monitor the degradation process of the target protein.
- Hela cells were used as an example to detect the process of the interstitial CX43 assembly in mammalian cells to form intercellular channels.
- the C-terminal of CX43 gene was fused with the HaloTag gene, and a Hela cell strain stably expressing the fusion protein CX43-HaloTag was constructed by lentiviral infection technique.
- the Hela-CX43-HaloTag cell line was planted in a 20 mm glass bottom cell culture culture dish 10 hours before the probe labeling. When labeling, firstly dilute the probe 61 to 2 ⁇ M with phenol red free DMEM medium and replace the original cell culture medium.
- the cells were incubated for 1 hour in a 37 ° C carbon dioxide incubator. The cells were then washed twice with fresh phenol red free DMEM medium and the unbound probe 61 was removed, 2 minutes apart. Then, the cells were labeled with DMEM without phenol red medium containing 1 ⁇ M probe 57, and the fluorescence intensity and position of the corresponding fluorescent channels of the probe cells and probe 57 were probed for a long time using a Leica SP8 confocal microscope. The variation process is shown in Figures 17A and 17B, respectively. A long column-shaped fluorescence signal between the two cells can be seen in the fluorescent channel of probe 61, as shown in Figure 17A, consistent with the results reported in the literature (Guido Gaietta et al.
- the probe 48-labeled HaloTag protein tag and the reference probe 85-labeled SNAP protein tag are used as examples ( Probe 48 and reference probe 85 have the same fluorescent dye moiety), and the purified HaloTag protein and SNAP protein were first diluted to 7.5 uM with PBS. The probe 48 and the reference probe 85 were separately diluted to 15 uM with PBS, 20 uL of probe 48 was added to 80 uL of HaloTag protein, and 20 uL of the reference probe 85 was added to 80 uL of SNAP protein and mixed, and monitored by a microplate reader.
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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
本发明涉及一种荧光探针及其制备方法和用途。
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且需要氧气的参与,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.)、HaloTag(Wood,Keith V et.al.WO 2004072232)等蛋白标签技术已经商业化。
虽然SNAP-tag、HaloTag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HaloTag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HaloTag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.)报道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部分相连,能够实现配体与标签蛋白结合激活荧光后的荧光亮度的显著增长,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,能够有效用于靶蛋白的标记、跟踪、定位和或定量。
提供一种荧光探针,包括配体部分A、任选的连接体部分B、荧光染料部分C,所述荧光染料部分C为粘度响应性荧光染料,包括电子供体部分D、共轭体系E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。
可选地,上述的荧光探针,其为具有如式(I)所示结构的化合物,或者其盐,
A-B-C
(I)
其中,
连接体部分B为任选存在的基团,选自亚烷基、改性亚烷基;
式(I)中的荧光染料部分C为式(I-R)所示结构部分,
其中:
式(I-R)中,与连接体部分B或配体部分A相连位置的氢被连接体部分B或配体部分A替代;
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选自氢、烷基、或改性烷基,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E为选自双键、叁键、芳香环、芳香杂环中的至少一种共轭连接而形成的基团,其为如下式(I-1)所示的结构,其中所含的各氢原子任选独立地被选自卤原子、硝基、亲水性基团、烷基和改性烷基的取代基取代,所述取代基任选地相互连接构成脂环或脂杂环;
任选地,所述式(I-1)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,
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、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;
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:
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;可选地,为C
1-C
10直链或支链烷基;可选地,为C
1-C
7直链或支链烷基;可选地,为C
1-C
5直链或支链烷基;可选地,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;可选地,为C
1-C
7直链或支链亚烷基;可选地,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)-、
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)-、
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元单环或稠合双环杂芳香基团;
可选地,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11~14元稠合三环杂芳香基团;
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5~7元双环亚环烃基;
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RCONR′基团;
所述“硫代酰胺基”为R(C=S)NR′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、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(可选地为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,可选地为1~50,可选地为1~30,可选地为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,所述杂芳环选自噻吩、呋喃、吡咯。
可选地,上述的荧光探针,其为具有如(I-a)、(I-b)所示结构的化合物,或者其盐:
电子受体部分具有如下式(I-2)、(I-2-i)所示的结构,
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,所述蛋白标签选自脱卤素酶;
可选地,所述蛋白标签为卤代烷脱卤素酶(HaloTag)或其突变体;
可选地,所述配体部分A来自于卤代烃类化合物;
可选地,适用于HaloTag的配体部分A来自于脱卤素酶底物;
可选地,所述配体部分A为卤代烷基;可选地为卤代乙基,可选地为下式结构:
其中:
X是卤素,可选地为氯;
可选地,所述连接体部分B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由一个或多个-O-置换;所述的碳原子被-O-置换,是指碳原子或碳原子与其上的氢原子一起被-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直链或支链烷基或亚烷基;
可选地,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;
可选地,X
1、X
2各自独立地为任选被一个或多个选自磺酸基、羧基的基团取代的由-HN(C=O)-O-改 性的C
4-30烷基或亚烷基;
可选地,-NX
1-X
2形成选自下式(I-i-1)、(I-i-2)的任一基团:
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,所述共轭体系E中两个相邻取代基相互连接构成饱和或不饱和的脂环或脂杂环;
可选地,所述共轭体系E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,所述共轭体系E中含有NH;可选地,所述NH上的H被烷基或改性烷基取代;
可选地,所述共轭体系E选自下式(I-1-1)~(I-1-38)中的结构:
可选地,所述共轭体系E与-NX
1-X
2形成如下(I-i-3)(I-i-7)所示的脂杂环:
或者,所述共轭体系E与-NX
1-X
2形成如下(I-i-8)所示的结构:
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:可选地为双环或三环稠芳香环或稠芳香杂环;
可选的,R
2或R
e的上述结构中CH上的H被卤原子、氰基、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-i)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-i)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)、式(I-2-b)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-2-38)中的一种:
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或其盐:
上述化合物的盐可以为其碱金属盐、碱土金属盐、阴离子盐,可选地为钠盐、钾盐、钙盐、盐酸盐、硫酸盐、磺酸盐、羧酸盐等。
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:
其中,D’反应后能够形成D-基团与连接基团或配体键合;或者R
2’反应后能够形成R
2-基团与连接基团或配体键合。
另一方面,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。
另一方面,还提供上述的荧光探针在蛋白荧光标记;蛋白的定量、检测或动力学研究中的用途;或者在细胞、组织、活体成像中的用途。
另一方面,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。
上述蛋白标签(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。
上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,可选地大于5,可选地大于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,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HaloTag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。
