Fluorescent probe and preparation method and use thereof

Abstract

Provided are a fluorescent probe and a preparation process and the use thereof. The fluorescent probe is sensitive and specific to viscosity, and can be used for specific fluorescent labeling of proteins, and can also be used for quantification, detection or kinetic studies of proteins, and the imaging of cells, tissues and living bodies.

Description

FIELD OF THE INVENTION

The present invention relates to a fluorescent probe and a preparation method and the use thereof.

BACKGROUND OF THE INVENTION

Fluorescence specific labeling is a powerful tool for studying protein function and quantification. Contrast to other study methods, fluorescent labels have the irreplaceable advantages, such as being sensitive, in situ, instant, and visual. Currently, the most often used method of fluorescent labeling proteins is expressing the fluorescent protein in situ on the target protein by gene fusion technology, thereby realizing specific illumination of the target protein and making it possible to perform tracking studies of the target protein in cells or tissues under a fluorescence microscope. Fluorescent protein technology has been developed for a long time, and this technology is relatively mature. However, there are a few shortcomings. For example, fluorescent proteins suffer from a long maturing and folding time, and tendency to aggregate. Once the fluorescent protein is expressed, it is difficult to carry out post-modification; in addition, most fluorescent proteins still have a shortcoming such as less photostability, etc. These shortcomings limit the application of fluorescent proteins in some extent.

In fact, the chromophores structure of fluorescent protein is relatively simple, which makes it very difficult to construct different types or functionalized fluorescent proteins. Furthermore, there is little guideline to follow, and sea screen can only be carried out by means of random mutation. In contrast, organic small molecule fluorescent dyes are rich in molecular structure, but small molecule fluorescent probes still have many defects in protein-specific labeling. Recently, the emergence of chemical tag technology has effectively solved this problem. With chemical tags, the target protein is tagged with a polypeptide or a protein tag with specific recognition function, and small-molecule fluorescent probe-specific protein labeling is achieved by the highly specific binding of the tag to the substrate. Thus, chemistry tag technology not only inherits advantages of gene fusion technology and sufficiently inherits advantages in all aspects of organic dye probes as compared to fluorescent proteins. Currently, SNAP-tag (K. Johnsson et. al. WO 2004031405), CLIP-tag (K. Johnsson et. al. WO 2008012296), Halo-tag (Wood, Keith V et. al. WO 2004072232), have been commercialized, especially SNAP-tag and CLIP-tag are the most widely used and have been recognized by the market.

Although chemical tag such as SNAP-tag and CLIP-tag are capable of specifically labeling their protein of interest during the labeling process, no matter free probes or labeled probes in the system all emit fluorescent. That is, the probes emits fluorescence within the system, no matter the probe binds to the target or not. This non-characteristic fluorescence emission is clearly a serious problem in current chemical labeling technology. Therefore, in a strict sense, this method still cannot achieve the same specificity as fluorescent protein. The only effective way to solve this problem is washing out the unlabeled probe. Apparently, the application of this technology will be severely limited in situations where it is needed quickly or the probe cannot be washed.

If a method for fluorescent-activated protein-specific labeling for SNAP-tag and CLIP-tag is designed, which remains dark or emits very weak fluorescence before labeling, and the fluorescence of the dye is sharply enhanced once it is bind to the protein. Undoubtedly, this kinds of probes will be able to achieve the same specificity as fluorescent proteins, which will not only eliminate the washing out procedure of free probes, but also greatly reduce the background interference of free probes, furthermore will widen the application of SNAP-tag and CLIP-tag technology. A method for designing a fluorescently activated protein-specific label suitable for this technique must consider a suitable fluorescence ON/OFF switching mechanism. The FRET mechanism is first applied to this design, which additionally adds the ligand with a fluorescence quenching group. Normally, the small molecule fluorescence is quenched by the quenching group; once the ligand binds to the chemical tag, the quenching group is released, to achieve fluorescence activation (T. Komatsu. et. al. J. Am. Chem. Soc. 2011, 133, 6745-6751). However, the introduction of the quenching group greatly increases the molecular volume of the probe, which greatly reduces the labeling speed, and severely limits the real-time tracking and detection of proteins in cells and tissues by the probe. Furthermore, there must be a good energy level match between the fluorescent probe and the quenching group, which makes the FRET design of long wavelength fluorescent probes become very difficult, for example, the red light emitting dye. In addition, some dyes that are extremely sensitive to fluorescence have also been used to design fluorescent probes (T K Liu. et. al. ACS Chem. Biol. 2014, 9, 2359-2365). These probes exhibit no fluorescence or weak fluorescence when the dyes are in polar fluids, such as cell fluid. When the ligand binds to the protein, the probe is placed in the non-polar pocket of the protein, result the probe emits stronger fluorescence. However, on the one hand, due to the presence of a more polar hydration layer on the surface of the protein, 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 greatly, which can lead to polar-sensitive probes with a high background in cell or tissue imaging. Recently, the literature (T Y Wang et. al. Chem Sci. 2016, 7, 301-307) reported a molecular rotor fluorescent probe with viscosity sensitivity, which is sterically hindered after protein ligands are covalently bound to proteins. The action reduces the molecular rotor's degree of freedom, thereby allowing fluorescent of the probe to be activated. However, in this literature, the fluorescence intensity of the probe after fluorescence activation is dim, and the fluorescence quantum yield is very low. Therefore, the method reported in this literature cannot be used as qualified fluorescent protein tags for labeling, tracking, localization and quantification of target proteins.

SUMMARY

We have found that by linking the ligand moiety to the electron donor moiety of the viscosity-sensitive fluorescent dye, the fluorescence intensity of the viscosity-sensitive fluorescent dye can be greatly enhanced after the ligand bond to the target protein, thereby obtaining a novel structure of fluorescence probes. These probes are sensitive to the viscosity and can be used for proteins specific labeling, which exhibit fast labeling speed, dramatic fluorescence enhancement and wide application range, and can be effectively used for labeling, tracking, localization and quantification of target proteins.

In view of the above, there is provided a fluorescent probe comprising: a ligand moiety A, an optional linker moiety C and a fluorescent dye moiety, the fluorescent dye moiety is a viscosity sensitive fluorescent dye group, which comprises an electron donor moiety D, a conjugated system moiety B and an electron acceptor moiety, said ligand moiety A being a group capable of specifically recognizing and labeling a target protein tagged with a protein tag or a fusion protein, and optionally, said ligand moiety A being capable of specifically recognizing and covalently labeling a target protein tagged with a protein tag or a fusion protein; wherein, the ligand moiety A is directly covalently attached to the electron donor moiety D of the fluorescent dye moiety, or is covalently attached to the electron donor moiety D of the fluorescent dye moiety via a linker moiety C.

Optionally, the fluorescent probe described above has a structure as shown in the Formula (I),

wherein the linker moiety C is an optionally present group, which is selected from the group consisting of an alkylene group, or a modified alkylene group;

the structural moiety represented by the Formula (I-R) in the Formula (I) is a fluorescent dye moiety,

wherein, the electron donor moiety -D- is —NX₁—X₂—, X₁ is selected from hydrogen, an alkyl group or a modified alkyl group, X₂ is selected from an alkylene group or a modified alkylene group, X₁, X₂ are optionally linked to each other, and form an aliphatic heterocycle with the nitrogen atom;

the conjugated system moiety B is formed by conjugating at least one selected from the group consisting of a double bond, a triple bond, an aromatic ring, and an aromatic hetero ring, and has a structure represented by the following Formula (I-1), optionally, the hydrogen atoms contained are independently replaced with one or more substituents selected from the group consisting of a halogen atom, nitro, a hydrophilic group, an alkyl group, and a modified alkyl group, and the substituents are optionally bonded to each other to form an alicyclic or heterocyclic ring;

Optionally, the structure of formula (I-1) is linked to X₁ and X₂ to form an aliphatic heterocyclic ring;

the electron acceptor moiety has a structure represented by the following Formula (I-2),

wherein,

R₁ is selected from the group consisting of hydrogen, a halogen atom, nitro, an alkyl group, an aryl group, a heteroaryl group, a hydrophilic group or a modified alkyl group;

R₂ is selected from the group consisting of cyano, carboxyl, a keto group, an ester group, an amide group, a phosphonic acid group, a phosphonate group, sulfo, a sulfonate group, sulfone, sulfoxide group, an aryl group, a heteroaryl group, an alkyl group or a modified alkyl group;

R₃ is cyano;

In certain embodiment, the electron acceptor moiety optionally forms a cyclic structure of the following Formulas (I-2-a), (I-2-b):

wherein, R_a, R_b are independently selected from the group consisting of hydrogen, a hydrophilic group, an alkyl group, and a modified alkyl group, and R_a and R_b are optionally bonded to each other to form an alicyclic or heterocyclic ring;

Y₁ is selected from the group consisting of —O—, —S—, —(S═O)—, and —(NR_i)—, wherein R_i is selected from hydrogen, an alkyl group or a modified alkyl group;

Y₂ is selected from the group consisting of ═O, ═S, ═S═O and ═NR_i wherein R_i is selected from hydrogen, an alkyl group or a modified alkyl group;

Y₃ is selected from the group consisting of ═O, ═S, ═S═O and ═NR_i wherein R_i is selected from hydrogen, an alkyl group or a modified alkyl group;

or, Y₃ is ═C(R_e)(CN);

R_e is selected from the group consisting of cyano, carboxyl, a keto group, an ester group, an amide group, a phosphonic acid group, a phosphonate group, sulfo, a sulfonate group, sulfone, sulfoxide, an aryl group, a heteroaryl group, an alkyl group or a modified alkyl group;

when R₂ or R_e is an aryl group or a heteroaryl group, optionally, the hydrogen atom on the ring is independently replaced with a substituent selected from a halogen atom, nitro, a hydrophilic group, an alkyl group or a modified alkyl group; optionally, the substituents are linked to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring;

wherein,

said alkyl group is a saturated aliphatic straight or branched alkyl group of 1 to 30 carbon atoms;

said alkylene group is a saturated aliphatic straight or branched alkylene group of 1 to 30 carbon atoms;

said modified alkyl group is an alkyl group wherein any carbon atom contained is independently replaced with one or more substituents selected from halogen atom, —O—, —OH, —CO—, —NO₂, —CN, —S—, —SO₂—, —(S═O),

phenyl, phenylene, a primary amino group, a secondary amino group, a tertiary amino group, a quaternary ammonium salt group, a saturated or unsaturated monocyclic or bicyclic cycloalkylene group, a bridged aliphatic heterocyclic ring; the modified alkyl group has 1 to 300 carbon atoms, the carbon-carbon single bond optionally being replaced independently by a carbon-carbon double bond or a carbon-carbon triple bond; said modified alkylene group is an alkylene group wherein any carbon atom contained is independently replaced with one or more substituents independently selected from halogen atom, —O—, —OH, —CO—, —NO₂, —CN, —S—, —SO₂—, —(S═O),

phenyl, phenylene, a primary amino group, a secondary amino group, a tertiary amino group, a quaternary ammonium salt group, a saturated or unsaturated monocyclic or bicyclic cycloalkylene group, a bridged aliphatic heterocyclic ring, the modified alkylene group has 1 to 30 carbon atoms, the carbon-carbon single bond optionally being replaced independently by a carbon-carbon double bond or a carbon-carbon triple bond;said alicyclic ring is a saturated or unsaturated 4- to 10-membered monocyclic or polycyclic alicyclic ring;

said heteroalicyclic ring is a saturated or unsaturated 4- to 10-membered monocyclic or polycyclic heterocyclic ring having at least one heteroatom selected from N, O, S or Si, on the heteroalicyclic ring. When the heteroalicyclic ring contains an S atom, it is optionally —S— or —SO₂—; the heterocyclic ring is optionally substituted with halogen atom, nitro, an alkyl group, an aryl group, a hydrophilic group, and a modified alkyl group;

said aryl or aromatic ring is 5 to 10 membered monocyclic or fused bicyclic ring;

the heteroaryl or aromatic heterocyclic ring is a 5- to 10-membered monocyclic or fused bicyclic ring containing at least one heteroatom selected from N, O, S or Si in the ring;

said halogen atoms are each selected independently from the group consisting of F, Cl, Br, I;

said hydrophilic group is hydroxyl, sulfo, sulfuric acid group, phosphoric acid group, primary amino group, secondary amino group or tertiary amino group and substituendum thereof;

said monocyclic cycloalkylene group is a 4- to 7-membered cycloalkylene group;

said bicyclic cycloalkylene group is a 5- to 7-membered bicyclic cycloalkylene group;

said bridged aliphatic heterocyclic ring is a 5- to 20-membered bridged aliphatic heterocyclic ring containing at least one heteroatom selected from N, O, or S in the ring.