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11分别为探针57、探针59、探针60、探针61、探针62、探针63、探针69、探针75、探针76、探针82的荧光强度对不同HaloTag蛋白标签浓度做的标准曲线;
图12为不同探针标记细胞荧光图谱,其中,(1)~(8)分别为探针57、探针59、探针60、探针61、探针69、探针75、探针76、探针82,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13为不同探针标记不同的细胞器,其中,A~F组分别为探针57、探针59、探针60、探针61、探针62、探针63,(1)~(6)分别为细胞骨架、线粒体、细胞核、高尔基体、全细胞、溶酶体;
图14为不同探针对相同细胞进行双色标记,其中,A为参比探针85标记的线粒体,B为探针57标记的细胞核,C为A和B的正交成像;
图15为探针77用于活体小鼠的标记,其中,A组为空白组,B组为对照组,C组为样品组;
图16为探针61在哺乳动物细胞中的荧光随蛋白降解的变化;
图17为不同探针示踪细胞间隙组装过程,其中,A表示探针61的荧光通道,B表示探针57的荧光通道,C表示探针61和探针57的叠加荧光通道;
图18为相同条件下,探针48标记HaloTag蛋白标签和参比探针85标记SNAP蛋白标签的标记速度和荧光强度的对比。
以下对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HaloTag蛋白标签的荧光激活型共价标记的探针1:
化合物1:
N-甲基-N-(2-羟乙基)-4-氨基苯醛(0.358g,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,即得黄色化合物1(0.49g,81%)。
1H-NMR(400MHz,DMSO-d
6):δ=8.01(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),4.79(bt,1H),3.59-3.55(m,4H),3.08(s,3H),1.50(s,9H)。
化合物2:
合成方法参照文献Craig M.Crews et.al.Nature Chemical Biology.2011,7,538-543.公开的方法进行合成。
1H-NMR(400MHz,CDCl
3)δ=6.47(brs,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
探针1:
化合物1(0.302g,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2(0.269g,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,即得探针10.42g,产率70%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.01(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.59-3.55(m,4H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.08(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的探针2:
化合物3:
合成方法参照文献J.Das et.al.Bioorg.Med.Chem.Lett.2005,15,337-343.公开的方法进行合成。
1H-NMR(400MHz,CDCl
3):δ=7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.36-7.42(m,2H),4.12(s,2H)。
化合物4的合成:
参照化合物1的合成方法。
1H-NMR(400MHz,DMSO-d
6):δ=8.11-8.07(m,2H),8.01-7.97(m,3H),7.70(d,1H,J=8.8Hz),7.46-7.43(m,1H),6.27(dd,1H,J=9.2,1.6Hz),6.02(s,1H),3.88(t,2H,J=5.6Hz),3.64(t,2H,J=5.6Hz),3.15(s,3H)。
探针2:
参照探针1的合成方法,产率75%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.11-8.07(m,2H),8.01-7.97(m,2H),7.70(d,1H,J=8.8Hz),7.46-7.43(m,1H),6.27(dd,1H,J=9.2,1.6Hz),6.02(s,1H),3.88(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.64(t,2H,J=5.6Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.15(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针3:
化合物5:
参照化合物1的合成方法合成,产率95%。
1H-NMR(400MHz,CDCl
3):δ=8.09(s,1H),8.02(d,1H,J=8.0Hz),7.98(d,2H,J=9.2Hz),7.86(d,1H,J=8.4Hz),7.48(t,1H,J=7.8Hz),7.36(t,1H,J=7.36Hz),6.73(d,2H,J=9.2Hz),3.88(t,2H,J=5.6Hz),3.64(t,2H,J=5.6Hz),3.15(s,3H)。
探针3:
参照探针1的合成方法合成,产率65%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.02(d,1H,J=8.0Hz),7.98(d,2H,J=9.2Hz),7.86(d,1H,J=8.4Hz),7.48(t,1H,J=7.8Hz),7.36(t,1H,J=7.36Hz),6.73(d,2H,J=9.2Hz),3.88(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.64(t,2H,J=5.6Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.15(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针4:
化合物6:
参照化合物1的合成方法,产率95%。1H-NMR(400MHz,DMSO-d6):δ=8.01(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),4.79(bt,2H),3.85(t,4H,J=5.6Hz),3.60(t,4H,J=5.6Hz),1.50(s,9H)。
探针4:
参照探针1的合成方法合成,产率35%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),4.79(bt,1H),3.85(t,4H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,4H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针5:
探针5:
探针4(0.581g,1.0mmol)和4-二甲氨基吡啶(0.146g,1.2mmol)于20ml的无水二甲基甲酰胺中溶解,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,加入3-氨基-丙磺酸钠(0.168g,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,残余物反相柱分离,即得探针0.397g,产率50%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,2H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.85(t,4H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,8H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,4H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针6:
化合物7:
参照文献(L.X.Wu,K.Burgess,J.Am.Chem.Soc.2008,130,4089-4096.)公开方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.63-7.48(m,5H),4.27(s,2H),3.13(s,3H)。
化合物8:
参照化合物1的合成方法,产率99%。
1H-NMR(400MHz,CDCl
3):δ=8.03(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),4.27(s,2H),3.55-3.59(m,4H),3.08(s,3H),3.13(s,3H)。
探针6:
参照探针1的合成方法,产率70%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.03(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),4.27(s,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.59-3.55(m,4H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J= 6.8Hz,2H),3.13(s,3H)3.12(t,J=4.9Hz,2H),3.08(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针7:
化合物9:
参照文献(L.X.Wu,K.Burgess,J.Am.Chem.Soc.2008,130,4089-4096.)公开方法合成。
1H-NMR(400MHz,CDCl
3):δ=3.98(q,2H,J=2.4Hz),3.01(s,3H),2.15(t,3H,J=2.4Hz)。
化合物10:
参照化合物1的方法合成,产率97%。
1H-NMR(400MHz,DMSO-d
6):δ=8.00(s,1H),7.97(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.98(q,2H,J=2.4Hz),3.55-3.59(m,4H),3.08(s,3H),3.01(s,3H),2.15(t,3H,J=2.4Hz)。
化合物11:
化合物10(0.546g,2mmol)、苯甲醛(0.530g,5mmol)和无水氯化锌(0.545g,4mmol)溶于100ml无水甲苯中,Ar保护条件下油浴加热回流48h,反应完毕,旋转蒸干溶剂,残余物溶于100ml二氯甲烷中,水洗三次,有机相用无水硫酸钠干燥,旋转蒸干溶剂,柱色谱分离,得红褐色固体0.181g,产率25%。
1H-NMR(400MHz,DMSO-d
6):δ=8.21(d,2H,J=8.8Hz),8.00(d,1H,J=16Hz),7.85(d,2H,J=8.0Hz),7.45-7.38(m,3H),7.24(s,1H),7.01(s,1H),6.92(d,2H,J=8.8Hz),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
探针7:
参照探针1的合成方法,产率66%。
1H-NMR(400MHz,DMSO-d
6):8.21(d,2H,J=8.8Hz),δ=8.18(t,1H),8.00(d,1H,J=16Hz),7.85(d,2H,J=8.0Hz),7.45-7.38(m,3H),7.24(s,1H),7.01(s,1H),6.92(d,2H,J=8.8Hz),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针8:
化合物12:
参照文献(Wang H.et al.Tetra Let.2007,48,3471-3474.)公开的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.05(m,2H),7.01(m,2H),1.83(s,6H)。
化合物13:
化合物12(0.279g,1mmol)溶于20ml无水吡啶中,加入N-甲基-N-羟乙基1ml,Ar保护条件下40℃油浴加热过夜,反应完毕,旋转蒸干溶剂,残余物柱色谱分离得红色产物0.187g,产率56%。
1H-NMR(400MHz,DMSO-d
6):δ=8.05(m,2H),7.01(m,2H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H),1.83(s,6H)。
探针8:
参照探针1的合成方法,产率56%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.05(m,2H),7.01(m,2H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.83(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针9:
化合物14:
参照化合物1的合成方法,产量95%。1H-NMR(400MHz,DMSO-d6):δ=8.07(s,1H),7.93(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.55-3.59(m,4H),3.08(s,3H)。
探针9:
参照探针1的合成方法,产量35%。1H-NMR(400MHz,DMSO-d6):δ=8.18(t,1H),8.07(s,1H),7.93(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.59-3.55(m,4H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.08(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针10:
化合物15:
参照公开方法(WO 2013142841(A1),2013.09.26。)合成。
1H-NMR(400MHz,CDCl
3):δ=7.92(s,1H),7.63(d,1H,J=5.2Hz),7.31(d,1H,J=5.2Hz)。
化合物16:
化合物15(0.438g,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85%。
1H-NMR(400MHz,CDCl
3):δ=7.92(s,1H),7.63(d,1H,J=5.2Hz),7.31(d,1H,J=5.2Hz),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
化合物17:
化合物16(0.426g,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0.285g,产率59%。
1H-NMR(400MHz,CDCl
3):δ=10.01(s,1H),7.92(s,1H),7.63(s,1H)3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
化合物18:
参照化合物1的合成方法,产率89%。
1H-NMR(400MHz,DMSO-d
6):δ=8.22(s,1H),8.02(s,1H),6.43(s,1H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H),1.49(s,9H)。
探针10:
参照探针1的方法合成,产率65%。
1H-NMR(400MHz,DMSO-d
6):δ=8.22(s,1H),8.18(t,1H),8.02(s,1H),6.43(s,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.49(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针11:
化合物19:
参照化合物1的合成方法,产率93%。
1H-NMR(400MHz,CDCl
3):δ=8.22(s,1H),8.02(s,1H),7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.36-7.42(m,2H),6.43(s,1H),3.75(t,2H,J=5.6Hz),3.55(t,2H,J=5.6Hz),3.10(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=8.22(s,1H),8.18(t,1H),8.02(s,1H),7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.36-7.42(m,2H),6.43(s,1H),3.75(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.55(t,2H,J=5.6Hz),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针12:
化合物20:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,DMSO-d
6):δ=8.22(s,1H),8.02(s,1H),6.43(s,1H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
探针12:
参照探针1的合成方法,产率39%。
1H-NMR(400MHz,DMSO-d
6):δ=8.22(s,1H),8.18(t,1H),8.02(s,1H),6.43(s,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光 探针13:
化合物21:
参照化合物16的合成方法,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22:
参照化合物17的合成方法,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,2H),3.34(t,J=8.0Hz,2H),3.10(s,3H)。
化合物23:
参照化合物1的合成方法,产率91%。
1H-NMR(400MHz,CDCl
3):δ=8.02(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,2H),3.34(t,J=8.0Hz,2H),3.11(s,3H),1.48(s,9H)。
探针13:
参照探针1的合成方法,产率66%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),8.02(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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,J=8.0Hz,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,J=8.0Hz,2H),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.48(s,9H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针14:
化合物24:
参照化合物4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,J=8.0Hz,2H),3.34(t,J=8.0Hz,2H),3.21(s,3H)。
探针14:
参照探针1的合成方法,产率71%。
1H-NMR(400MHz,DMSO-d
6):δ=8.45(s,1H),8.18(t,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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,J=8.0Hz,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,J=8.0Hz,2H),3.21(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针15:
化合物25:
参照化合物16的合成方法,产率67%。
1H-NMR(400MHz,CDCl
3):δ=7.62(d,1H,J=8.8Hz),7.15(d,1H,J=5.6Hz),7.08-7.01(m,2H),6.81(d,1H,J=2.4Hz),3.62(t,4H,J=8.0Hz),3.35(t,4H,J=8.0Hz)。
化合物26:
参照化合物17的合成方法,产率67%。
1H-NMR(400MHz,CDCl
3):δ=9.99(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0Hz),6.81(m,1H),3.62(t,4H,J=8.0Hz),3.35(t,4H,J=8.0Hz)。
化合物27:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=8.00(s,1H),7.83(s,1H),7.69(d,1H,J=9.0Hz),6.92(d,1H,J=2.0Hz),6.81(m,1H),3.62(t,4H,J=8.0Hz),3.35(t,4H,J=8.0Hz),1.49(s,9H)。
探针15:
参照探针1的合成方法,产率44%。1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),8.00(s,1H),7.83(s,1H),7.69(d,1H,J=9.0Hz),6.92(d,1H,J=2.0Hz),6.81(m,1H),3.69(t,J=4.9Hz,2H),3.62(t,4H,J=8.0Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(t,4H,J=8.0Hz),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.49(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针16:
探针16:
参照探针5的合成方法,产率44%。1H-NMR(400MHz,CDCl3):δ=8.18(m,2H),8.00(s,1H), 7.83(s,1H),7.69(d,1H,J=9.0Hz),6.92(d,1H,J=2.0Hz),6.81(m,1H),3.69(t,J=4.9Hz,2H),3.62(t,4H,J=8.0Hz),3.63-3.60(m,6H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(t,4H,J=8.0Hz),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.49(s,9H),1.46-1.39(m,4H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针17:
化合物28:
参照化合物10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),3.00(s,3H),2.15(t,3H,J=2.4Hz),1.48(s,9H)。
探针17:
参照探针1的合成方法,产率55%。
1H-NMR(400MHz,CDCl
3):8.22(s,1H),δ=8.18(t,1H),8.02(s,1H),6.43(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,J=8.0Hz,3H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,J=8.0Hz,3H),3.12(t,J=4.9Hz,2H),3.11(s,3H),3.00(s,3H),2.15(t,3H,J=2.4Hz),1.79-1.71(m,2H),1.60-1.53(m,2H),1.48(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针18:
化合物29:
参照化合物14的合成方法,产率98%。
1H-NMR(400MHz,CDCl
3):δ=7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
探针18:
参照探针99的合成方法,产率91%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H), 1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针19:
化合物30:
参照化合物16的合成方法,产率78%。
1H-NMR(400MHz,CDCl
3):δ=8.07(s,1H),7.77(d,1H,J=1.6Hz),7.66(d,1H,J=8.4Hz),7.50(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H)。
化合物31:
参照化合物17的合成方法,产率81%。
1H-NMR(400MHz,CDCl
3):δ=9.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H)。
化合物32:
参照化合物1的合成方法,产率98%。
1H-NMR(400MHz,CDCl
3):δ=8.01(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H),1.50(s,9H)。
探针19:
参照探针1的合成方法,产率66%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),8.01(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针20:
化合物33:
参照化合物1的合成方法,产率97%。
1H-NMR(400MHz,CDCl
3):δ=7.99(s,1H),7.61(d,1H,J=1.6Hz),7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.49(d,1H,J=8.4Hz),7.36-7.42(m,3H),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H)。
探针20:
参照探针1的合成方法,产率65%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),7.99(s,1H),7.61(d,1H,J=1.6Hz),7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.49(d,1H,J=8.4Hz),7.36-7.42(m,3H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针21:
化合物34:
参照化合物1的合成方法,产率87%。
1H-NMR(400MHz,CDCl
3):δ=7.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H)。
探针21:
参照探针1的合成,产率31%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针22:
化合物35:
参照化合物1的合成方法,产率91%。
1H-NMR(400MHz,CDCl
3):δ=7.98(s,1H),7.64-7.48(m,7H),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.13(s,3H),3.11(s,3H)。
探针22:
参照探针1的合成方法,产率67%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),7.98(s,1H),7.64-7.48(m,7H),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m, 2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.13(s,3H),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针23:
化合物36:
参照化合物1的合成方法,产率97%。
1H-NMR(400MHz,CDCl
3):δ=8.04(d,1H,J=8.0Hz),7.90(d,1H,J=8.0Hz),7.99(s,1H),7.61(d,1H,J=1.6Hz),7.53(t,1H,J=8.0Hz),7.49(d,1H,J=8.4Hz),7.45(t,1H,J=8.0Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.11(s,3H)。
探针23:
参照探针1的合成方法,产率61%。
1H-NMR(400MHz,CDCl
3):δ=8.18(t,1H),8.04(d,1H,J=8.0Hz),7.90(d,1H,J=8.0Hz),7.99(s,1H),7.61(d,1H,J=1.6Hz),7.53(t,1H,J=8.0Hz),7.49(d,1H,J=8.4Hz),7.45(t,1H,J=8.0Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针24:
化合物37:
参照化合物16的合成方法,产率81%。
1H-NMR(400MHz,CDCl
3):δ=8.67(s,1H),7.95(d,1H,J=10.0Hz),7.15(d,1H,J=2.4Hz),7.00(dd,1H,J
1=8.8Hz,J
2=2.4Hz),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.10(s,3H)。
化合物38:
参照化合物17的合成方法,产率56%。
1H-NMR(400MHz,CDCl
3):δ=10.06(s,1H),8.03(d,1H,J=10.0Hz),7.07-7.04(m,2H),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.10(s,3H)。
化合物39:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=8.03(d,1H,J=10.0Hz), 7.95(s,1H),7.07-7.04(m,2H),3.61(t,2H,J=8.0Hz),3.34(t,2H,J=8.0Hz),3.10(s,3H),1.50(s,9H)。
探针24:
参照探针1的合成方法,产率61%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.03(d,1H,J=10.0Hz),7.95(s,1H),7.07-7.04(m,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H).
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针25:
化合物40:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=8.06(s,1H),8.03(d,1H,J=10.0Hz),7.07-7.04(m,2H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.10(s,3H)。
探针25:
参照探针1的合成方法,产率89%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.06(s,1H),8.03(d,1H,J=10.0Hz),7.07-7.04(m,2H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针26:
化合物41:
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42:
参照探针1的合成方法,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)。
探针26:
参照探针1的合成方法,产率82%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(s,1H),7.83-7.89(m,2H),7.25-7.34(m,1H),7.13(d,1H),6.73(d,1H),3.69(t,J=4.9Hz,2H),3.68(t,2H,J=5.6Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.53(t,2H,J=5.6Hz),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),,3.08(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.52(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针27:
化合物43:
参照探针1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27:
参照探针1的合成方法,产率88%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(t,2H,J=8.0Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.34(t,2H,J=8.0Hz),3.12(t,J=4.9Hz,2H),3.11(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针28:
参照文献(M.Klikar et.al.New.J.Chem.2017.41.1459-1472)的方法合成(实际上是探针的中间体的制备参考该文献中的制备方法。下同)。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.01(s,1H),7.47(d,2H,J=8.8Hz),6.76(d,2H,J=8.8Hz),3.78(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.32(t,2H,J=4.80Hz),3.12(t,J=4.9Hz,2H),3.02(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针29:
参照文献(Ruikui Chen et.al.Chem.Mater.2007.19.4007-4015)以及探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.01(s,1H),7.84(s,1H),7.24(s,1H),3.78(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(m,4H),3.12(t,J=4.9Hz,2H),3.02(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针30:
参照探针29的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(1H,d,J=8.0Hz),8.01(s,1H),7.90(1H,d,J=8.0Hz),7.84(s,1H),7.53(1H,t,J=8.0Hz),7.45(1H,t,J=8.0Hz),7.24(s,1H),3.78(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(m,4H),3.12(t,J=4.9Hz,2H),3.02(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针31:
参照探针29的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(1H,d,J=8.0Hz),8.01(s,1H),7.90(1H,d,J=8.0Hz),7.84(s,1H),7.53(1H,t,J=8.0Hz),7.45(1H,t,J=8.0Hz),7.24(s,1H),3.78(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(m,4H),3.12(t,J=4.9Hz,2H),3.02(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
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HaloTag蛋白标签的荧光激活型共价标记的荧光 探针32:
参照探针29的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.01(s,1H),7.87(m,1H),7.71(m,1H),7.51(d,J=5.4Hz,1H),7.32(d,J=5.4Hz,1H),3.78(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(m,4H),3.12(t,J=4.9Hz,2H),3.02(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.49(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例33
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针33:
参照文献(ShengboZhu et.al.2015.16.146-154)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.01(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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56(t,2H,J=4.80Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例34
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针34:
参照探针33的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.31(s,1H),8.22(bt,1H),8.18(t,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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.57(t,2H,J=5.20Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.48-3.41(m,6H),3.38(t,2H,J=5.20Hz),3.27(s,3H),3.12(t,J=4.9Hz,2H),3.01(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例35
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光 探针35:
参照探针33的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.00(s,1H),7.68(d,1H,J=4.0Hz),7.56(d,2H,J=9.0Hz),7.24(d,1H,J=4.0Hz),6.72(d,2H,J=9.0Hz),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
实施例36
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针36:
参照探针29的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,2H),7.59(s,1H),7.27(s,1H),7.05(s,1H),3.86(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56(t,2H,J=4.80Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例37
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针37:
参照文献(Sangwon Ko et.al.J.Mater.Chem.2010.20.2391-2399)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.83(s,1H),7.11(s,1H),3.85(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.46(t,2H,J=4.80Hz),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H),0.46(s,6H)
实施例38
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针38:
参照探针37的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.83(s,1H),7.11(s,1H),3.85(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.46(t,2H,J=4.80Hz),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H),δ=0.42(s,6H)。
实施例39
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针39:
参照探针37的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.11(s,1H),6.50(s,1H),3.85(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.46(t,2H,J=4.80Hz),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H),0.46(s,6H)
实施例40
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针40:
参照文献(Kassem Amro et.al.2014.70.1903-1909)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),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),3.69(t,J=4.9Hz,2H),3.67(t,2H,J=5.60Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(t,2H,J=5.60Hz),3.12(t,J=4.9Hz,2H),3.13(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例41
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光 探针41:
参照探针40的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=12.42(s,1H),10.01(s,1H),8.18(t,1H),8.00(s,1H),7.81(s,1H),7.57(d,1H,J=4.00Hz),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),3.69(t,J=4.9Hz,2H),3.67(t,2H,J=5.60Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.48-3.52(m,6H),3.44(t,J=6.8Hz,2H),3.38(s,3H),3.35(t,2H,J=5.60Hz),3.13(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
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HaloTag蛋白标签的荧光激活型共价标记的荧光探针42:
参照探针40的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.00(s,1H),7.74(d,1H,J=4.0Hz),7.57(d,1H,J=4.00Hz),7.51(d,1H,J=4.0Hz),7.36-7.42(m,2H),7.13(d,1H,J=4.00Hz),6.95(d,1H,J=4.00Hz),5.81(d,1H,J=4.00Hz),3.69(t,J=4.9Hz,2H),3.67(t,2H,J=5.60Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(t,2H,J=5.60Hz),3.13(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例43
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针43:
参照探针40的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.57(d,1H,J=4.0Hz),7.13(d,1H,J=4.0Hz),6.95(d,1H,J=4.0Hz),5.81(d,1H,J=4.0Hz),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.60(t,2H,J=5.6Hz),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s, 3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例44
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针44:
参照文献(JingJing et.al.Chem.Sci.2012.3.3315-3320)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.21(s,1H),6.71(d,1H,J=8.0Hz),6.50(m,1H)4.71(t,1H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.62(m,2H),3.63-3.60(m,2H),3.56(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(m,2H),3.12(t,J=4.9Hz,2H),3.00(m,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.49(s,9H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例45
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针45:
参照探针44的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.74(d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.36-7.42(m,2H),7.21(s,1H),6.71(d,1H,J=8.0Hz),6.50(m,1H)4.71(t,1H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.62(m,2H),3.56-3.53(m,2H),3.56(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.35(m,2H),3.12(t,J=4.9Hz,2H),3.00(m,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例46
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针46:
参照文献(Kaijun Tian et.al.Chem.Commun.2011.47.10052-10054)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.03(s,1H),7.36(d,1H,J=7.6Hz),6.78(t,1H,J=7.2Hz),6.49(d,1H,7.8Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52 (t,J=6.6Hz,2H),3.46(m,4H),3.44(t,J=6.8Hz,2H),2.35(s,3H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,9H),1.46-1.39(m,2H),1.36(s,6H),1.36-1.28(m,2H)
实施例47
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针47:
参照探针46的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.03(s,1H),7.36(d,1H,J=7.6Hz),6.78(t,1H,J=7.2Hz),6.49(d,1H,7.8Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.11(m,4H),2.35(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36(s,6H),1.36-1.28(m,2H)
实施例48
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针48:
参照文献(Chun-GueyWu et.al.2013.99.1091-1100)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(m,15H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例49
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针49:
参照探针48的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.74 (d,1H,J=4.0Hz),7.55(d,1H,J=4.0Hz),7.36-7.42(m,2H),7.18(s,1H),6.96(d,1H),4.12(s,2H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例50
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针50:
参照探针48的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例51
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针51:
化合物44:
参照文献(Gen-ichi Konishi.et.al.Tetrahedron.2014,70,7551-7559)的方法合成。
1H-NMR(400MHz,CDCl
3):δ=9.92(s,1H),7.82(s,1H),7.71(m,1H),7.60(m,2H),6.78(s,2H),3.82(t,2H,J=5.6Hz),3.54(t,2H,J=5.6Hz),3.10(s,3H),1.42(s,6H)
化合物45:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=8.07(s,1H),7.82(s,1H),7.71(m,1H),7.60(m,2H),6.78(s,2H),3.82(t,2H,J=5.6Hz),3.54(t,2H,J=5.6Hz),3.10(s,3H),1.50(s,9H),1.42(s,6H)。
探针51:
参照探针1的合成方法,产率85%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(s,1H),7.82(s,1H),7.71(m,1H),7.60(m,2H),6.78(s,2H),3.82(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.54(t,2H,J=5.6Hz),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53 (m,2H),1.50(s,9H),1.46-1.39(m,2H),1.42(s,6H),1.36-1.28(m,2H)
实施例52
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针52:
化合物46:
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=6.52(s,2H),3.48-3.52(m,4H),3.38(s,3H)
化合物47:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.52-3.48(m,4H),3.38(s,3H),3.10(s,3H),1.50(s,6H)
探针52:
参照探针1的合成方法,产率85%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.52-3.48(m,4H),3.44(t,J=6.8Hz,2H),3.38(s,3H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例53
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针53:
化合物48:
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49:
参照化合物1的合成方法,产率96%。
1H-NMR(400MHz,CDCl
3):δ=7.92(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.42(s,3H),3.10(s,3H),1.50(s,6H)
探针53:
参照探针1的合成方法,产率85%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.92(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m, 2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.42(s,3H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例54
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针54:
化合物50:
参照化合物47的合成方法,产率86%。
1H-NMR(400MHz,CDCl
3):δ=7.83(s,1H),7.11(s,1H),3.52-3.48(m,4H),3.85(t,2H,J=4.80Hz),3.46(t,2H,J=4.80Hz),3.38(s,3H),3.06(s,3H),0.46(s,6H)
探针54:
参照探针1的合成方法,产率85%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.83(s,1H),7.11(s,1H),3.52-3.48(m,4H),3.85(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.46(t,2H,J=4.80Hz),3.44(t,J=6.8Hz,2H),3.38(s,3H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)0.46(s,6H)
实施例55
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针55:
化合物51:
参照化合物1的合成方法,产率77%。
1H-NMR(400MHz,CDCl
3):δ=7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.60(t,2H,J=5.6Hz),3.48(s,3H),3.10(s,3H),1.50(s,6H)
探针55:
参照探针1的合成方法,产率82%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(d,1H),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.48(s,3H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(s,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例56
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光 探针56:
化合物52:
参照化合物1的合成方法,产率77%。
1H-NMR(400MHz,CDCl
3):δ=7.93(s,1H),7.11(s,1H),6.42(s,1H),3.85(t,2H,J=4.80Hz),3.46(t,2H,J=4.80Hz),3.32(s,3H),3.06(s,3H),0.42(s,6H)
探针56:
参照探针1的合成方法,产率82%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.93(s,1H),7.11(s,1H),6.42(s,1H),3.85(t,2H,J=4.80Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.46(t,2H,J=4.80Hz),3.44(t,J=6.8Hz,2H),3.32(s,3H),3.12(t,J=4.9Hz,2H),3.06(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H),0.42(s,6H)
实施例57
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针57:
化合物53:
参照化合物1的合成方法,产率65%。
1H-NMR(400MHz,CDCl
3):δ=8.11(s,1H),7.97(d,J=9.0Hz,2H),6.69(d,J=9.5Hz,2H),3.38(s,6H)
探针57:
化合物53(0.216g,1mmol)和化合物2(0.223g,1mmol)溶于150ml无水DMF中,加入六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(0.512g,1mmol)和0.1mL的三乙胺,Ar保护条件下反应半小时,旋转蒸干溶剂,残余物柱色谱分离得黄色固体0.156g,产率72%。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.11(s,1H),7.97(d,J=9.0Hz,2H),6.69(d,J=9.5Hz,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.38(s,6H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例58
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针58:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(s,1H),7.93(d,2H,J=9.2Hz),6.85(d,2H,J=9.2Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,6H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.08(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例59
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针59:
化合物54:
参照化合物1的合成方法,产率65%。
1H-NMR(400MHz,CDCl
3):δ=7.92(s,1H),7.49(s,2H),3.37-3.26(m,4H),2.80-2.67(m,4H),2.07-1.85(m,4H)
探针59:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.92(s,1H),7.49(s,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.37-3.26(m,4H),3.12(t,J=4.9Hz,2H),2.80-2.67(m,4H),2.07-1.85(m,4H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例60
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针60:
化合物55:
参照探针44的方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.84(s,1H),7.71(m,1H),7.55(d,1H),6.73(d,1H),3.90-3.86(m,4H),3.61(s,3H)
探针60:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),δ=7.84(s,1H),7.71(m,1H),7.55(d,1H),6.73(d,1H),3.90-3.86(m,4H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.61(s,3H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例61
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针61:
化合物56:
参照探针10的方法合成。
1H-NMR(400MHz,CDCl
3):δ=8.22(s,1H),8.02(s,1H),6.43(s,1H),3.10(s,6H)
探针61:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.22(s,1H),8.18(t,1H),8.02(s,1H),6.43(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例62
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针62:
化合物57:
参照化合物42的方法。
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),3.08(s,6H)
探针62:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.07(s,1H),7.83-7.89(m,2H),7.25-7.34(m,1H),7.13(d,1H),6.73(d,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.08(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例63
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针63:
化合物58:
参照化合物23的方法。
1H-NMR(400MHz,CDCl
3):δ=7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.10(s,6H)
探针62:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例64
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针64:
化合物59:
参照文献(Eric T.Kool et.al.Bioconjug Chem.2016.27.2839-2843)的方法合成。
1H-NMR(400MHz,CDCl
3):δ=3.74-3.70(m,2H),3.69-3.65(m,2H),3.63-3.56(m,4H),3.53(t,J=6.7Hz,2H),3.47(t,J=6.7Hz,2H),2.42(s,1H),1.82-1.72(m,2H),1.66-1.55(m,2H),1.50-1.31(m,4H)
探针64:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.74-3.70(m,2H),3.69-3.65(m,2H),3.63-3.56(m,4H),3.53(t,J=6.7Hz,2H),3.47(t,J=6.7Hz,2H),3.10(s,6H),2.42(s,1H),1.82-1.72(m,2H),1.66-1.55(m,2H),1.50-1.31(m,4H)
实施例65
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针65:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.85(t,2H,J=5.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.60(t,2H,J=5.6Hz),3.56-3.53(m,2H),3.10(s,3H)3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例66
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针66:
参照探针63的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,3H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例67
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针67:
化合物60:
参照文献(Simon J.A.Pope.et.al.Chem.Commun.2009.4278-4280)的方法合成。
1H-NMR(400MHz,CDCl
3):δ=3.61(t,2H),3.08(t,2H),2.07-1.82(m,8H)
探针67:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.16(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.61(t,2H),3.10(s,6H),3.08(t,2H),2.07-1.82(m,8H)
实施例68
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针68:
化合物61:
参照文献(Simon J.A.Pope.et.al.Chem.Commun.2009.4278-4280)的方法合成。
1H-NMR(400MHz,CDCl
3):δ=3.85(t,J=5.2Hz,2H),3.81(t,J=5.7Hz,2H),3.72(t,J=5.7Hz,2H),3.27(sext,J=5.2Hz,2H)
探针68:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.19(t,1H),7.95(s,1H),7.81(s,1H),7.68(d,1H,J=9.0Hz),6.92(d,1H,J=2.0),6.82(d,1H,J=9.2Hz),3.85(t,J=5.2Hz,2H),3.81(t,J=5.7Hz,2H),3.72(t,J=5.7Hz,2H),3.27(sext,J=5.2Hz,2H),3.10(s,6H)
实施例69
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针69:
化合物62:
参照化合物34的合成方法。
1H-NMR(400MHz,CDCl
3):δ=7.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.11(s,6H)
探针69:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.11(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例70
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针70:
参照探针64的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=7.99(s,1H),7.61(d,1H,J=1.6Hz),7.49(d,1H,J=8.4Hz),7.40(dd,1H,J
1=8.4Hz,J
2=1.6Hz),3.74-3.70(m,2H),3.69-3.65(m,2H),3.63-3.56(m,4H),3.53(t,J=6.7Hz,2H),3.47(t,J=6.7Hz,2H),3.11(s,3H),2.42(s,1H),1.82-1.72(m,2H),1.66-1.55(m,2H),1.50-1.31(m,4H)