Optionally, 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;

Optionally, the protein tag is O₆-alkylguanine-DNA alkyltransferase (SNAP-tag) or a mutant thereof, alkylcytosine transferase (CLIP-tag) or a mutant thereof;

Optionally, the mutant of the O₆-alkylguanine-DNA alkyltransferase is selected from the group consisting of SNAP F33G or SNAP V164E;

Optionally, the protein tag is O₆-alkylguanine-DNA alkyltransferase (SNAP-tag) or a mutant thereof;

The ligand moiety A is derived from an O₆-alkylguanine derivative, an alkyl 4-chloropyrimidine derivative or an alkylcytosine derivative;

Optionally, the ligand moiety A suitable for the SNAP-tag is derived from an O₆-alkylguanine derivative or an alkyl 4-chloropyrimidine derivative; optionally, the ligand moiety A suitable for the CLIP-tag is derived from an alkylcytosine derivative;

Optionally, the ligand moiety A- is selected from the following structures:

Optionally, the modified alkyl group or the modified alkylene group is independently a group containing one or more substituents selected from —OH, —O—, -ethylene glycol moiety (i.e. —(CH₂CH₂O)n-), C₁ to C₈ alkyl groups, C₁ to C₈ alkoxy, C₁ to C₈ acyloxy, C₁ to C₈ haloalkyl, monosaccharide moiety, disaccharide moiety, polysaccharide moiety, —O—CO—, —NH—CO—, —(—NH—CHR—CO—)_n, —SO₂—O—, —SO—, —SO₂—NH—, —S—S—, —CH═CH—, —C≡C—, a halogen atom, cyano, nitro, o-nitrophenyl, phenacyl, a phosphate group or a phosphonate group, wherein n is from 1 to 100, and R is selected from H or an α-amino acid residue;

Optionally, the C₁ to C₈ alkyl group is methyl, ethyl, propyl, isopropyl, and the C₁ to C₈ alkoxy group is methoxy, ethoxy, propoxy, isopropoxy, the C₁ to C₈ acyloxy is acetoxy, propionyloxy, isopropionyloxy, C₁ to C₈ haloalkyl is trifluoromethyl, chloromethyl, bromomethyl;

Optionally, the aliphatic heterocyclic ring is azetidine, pyrrolidine, piperidine, tetrahydrofuran, tetrahydropyran, morpholine, thiomorpholine;

Optionally, the heteroaryl ring is thiophene, furan, and pyrrole.

Optionally, the linker moiety is selected from the group consisting of —(C═O)—, —(CH₂CH₂O)_n—, wherein n is 1 to 20;

Optionally, X₁ is a C_1-50 straight or branched alkyl group optionally substituted by one or more groups selected from hydroxyl, cyano, halogen atom, carboxyl, and quaternary ammonium group, or a C_2-50 ether chain group having 1 to 10 oxygen atoms and optionally substituted by one or more groups selected from a sulfonic acid group or carboxyl, and X₂ is a C_1-50 straight or branched alkylene groups optionally substituted by one or more groups selected from hydroxyl, cyano, halogen atom, carboxyl, quaternary ammonium group; or a C_2-50 ether chain group having 1 to 10 oxygen atoms and optionally substituted by one or more groups selected from a sulfonic acid group or carboxyl; or, —NX₁—X₂— forms any one of groups of the following Formulas (I-1-1) to (I-1-2):

Optionally, X₁ is a C_1-10 straight or branched alkyl group optionally substituted with one or more groups selected from hydroxyl, cyano, halogen atom, carboxyl, and quaternary ammonium group, and X₂ is a C_1-10 straight or branched alkylene group optionally substituted with one or more groups selected from hydroxyl, cyano, a halogen atom, carboxyl, and a quaternary ammonium group.

Optionally, the above fluorescent probe is characterized in that two adjacent substituents in the conjugated system moiety B are linked to each other to form a saturated or unsaturated alicyclic ring or aliphatic heterocyclic ring;

Optionally, H of the CH in the conjugated system moiety B is replaced with halogen atom, nitro, hydrophilic group, alkyl group or modified alkyl group;

Optionally, the conjugated system moiety B contains NH; optionally, H on the NH is replaced with an alkyl group or a modified alkyl group;

Optionally, the conjugated system moiety B is selected from the structures of the following Formulas (I-1-1) to (I-1-28):

Optionally, the conjugated system moiety B and X₁ are linked to each other to form an alicyclic heterocyclic ring as shown below.

wherein, X₂ is as defined in claim 2 or 3.

Optionally, the fluorescent probe described above, wherein R_a, R_b in the Formula (I-2-a) form

with the bonded carbon atoms;

Optionally, the R₂ and R_e are independently selected from the following structures, or a bicyclic or polycyclic fused aromatic ring or fused aromatic heterocyclic ring formed by the following structures themselves or fused with each other: preferably are a bicyclic or a tricyclic fused aromatic ring or fused aromatic heterocyclic ring;

Optionally, H of CH in the above structures of R₂ or R_e is replaced by halogen atom, nitro, hydrophilic group, alkyl group or modified alkyl group; optionally, R₂ or R_e is a group containing NH selected from the above structure, and optionally, the H on the NH is replaced with an alkyl group or a modified alkyl group;

Alternatively, the R₂ and R_e are independently selected from modified alkyl group: the modified alkyl group contains a keto group, an ester group or an amide group, and is bonded to alkenyl carbon of the Formula (I-2) or of formula (I-2-a) via a carbonyl group in the keto, ester or amide group;

Optionally, the structure of the Formula (I-2) is selected from the group consisting of the following Formulas (I-2-1) to (I-2-19):

Optionally, the fluorescent probe described above is selected from the following structures:

In another aspect, there is provided a process for preparing the fluorescent probe as described above, comprising a step of reacting a fluorescent dye of formula (II) with a ligand and an optionally linker:

wherein, after reaction, D′ forms a D-group that bonds to the linker moiety or the ligand moiety.

In another aspect, there is also provided a fluorescent activated protein specific labeling method, comprising the following steps: contacting the fluorescent probe described above with a target protein of a protein tag or a fusion protein tag, labeling reaction taking place at the ligand moiety of the fluorescent probe with the protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is covalently labeled on the protein tag;

Optionally, a reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or an in situ medium in which the target protein of the protein tag or the fused protein tag is located; optionally, the in situ medium is intracellular media, organelle media, living tissue media, blood or body fluids.

In another aspect, there are further provided uses of the above-mentioned fluorescent probe for protein fluorescent labeling, quantification, detection or kinetic studies of proteins, and use in imaging of cells, tissues, and living bodies.

In another aspect, a probe kit is provided, comprising the fluorescent probe described above.

Optionally, the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is at least one selected from dimethyl sulfoxide, a buffer, and physiological saline; optionally, the buffer includes phosphate buffer.

The above-described protein tag or the target protein moiety to which the tag is fused can be prepared by existing genetic engineering techniques.

The above-mentioned viscosity-sensitive fluorescent dyes mean that the fluorescence intensity of the dye responds to the viscosity of the solution, and as the viscosity of the solution increases, the fluorescence intensity increases. Optionally, the viscosity-sensitive fluorescent dye is an organic dye molecule which, under the same concentration and excitation wavelength, at 25° C. has a ratio of the maximum fluorescence emission intensity of the dye in glycerol to the fluorescence intensity in methanol of greater than 2, preferably more than 5, more preferably more than 10. The concentration of the viscosity-sensitive dye ranges from 1×10⁻⁷ M to 1×10⁻⁵ M.

Depending on the circumstances, the person skilled in the art can select the corresponding label and ligand as needed.

Those skilled in the art can use correspondingly instrumentation equipment to track and monitor the target protein of the protein tag or the fusion protein tag. The instrument equipment used, as needed, includes equipment and facilities capable of testing or displaying fluorescent, such as fluorescence spectrometer and fluorescent microscopes, confocal fluorescence microscopes, microplate readers, flow cytometers, and in vivo imagers.

The operator can select different types of dyes with different emission/excitation wavelengths as needed.

According to an embodiment of one aspect of the present invention, the fluorescent probe has a wide range of fluorescence emission wavelengths.

According to an embodiment of another aspect of the present invention, the fluorescence intensity of the fluorescent probe is enhanced as the viscosity of the environment increase, is sensitive to viscosity, and has viscosity responsiveness.

According to an embodiment of another aspect of the present invention, the fluorescent probe can be used for a specific label of a target protein of a protein tag or a fusion protein tag, and fluorescence can be activated after the fluorescent probe binds to the protein tag, the fluorescent probe has good fluorescent molecular switching properties, and the fluorescence activation factor is high and the fluorescence activation brightness is high.

According to an embodiment of another aspect of the present invention, the fluorescent probe has a good linear relationship between the fluorescence intensity and the protein tag concentration, which can be used for quantitative detection of a target protein.

According to an embodiment of another aspect of the present invention, the fluorescent probe can achieve specific labeling intracellular protein tags and achieve fluorescence-specific imaging, and the probe fluorescence is not affected by the intracellular environment.

According to an embodiment of another aspect of the present invention, a fluorescent probe can be used as a powerful tool for labeling cell subcellular organelle marker, such as labeling nucleus, mitochondria, Golgi, endoplasmic reticulum, whole cells, cytoskeleton, extracellular membrane, lysosomes, cell inner membrane or the like.

According to an embodiment of another aspect of the present invention, the spectra of fluorophores of different fluorescent probes do not interfere with each other, and the fluorescent probes of different color systems of the present invention can perform multi-color labeling on samples, and can simultaneously perform orthogonal labeled imaging.

According to an embodiment of another aspect of the present invention, the fluorescence of the fluorescent probe is not affected by the internal environment of the animal, and can be applied to living animal, for example, specifically labels a SNAP protein tag expressed in liver, and exhibits a strong fluorescent signal.

According to an embodiment of another aspect of the present invention, a fluorescent probe can be used to track and monitor the degradation process of a target protein.

According to an embodiment of another aspect of the present invention, the fluorescent probe monitors the assembly and degradation processes of biological macromolecules in mammalian cells in real time.

According to an embodiment of another aspect of the present invention, 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.

According to an embodiment of another aspect of the present invention, the fluorescent probe does not exhibit any detection signal when the fluorescent probe does not label the target protein of the protein tag or the fusion protein tag, and does not interfere with the detection of the sample, and can realize rapid quantitative detection of target protein in complex samples, and can also track the dynamics of the labeling reaction process.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The fluorescence emission diagram of activated fluorescence of different wavelengths after different probes are bound to protein tags;

FIG. 2 to FIG. 11 are standard curves of the corresponding fluorescence intensity versus different SNAP protein tag concentrations for probe 1, probe 14, probe 21, probe 30, probe 43, probe 48, probe 56, probe 63, probe 70, and probe 88, respectively;

FIG. 12 is a fluorescence spectrum of cell labeled with different probes, wherein (1) to (8) are probe 1, probe 15, probe 21, probe 30, probe 56, probe 60, probe 63, Probe 88, respectively, group A is a HeLa cell with a protein tag expressed, and group B is a HeLa-WT cell (original HeLa cell, without a protein tag expressed);

FIG. 13 shows micrographs of different organelles labeled with different probes, wherein group A to group F are probe 1, probe 21, probe 48, probe 60, probe 66, probe 77 respectively, (1) to (9) are the nucleus, mitochondria, Golgi, endoplasmic reticulum, whole cells, cytoskeleton, extracellular membrane, lysosome, and intracellular membrane, respectively;

FIG. 14 shows the two-color labeling of the same cell by different probes, wherein A is probe 1 labeled mitochondrion, B is probe 43 labeled nucleus, and C is an orthogonal imaging of A and B;

FIG. 15 shows labeling with probe 88 in living mice, wherein group A is a blank group, group B is a control group, group C is a sample group, (1) is a liver, and (2) is a kidney;

FIG. 16 shows the fluorescence changes of probe 21 in mammalian cells with the protein degradation;

FIG. 17 shows tracing cell gap assembly process with different probes, wherein, A is the fluorescent channel of probe 21, B is the fluorescent channel of probe 1, and C is the superimposed fluorescent channel of probe 21 and probe 1.