实施例71
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针71:
化合物62:
参照化合物34的合成方法。
1H-NMR(400MHz,CDCl
3):δ=8.06(s,1H),8.03(d,1H),7.07-7.04(m,2H),3.10(s,6H)
探针69:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.06(s,1H),8.03(d,1H),7.07-7.04(m,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例72
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针72:
参照探针70的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.06(s,1H),8.03(d,1H),7.07-7.04(m,2H),3.74-3.70(m,2H),3.69-3.65(m,2H),3.63-3.56(m,4H),3.53(t,J=6.7Hz,2H),3.47(t,J=6.7Hz,2H),3.10(s,6H),2.42(s,1H),1.82-1.72(m,2H),1.66-1.55(m,2H),1.50-1.31(m,4H)
实施例73
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针73:
化合物64:
参照探针33的方法合成。
1H-NMR(400MHz,CDCl
3):δ=8.00(s,1H),7.68(d,1H,J=4.0Hz),7.56(d,2H,J=9.0Hz),7.24(d,1H,J=4.0Hz),6.72(d,2H,J=9.0Hz),3.10(s,6H)
探针73:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),8.00(s,1H),7.68(d,1H,J=4.0Hz),7.56(d,2H,J=9.0Hz),7.24(d,1H,J=4.0Hz),6.72(d,2H,J=9.0Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例74
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针74:
化合物65:
参照探针40的方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.99(s,1H),7.57(d,1H,J=4.0Hz),7.13(d,1H,J=4.0Hz),6.95(d,1H,J=4.0Hz),5.81(d,1H,J=4.0Hz),3.00(s,6H)
探针74:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),δ=7.99(s,1H),7.57(d,1H,J=4.0Hz),7.13(d,1H,J=4.0Hz),6.95(d,1H,J=4.0Hz),5.81(d,1H,J=4.0Hz),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.00(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例75
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针75:
参照探针29的方法合成。
1H-NMR(400MHz,CDCl
3):δ=8.01(s,1H),7.84(s,1H),7.24(s,1H),3.02(s,6H)
探针75:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):8.18(t,1H),8.01(s,1H),7.84(s,1H),7.24(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.02(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例76
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针76:
化合物67:
参照探针48的方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.89(s,1H),7.18(s,1H),6.96(s,1H),3.10(s,6H),1.50(m,6H)
探针76:
参照探针57的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(m,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)。
实施例77
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针77:
参照探针64的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=7.89(s,1H),7.18(s,1H),6.96 (s,1H),3.74-3.70(m,2H),3.69-3.65(m,2H),3.63-3.56(m,4H),3.53(t,J=6.7Hz,2H),3.47(t,J=6.7Hz,2H),3.10(s,6H),2.42(s,1H),1.82-1.72(m,2H),1.66-1.55(m,2H),1.50(m,6H),1.50-1.31(m,4H)
实施例78
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针78:
化合物68:
参照化合物1的方法合成。
1H-NMR(400MHz,CDCl
3):δ=8.01(s,1H),7.18(s,1H),6.96(s,1H),3.10(s,6H),1.50(m,6H)
探针78:
化合物68(0.376g,1.0mmol)和化合物2(0.446g,2mmol)于10mL DMF中,加入0.1mL的三乙胺,室温下搅拌两天,反应完成后旋干溶剂,柱层析分离得到产物0.124g,
1H-NMR(400MHz,DMSO-d
6):δ=8.01(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(m,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例79
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针79:
参照探针78的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=7.88(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(m,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例80
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针80:
化合物69:
参照化合物1的方法合成。
1H-NMR(400MHz,CDCl
3):δ=8.01(s,1H),7.18(s,1H),6.96(s,1H),3.10(s,6H),0.41(m,6H)
探针80:
参照探针78的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.01(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H),0.42(m,6H)
实施例81
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针81:
化合物70:
参照化合物1的方法合成。
1H-NMR(400MHz,CDCl
3):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),3.13(s,6H)
探针81:
参照探针78的合成方法,
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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.13(s,6H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例82
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针82:
化合物71:
参照化合物49的方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.89(s,1H),7.18(s,1H),6.96(s,1H),3.47(s,3H),3.10(s,6H),1.50(m,6H)
探针82:
参照探针78的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.50(m,6H),1.46-1.39(m,2H),1.36-1.28(m,2H)
实施例83
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针83:
化合物72:
参照化合物50的方法合成。
1H-NMR(400MHz,CDCl
3):δ=7.89(s,1H),7.18(s,1H),6.96(s,1H),3.47(s,3H),3.10(s,6H),0.42(m,6H)
探针83:
参照探针78的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.89(s,1H),7.18(s,1H),6.96(s,1H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.12(t,J=4.9Hz,2H),3.10(s,6H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H),0.41(m,6H)
实施例84
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针84:
化合物73:
参照化合物49的方法合成。
1H-NMR(400MHz,CDCl
3):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),3.47(s,3H),3.13(s,6H)
探针84:
参照探针78的合成方法,
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),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),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.13(s,6H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.46-1.39(m,2H),1.36-1.28(m,2H)
对比实施例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参比荧光探针85:
参照探针1的合成方法,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)。
实施例85
以分子转子作为粘度响应性荧光染料,构建适用于HaloTag蛋白标签的荧光激活型共价标记的荧光探针86:
参照文献(Kimin Lim et.al.2011.115.22640-22646)和探针10的方法合成。
1H-NMR(400MHz,DMSO-d
6):δ=8.18(t,1H),7.48(s,1H),7.41(s,1H),7.32(m,2H),3.69(t,J=4.9Hz,2H),3.63-3.60(m,2H),3.56-3.53(m,2H),3.52(t,J=6.6Hz,2H),3.44(t,J=6.8Hz,2H),3.13(s,6H),3.12(t,J=4.9Hz,2H),1.79-1.71(m,2H),1.60-1.53(m,2H),1.48(s,6H),1.46-1.39(m,2H),1.36-1.28(m,2H).
实施例86
将探针与对应的HaloTag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。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。
表1不同探针荧光发射图谱检测结果
实施例87
将5uM HaloTag蛋白标签分别加入3uM的探针57、探针60、探针61、探针63、探针75、探针76、探针79溶液中,制得HaloTag蛋白标签和探针混合样品溶液,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发射光谱,结果分别如图1所示。从图1可以看出,上述探针可以实现从发射波长480nm到730nm的光谱覆盖。
实施例88
将HaloTag蛋白标签分别加入30uM的探针57、探针59、探针60、探针61、探针62、探针63、探针69、探针75、探针76、探针82的溶液中,制得HaloTag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HaloTag蛋白标签浓度与荧光强度关系图,结果分别如图2~图11所示。
从图2中可以看出,HaloTag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89
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HaloTag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57、探针59、探针60、探针61、探针69、探针75、探针76、探针82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12B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12A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200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90