EXAMPLES

Specific examples of the present invention will be described in detail below. It should be understood that the specific examples described herein are merely illustrative description of the present invention and are not intended to limit the present invention.

Example 1

A fluorescence-activated covalently labeling fluorescent probe 1 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 1

To a solution of N-methyl-N-(2-hydroxyethyl)-4-amino-benzaldehyde (0.358 g, 2 mmol) and tert-butyl cyanoacetate (0.338 g, 2.4 mmol) in 50 mL anhydrous ethanol, a catalytic amount of anhydrous zinc chloride was added. The obtained mixture was heated in an oil bath for 5 hrs under the protection of Ar. After the reaction was completed, the solution was cooled at room temperature, a part of the solvent was removed by rotary evaporation. A large amount of precipitate was formed. The precipitate was separated by filtration, thoroughly washed with cooled ethanol twice and finally dried in vacuo to give a pure yellow compound 1 (0.49 g, 81%). ¹H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 4.79 (bt, 1H), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 1.50 (s, 9H).

Compound 2

The synthesis was carried out with reference to the method disclosed in Antje Keppler et. al. Nat Biotechnology. 2002, 21, 86-89. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.82 (s, 1H), 7.39 (m, 4H), 6.27 (s, 2H), 5.45 (s, 2H), 3.71 (s, 2H).

Probe 1

Compound 1 (0.302 g, 1.0 mmol) and 4-dimethylaminopyridine (0.146 g, 1.2 mmol) were dissolved in 20 mL anhydrous dichloromethane, p-nitro phenyl chloroformate (0.242 g, 1.2 mmol) in 10 mL anhydrous dichloromethane was added dropwise under the protection of Ar. The obtained mixture was stirred and kept at room temperature for 1 hr under the protection of Ar. After the reaction was completed, the solvent was completely removed by rotary evaporation. The residue was re-dissolved in anhydrous N,N-dimethylformamide (DFM), compound 2 (0.324 g, 1.2 mmol) was added in the presence of anhydrous triethylamine (TEA) (0.16 ml, 1.2 mmol). The obtained mixture was stirred at room temperature for another 30 min under the protection of Ar. After the reaction was completed, the solvent was completely removed by rotary evaporation. The residue was purified by gel silica gel column chromatography to give a pure Probe 1, at a yield of 70%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.79 (bt, 1H), 4.40 (d, 2H, J=4.90 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 1.50 (s, 9H).

Example 2

A fluorescence-activated covalently labeling fluorescent probe 2 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 3

The synthesis was carried out with reference to the method disclosed in J. Das et. al. Bioorg. Med. Chem. Lett. 2005, 15, 337-343. ¹H-NMR (400 MHz, CDCl₃): δ=7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 4.12 (s, 2H).

Compound 4

This compound was obtained by following the general procedure for compound 1. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.11-8.07 (m, 2H), 8.01-7.97 (m, 3H), 7.70 (d, 1H, J=8.8 Hz), 7.46-7.43 (m, 1H), 6.27 (dd, 1H, J=9.2, 1.6 Hz), 6.02 (s, 1H), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Probe 2

This probe was obtained by following the general procedure for probe 1, and the yield was 75%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.41 (s, 1H), 10.01 (s, 1H), 8.11-8.07 (m, 2H), 8.01-7.97 (m, 3H), 7.81 (s, 1H), 7.70 (d, 1H, J=8.8 Hz), 7.46-7.43 (m, 1H), 7.41 (m, 4H), 6.29 (s, 2H), 6.27 (dd, 1H, J=9.2, 1.6 Hz), 6.02 (s, 1H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.9 Hz), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 3

A fluorescence-activated covalently labeling fluorescent probe 3 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 5

This compound was obtained by following the general procedure for compound 1, and the yield was 95%. ¹H-NMR (400 MHz, CDCl₃): δ=8.09 (s, 1H), 8.02 (d, 1H, J=8.0 Hz), 7.98 (d, 2H, J=9.2 Hz), 7.86 (d, 1H, J=8.4 Hz), 7.48 (t, 1H, J=7.8 Hz), 7.36 (t, 1H, J=7.36 Hz), 6.73 (d, 2H, J=9.2 Hz), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Probe 3

This probe was obtained by following the general procedure for probe 1, and the yield was 65%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.40 (s, 1H), 10.02 (s, 1H), 8.09 (s, 1H), 8.02 (d, 1H, J=8.0 Hz), 7.98 (d, 2H, J=9.2 Hz), 7.86 (d, 1H, J=8.4 Hz), 7.81 (s, 1H), 7.48 (t, 1H, J=7.8 Hz), 7.40 (m, 4H), 7.36 (t, 1H, J=7.36 Hz), 6.73 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 1H, J=4.8 Hz), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 4

A fluorescence-activated covalently labeling fluorescent probe 4 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 6

The synthesis was carried out with reference to the method disclosed in the literature (Srikun D 1 et. al JACS 2010, 132, 4455-4465). ¹H-NMR (400 MHz, DMSO-d₆): δ=7.33 (d, 2H, J=8.0 Hz), 7.31 (d, 2H, J=8.0 Hz), 7.10 (brs, 2H), 6.10 (s, 1H), 5.25 (s, 2H), 3.68 (s, 2H).

Probe 4

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): ¹H-NMR (400 MHz, DMSO-d₆): δ=9.99 (brs, 1H), 8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.39 (d, 2H), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.85 (d, 2H, J=9.2 Hz), 6.10 (s, 1H), 5.26 (s, 2H), 4.79 (bt, 1H), 4.36 (s, 2H), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 1.50 (s, 9H).

Example 5

A fluorescence-activated covalently labeling fluorescent probe 5 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 5

This probe was obtained by following the general procedure for probe 1, and the yield was 59%. 1H-NMR (400 MHz, DMSO-d6): δ=9.97 (s, 1H), 8.11-8.07 (m, 2H), 8.01-7.97 (m, 3H), 7.70 (d, 1H, J=8.8 Hz), 7.46-7.43 (m, 1H), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.10 (s, 2H), 6.27 (dd, 1H, J=9.2, 1.6 Hz), 6.10 (s, 1H), 6.02 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 6

A fluorescence-activated covalently labeling fluorescent probe 6 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 6

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, CDCl₃): δ=9.98 (brs, 1H), 8.09 (s, 1H), 8.02 (d, 1H, J=8.0 Hz), 7.98 (d, 2H, J=9.2 Hz), 7.86 (d, 1H, J=8.4 Hz), 7.48 (t, 1H, J=7.8 Hz), 7.39 (d, 2H, J=8.4 Hz), 7.36 (t, 1H, J=8.4 Hz), 7.26 (d, 2H, J=8.0 Hz), 7.09 (s, 2H), 6.73 (d, 2H, J=9.2 Hz), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 7

A fluorescence-activated covalently labeling fluorescent probe 7 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 7

The synthesis was carried out with reference to the method disclosed in the literature. ¹H-NMR (400 MHz, CD₃OD): δ=7.84 (d, 1H, J=6.0 Hz), 7.40 (d, 2H, J=8.0 Hz), 7.31 (d, 2H, J=8.0 Hz), 6.14 (d, 1H, J=6.0), 5.29 (s, 2H), 3.78 (s, 2H).

Probe 7

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.93 (d, 2H, J=8.0 Hz), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz) 6.85 (d, 2H, J=9.2 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 1.50 (s, 9H).

Example 8

A fluorescence-activated covalently labeling fluorescent probe 8 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 8

This probe was obtained by following the general procedure for probe 1, and the yield was 60%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.11-8.07 (m, 2H), 8.01-7.97 (m, 3H), 7.93 (d, 1H, J=5.6 Hz), 7.75 (s, 1H), 7.70 (d, 1H, J=8.8 Hz), 7.46-7.43 (m, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.27 (dd, 1H, J=9.2, 1.6 Hz), 6.10 (d, 1H, J=5.6 Hz), 6.02 (s, 1H), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 9

A fluorescence-activated covalently labeling fluorescent probe 9 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 9

This probe was obtained by following the general procedure for probe 1, and the yield was 65%. 1H-NMR (400 MHz, CDCl3): δ=8.09 (s, 1H), 8.02 (d, 1H, J=8.0 Hz), 7.98 (d, 2H, J=9.2 Hz), 7.93 (d, 1H, J=5.6), 7.86 (d, 1H, J=8.4 Hz), 7.75 (s, 1H), 7.48 (t, 1H, J=7.8 Hz), 7.36 (t, 1H, J=7.36 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.73 (d, 2H, J=9.2 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.88 (t, 2H, J=5.6 Hz), 3.64 (t, 2H, J=5.6 Hz), 3.15 (s, 3H).

Example 10

A fluorescence-activated covalently labeling fluorescent probe 10 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 8

This compound was obtained by following the general procedure for compound 1, and the yield was 95%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 4.79 (bt, 1H), 3.85 (t, 4H, J=5.6 Hz), 3.60 (t, 4H, J=5.6 Hz), 1.50 (s, 9H).

Probe 10

This probe was obtained by following the general procedure for probe 1, and the yield was 35%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.79 (bt, 1H), 4.40 (d, 2H, J=4.90 Hz), 3.87 (m, 4H), 3.61 (m, 4H), 1.51 (s, 9H).

Example 11

A fluorescence-activated covalently labeling fluorescent probe 11 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 11

Compound 1 (0.629 g, 1.0 mmol) and 4-dimethylaminopyridine (0.146 g, 1.2 mmol) were dissolved in 20 mL anhydrous DMF, p-nitrophenylchloroformate (0.242 g, 1.2 mmol) in 10 mL anhydrous dichloromethane was added dropwise under the protection of Ar. The obtained mixture was stirred and kept at room temperature for 1 hr. Then, 3-amino-propanesulfonic acid (0.168 g, 1.2 mmol) was added in the presence of TEA (0.16 ml, 1.2 mmol). The obtained mixture was stirred at room temperature for another 30 min under the protection of Ar. After the reaction was completed, the solvent was completely removed by rotary evaporation, and the residue was separated by a reverse phase column to give a pure probe 11 of 0.397 g, and the yield was 50%. 1H-NMR (400 MHz, DMSO-d6): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.79 (bt, 1H), 4.40 (d, 2H, J=4.90 Hz), 3.81 (t, 4H, J=6.0 Hz), 3.60 (t, 4H, J=6.0 Hz), 3.21 (t, 2H, 5.6 Hz), 2.71 (t, 2H, 5.6 Hz), 2.31 (m, 2H), 1.51 (s, 9H).

Example 12

A fluorescence-activated covalently labeling fluorescent probe 12 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 9

The synthesis was carried out with reference to the method disclosed in the literature (Tetsuaki Fujhara et al. Chem Comm, 2015, 51, 17382-17385). ¹H-NMR (400 MHz, CDCl₃): δ=7.75 (d, 2H, J=8.2 Hz), 7.30 (d, 2H, J=7.8 Hz), 4.11 (t, 2H, J=4.8 Hz), 3.52-3.65 (m, 16H), 3.37 (s, 3H), 2.50 (s, 3H).