为验证探针57、探针59、探针60、探针61、探针62、探针63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器HaloTag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HaloTag蛋白标签在不同细胞器上定位的质粒,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13显示,探针可以在免洗的情况下清晰展示细胞骨架、线粒体、细胞核、高尔基体、全细胞、溶酶体等亚细胞器结构。
以上结果说明的探针可以作为多种细胞亚细胞器标记的有力工具。
实施例91
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-SNAP-NLS、pcdna3.1-mito-HaloTag(HaloTag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的参比探针85和探针57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14显示,参比探针85与探针57可以在同时在免洗的情况下分别清晰展示线粒体和细胞核结构,且参比探针85标记的细胞核荧光与探针57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92
首先,将表达HaloTag蛋白标签的质粒pcdna3.1-HaloTag(样品组)和未表达HaloTag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DNA,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77通过尾静脉注射方法注射到小鼠体内标记HaloTag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15显示,注射探针77的对照质粒pcdna3.1-CAT的小鼠肝脏荧光很低,接近为未注射探针的空白肝脏的本底荧光水平,而注射探针77的HaloTag质粒pcdna3.1-HaloTag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HaloTag蛋白标签,并产生较强荧光信号。
实施例93
为验证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HaloTag蛋白为例,在Hela细胞中以AID降解系统为例检测了结合HaloTag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-HaloTag-IAA17-H2B质粒。细胞转染后24小时,使用含有1μM探针61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HaloTag-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16所示,HaloTag-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,HaloTag-IAA17-H2B蛋白荧光信号逐渐降低,加入吲哚乙酸90min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94
为验证探针可以用于实时监测哺乳动物细胞内生物大分子的组装与降解过程,以Hela细胞为例检测了探针在哺乳动物细胞内示踪细胞间隙蛋白CX43组装形成细胞间隙通道的过程。CX43基因C端同HaloTag基因融合,并通过慢病毒感染技术构建并获得了稳定表达融合蛋白CX43-HaloTag的Hela细胞株。探针标记前10小时,将Hela-CX43-HaloTag细胞株种植于20mm玻璃底细胞培养培养皿中。标记时首先使用无酚红DMEM培养基稀释探针61至2μM并替换原有细胞培养基。细胞置于37℃二氧化碳培养箱孵育1小时。之后使用新鲜无酚红DMEM培养基清洗细胞两次,去除未结合探针61,每次间隔2分钟。然后,加入含有1μM探针57的DMEM无酚红培养基标记细胞,使用Leica SP8共聚焦显微镜长时间监测标记细胞样品在探针61、探针57两个探针相应荧光通道的荧光强度及位置变化过程分别如图 17A和17B所示。在探针61荧光通道可见在2个细胞之间长柱形特异性荧光信号,如图17A所示,与文献(Guido Gaietta et al.Science 2002,296,503-507.)报道的结果一致。刚刚加入探针57时在其相应荧光通道并不能检测到荧光信号,如图17B所示,说明探针61标记了细胞内全部存在的CX43蛋白。随着培养时间延长,不断有新的CX43-HaloTag蛋白合成,并被探针57标记。标记1小时后,在被探针61标记的原有细胞间隙通道边缘出现探针57荧光信号出现并逐渐增强,而旧的细胞间隙通道探针61荧光信号逐渐降低,说明细胞间隙通道中新合成的CX43-HaloTag蛋白是从周围逐渐向中心替换原有CX43-HaloTag蛋白,叠加通道如图17C所示,与文献报道的结果一致。以上的实验结果证明HaloTag一系列化合物可以适用于实时监测细胞内生物大分子的组装与降解过程。
实施例95
为验证探针标记HaloTag蛋白标签相比探针标记SNAP蛋白标签拥有更快的标记速度和更高的荧光强度,以探针48标记HaloTag蛋白标签和参比探针85标记SNAP蛋白标签为例(探针48和参比探针85具有相同的荧光染料部分),标记时首先将纯化的HaloTag蛋白和SNAP蛋白用PBS稀释至7.5uM。分别将探针48和参比探针85用PBS稀释至15uM,取20uL探针48加入80uL HaloTag蛋白中混匀,取20uL参比探针85加入80uL SNAP蛋白中混匀,使用酶标仪监测两种溶液在620nm处激发、650nm处发射的荧光强度的变化,持续两个小时。如图18所示,探针48标记HaloTag蛋白标签的t
1/2小于2s,而参比探针85标记SNAP蛋白标签的t
1/2大约为7min,且荧光强度比前者低一倍以上。以上的实验结果证明探针标记HaloTag蛋白标签相比探针标记SNAP蛋白标签拥有更快的标记速度和更高的荧光强度。
Claims (9)
- 一种荧光探针,包括配体部分A、任选的连接体部分B、荧光染料部分C,所述荧光染料部分C为粘度响应性荧光染料,包括电子供体部分D、共轭体系E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。
- 根据权利要求1所述的荧光探针,其为如式(I)所示结构的化合物,或者其盐,A-B-C(I)其中,连接体部分B为任选存在的基团,选自亚烷基、改性亚烷基;式(I)中的荧光染料部分C为式(I-R)所示结构部分,其中:式(I-R)中,与连接体部分B或配体部分A相连位置的氢被连接体部分B或配体部分A替代;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选自氢、烷基、或改性烷基,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E为选自双键、叁键、芳香环、芳香杂环中的至少一种共轭连接而形成的基团,其为如下式(I-1)所示的结构,其中所含的各氢原子任选独立地被选自卤原子、硝基、亲水性基团、烷基和改性烷基的取代基取代,所述取代基任选地相互连接构成脂环或脂杂环;任选地,所述式(I-1)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,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、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;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: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;可选地,为C 1-C 10直链或支链烷基;可选地,为C 1-C 7直链或支链烷基;可选地,为C 1-C 5直链或支链烷基;可选地,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;可选地,为C 1-C 7直链或支链亚烷基;可选地,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)-、 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)-、 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元单环或稠合双环杂芳香基团;可选地,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11~14元稠合三环杂芳香基团;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5~7元双环亚环烃基;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RCONR′基团;所述“硫代酰胺基”为R(C=S)NR′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、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(可选地为C 1-C 6)烷基或亚烷基的任意一个碳原子被选自-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,可选地为1~50,可选地为1~30,可选地为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,所述杂芳环选自噻吩、呋喃、吡咯。
- 根据权利要求1-3中任一项所述的荧光探针,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,所述蛋白标签选自脱卤素酶;可选地,所述蛋白标签为卤代烷脱卤素酶(HaloTag)或其突变体;可选地,所述配体部分A来自于卤代烃类化合物;可选地,适用于HaloTag的配体部分A来自于脱卤素酶底物;可选地,所述配体部分A为卤代烷基;可选地为卤代乙基,可选地为下式结构:其中:X是卤素,可选地为氯;可选地,所述连接体部分B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由一个或多个-O-置换;所述的碳原子被-O-置换,是指碳原子或碳原子与其上的氢原子一起被-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直链或支链烷基或亚烷基;可选地,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;可选地,X 1、X 2各自独立地为任选被一个或多个选自磺酸基、羧基的基团取代的由-HN(C=O)-O-改性的C 4-30烷基或亚烷基;可选地,-NX 1-X 2形成选自下式(I-i-1)、(I-i-2)的任一基团: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,所述共轭体系E中两个相邻取代基相互连接构成饱和或不饱和的脂环或脂杂环;可选地,所述共轭体系E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,所述共轭体系E中含有NH;可选地,所述NH上的H被烷基或改性烷基取代;可选地,所述共轭体系E选自下式(I-1-1)~(I-1-38)中的结构:可选地,所述共轭体系E与-NX 1-X 2形成如下(I-i-3)(I-i-7)所示的脂杂环:或者,所述共轭体系E与-NX 1-X 2形成如下(I-i-8)所示的结构: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:可选地为双环或三环稠芳香环或稠芳香杂环;可选的,R 2或R e的上述结构中CH上的H被卤原子、氰基、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-i)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-i)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)、式(I-2-b)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-2-38)中的一种:
- 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1-5中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。
- 权利要求1-5中任一项所述的荧光探针在蛋白荧光标记、蛋白的定量、检测或动力学研究中的用途;或者在细胞、组织、活体成像中的用途。
- 一种探针试剂盒,其特征在于,包括权利要求1-5中任一项所述的荧光探针;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。
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US20200248069A1 (en) | 2020-08-06 |
EP3690002A4 (en) | 2021-06-23 |
EP3690002A1 (en) | 2020-08-05 |
CN109574880B (zh) | 2022-06-17 |
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