Compound 10

Compound 8 (0.664 g, 2 mmol) was dissolved in 10 mL anhydrous DMF, cooled to 0° C. under the protection of Ar, 60% NaH (0.088 g, 2.2 mmol) was added. The obtained mixture was stirred for 5 min under the protection of Ar. Then, Compound 9 (0.781 g, 2 mmol) was added. After slowly returning to room temperature and stirring for 3.5 hrs, 0.5 ml water was added to quench the reaction. The solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a pale yellow oil of 0.396 g, and the yield was 36%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 3.56-3.68 (m, 24H), 3.38 (s, 3H), 1.50 (s, 9H).

Probe 12

This probe was obtained by following the general procedure for probe 1, and the yield was 71%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.56-3.68 (m, 24H), 3.38 (s, 3H), 1.50 (s, 9H).

Example 13

A fluorescence-activated covalently labeling fluorescent probe 13 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 11

The synthesis was carried out by the method disclosed in the literature (Yang W et al. J. Photochem. Photobiol. A. 2011, 222, 228-235). ¹H-NMR (400 MHz, D₂O): δ=9.43 (s, 1H), 7.72 (d, 2H, J=9.0 Hz), 6.81 (d, 2H, J=9.0 Hz), 3.71 (t, 2H, J=7.36 Hz), 3.03 (s, 3H), 2.44 (t, 2H, J=7.2 Hz).

Compound 12

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, D₂O): δ=8.01 (s, 1H), 7.72 (d, 2H, J=9.0 Hz), 6.81 (d, 2H, J=9.0 Hz), 3.71 (t, 2H, J=7.36 Hz), 3.03 (s, 3H), 2.44 (t, 2H, J=7.2 Hz), 1.49 (s, 9H).

Probe 13

Compound 12 (0.33 g, 1 mmol), benzotriazole-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (0.625 g, 1.2 mmol) and compound 2 (0.324 g, 1.2 mmol) were put in a 50 ml round-bottom flask, to which 15 ml anhydrous DMF and 0.3 nil of triethylamine were added. The obtained mixture was stirred and kept at room temperature for 0.5 hr under the protection of Ar. After the reaction was completed, the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a pale yellow solid, 0.524 g. The yield was 90%. ¹H-NMR (400 MHz, D₂O): δ=12.43 (s, 1H), 10.00 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.72 (d, 2H, J=9.0 Hz), 7.40 (m, 4H), 6.81 (d, 2H, J=9.0 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.71 (t, 2H, J=7.36 Hz), 3.03 (s, 3H), 2.47 (t, 2H, J=7.2 Hz), 1.49 (s, 9H).

Example 14

A fluorescence-activated covalently labeling fluorescent probe 14 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 13

The synthesis was carried out by the method disclosed in literature (L. X. Wu, K. Burgess, J. Am. Chem. Soc. 2008, 130, 4089-4096). ¹H-NMR (400 MHz, CDCl₃): δ=7.63-7.48 (m, 5H), 4.27 (s, 2H), 3.13 (s, 3H).

Compound 14

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 4.27 (s, 2H), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 3.13 (s, 3H).

Probe 14

This probe was obtained by following the general procedure for probe 1, and the yield was 70%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.96 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.63-7.48 (m, 5H), 7.40 (m, 4H), 6.84 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 4.27 (s, 2H), 3.55-3.59 (m, 4H), 3.13 (s, 3H), 3.03 (s, 3H).

Example 15

A fluorescence-activated covalently labeling fluorescent probe 15 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 15

The synthesis was carried out by the method disclosed in literature (L. X. Wu, K. Burgess, J. Am. Chem. Soc. 2008, 130, 4089-4096). ¹H-NMR (400 MHz, CDCl₃): δ=3.98 (q, 2H, J=2.4 Hz), 3.01 (s, 3H), 2.15 (t, 3H, J=2.4 Hz).

Compound 16

This compound was obtained by following the general procedure for compound 1, and the yield was 97%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.97 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 3.98 (q, 2H, J=2.4 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H), 3.01 (s, 3H), 2.15 (t, 3H, J=2.4 Hz).

Compound 17

Compound 16 (0.546 g, 2 mmol), benzaldehyde (0.530 g, 5 mmol) and anhydrous zinc chloride (0.545 g, 4 mmol) were dissolved in 100 ml anhydrous toluene. The mixture was heated to reflux in an oil bath for 48 hrs, After the reaction was completed, the solvent was completely removed by rotary evaporation, and the residue was dissolved in 100 ml of methylene dichloride, washed with water three times, and the organic phase is dried over anhydrous sodium sulfate, and the solvent was was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a red-brown solid 0.181 g. The yield was 25%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.21 (d, 2H, J=8.8 Hz), 8.00 (d, 1H, J=16 Hz), 7.85 (d, 2H, J=8.0 Hz), 7.45-7.38 (m, 3H), 7.24 (s, 1H), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 15

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.21 (d, 2H, J=8.8 Hz), 8.00 (d, 1H, J=16 Hz), 7.85 (d, 2H, J=8.0 Hz), 7.81 (s, 1H), 7.45-7.38 (m, 7H), 7.24 (s, 1H), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 16

A fluorescence-activated covalently labeling fluorescent probe 16 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 16

This probe was obtained by following the general procedure for probe 1, and the yield was 78%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.99 (brs, 1H), 8.21 (d, 2H, J=8.8 Hz), 8.00 (d, 1H, J=16 Hz), 7.85 (d, 2H, J=8.0 Hz), 7.45-7.39 (m, 3H), 7.35 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.24 (s, 1H), 7.09 (s, 2H), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 17

A fluorescence-activated covalently labeling fluorescent probe 17 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 17

This probe was obtained by following the general procedure for probe 1, and the yield was 71%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.21 (d, 2H, J=8.8 Hz), 8.00 (d, 1H, J=16 Hz), 7.93 (d, 1H, J=5.6), 7.85 (d, 2H, J=8.0 Hz), 7.75 (s, 1H), 7.45-7.38 (m, 3H), 7.33 (d, 2H, J=8.0 Hz), 7.24 (s, 1H), 7.19 (d, 2H, J=8.0 Hz), 7.01 (s, 1H), 6.92 (d, 2H, J=8.8 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 18

A fluorescence-activated covalently labeling fluorescent probe 18 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 18

The synthesis was carried out by the method disclosed in the literature (Wang H. et al. Tetra Let, 2007, 48, 3471-3474). ¹H-NMR (400 MHz, DMSO-d₆): δ=8.05 (m, 2H), 7.01 (m, 2H), 1.83 (s, 6H).

Compound 19

Compound 18 (0.279 g, 1 mmol) was dissolved in 20 ml of anhydrous pyridine, ml of N-methyl-N-hydroxyethyl was added, and heated in an oil bath at 40° C. overnight under the protection of Ar, After the reaction was completed, the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a red product 0.187 g. The yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.05 (m, 2H), 7.01 (m, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.83 (s, 6H).

Probe 18

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.05 (m, 2H), 7.01 (m, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.83 (s, 6H).

Example 19

A fluorescence-activated covalently labeling fluorescent probe 19 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 19

This probe was obtained by following the general procedure for probe 1, and the yield was 75%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.90 (brs, 1H), 8.05 (m, 2H), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 7.01 (m, 2H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.83 (s, 6H).

Example 20

A fluorescence-activated covalently labeling fluorescent probe 20 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 20

This probe was obtained by following the general procedure for probe 1, and the yield was 71%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.05 (m, 2H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 7.01 (m, 2H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.83 (s, 6H).

Example 21

A fluorescence-activated covalently labeling fluorescent probe 21 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 20

The synthesis was carried out by the method disclosed in the literature (WO 2013142841 (A1) 2013 Sep. 26). ¹H-NMR (400 MHz, CDCl₃): δ=7.92 (s, 1H), 7.63 (d, 1H, J=5.2 Hz), 7.31 (d, 1H, J=5.2 Hz).

Compound 21

Compound 20 (0.438 g, 2 mmol) was dissolved in 15 mL of N-methyl-N-hydroxyethylamine, copper powder (6.4 mg, 0.01 mmol), cuprous iodide (19 mg, 0.01 mmol), tripotassium phosphate (0.850 g, 4 mmol) were added, and heated in an oil bath at 80° C. overnight under the protection of Ar. After the reaction was completed, cooled at room temperature, the system was poured into 50 mL of water and was extracted three times with 50 ml dichloromethane; the organic phases were combined, and the solvent was completely removed by rotary evaporation. After separation by column chromatography, 0.362 g of a yellow product was obtained, and the yield was 85%. ¹H-NMR (400 MHz, CDCl₃): δ=7.92 (s, 1H), 7.63 (d, 1H, J=5.2 Hz), 7.31 (d, 1H, J=5.2 Hz), 3.85 (t, 2H, J=5.6 Hz), 3H, J=5.6 Hz), 3.10 (s, 3H).

Compound 22

Compound 21 (0.426 g, 2 mmol) was dissolved in 50 ml of anhydrous dichloromethane, 1 ml of triethylamine was added, and acetic anhydride (0.3 ml, 3 mmol) was slowly added dropwise under an ice bath. After the temperature was raised to room temperature, the system was stirred for 3 hrs. After the reaction was completed, 100 ml water was added. The organic phase was separated, and the aqueous phase was extracted twice with 50 ml dichloromethane. The organic phase was combined and dried over anhydrous sodium sulfate. The solvent was completely removed by rotary evaporation. The residue was directly used in the next step without being further purified.

The residue was dissolved in 50 nil of dichloromethane. 5 ml of DMF was added, 2 ml of phosphorus oxychloride was added under an ice bath, and the mixture was stirred under the protection of Ar for 0.5 h. The system was slowly warmed at room temperature and stirring was continued for 5 hrs; after the reaction was completed, saturated sodium carbonate solution was added to adjust pH to 10.0; stirring was carried out at room temperature overnight, and the organic phase was separated the next day, and the aqueous phase was extracted three times with 50 ml of dichloromethane, and the organic phase was combined and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate. The solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a yellow solid 0.285 g, and the yield was 59%. ¹H-NMR (400 MHz, CDCl₃): δ=10.01 (s, 1H), 7.92 (s, 1H), 7.63 (s, 1H) 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Compound 23

This compound was obtained by following the general procedure for compound 1, and the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.02 (s, 1H), 6.43 (s, 1H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.49 (s, 9H).

Probe 21

This probe was obtained by following the general procedure for probe 1, and the yield was 65%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.22 (s, 1H), 10.01 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 6.43 (s, 1H), 6.28 (s, 2H), 5.45 (s, 2H), 4.41 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 1.49 (s, 9H).

Example 22

A fluorescence-activated covalently labeling fluorescent probe 22 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 24

This compound was obtained by following the general procedure for compound 1, and the yield was 93%. ¹H-NMR (400 MHz, CDCl₃): δ=8.22 (s, 1H), 8.02 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 6.43 (s, 1H), 3.75 (t, 2H, J=5.6 Hz), 3.55 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 22

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.02 (s, 1H), 10.00 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 6H), 6.43 (s, 1H), 6.28 (s, 2H), 5.45 (s, 2H), 4.41 (d, 2H, J=4.8 Hz), 3.75 (t, 2H, J=5.6 Hz), 3.55 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 23

A fluorescence-activated covalently labeling fluorescent probe 23 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 25

This compound was obtained by following the general procedure for compound 1, and the yield was 88%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.09 (d, 1H, J=8.0 Hz), 8.02 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 6.43 (s, 1H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 23

This Probe was obtained by following the general procedure for Probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.32 (s, 1H), 10.03 (s, 1H), 8.22 (s, 1H), 8.09 (d, 1H, J=8.0 Hz), 8.02 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.81 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (m, 4H), 6.43 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 24

A fluorescence-activated covalently labeling fluorescent probe 24 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 24

This probe was obtained by following the general procedure for probe 1, and the yield was 58%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.98 (brs, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.43 (s, 1H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.49 (s, 9H).

Example 25

A fluorescence-activated covalently labeling fluorescent probe 25 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 25

This probe was obtained by following the general procedure for probe 1, and the yield was 63%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.99 (brs, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.44 (d, 2H, J=8.4 Hz), 7.36-7.42 (m, 2H), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.43 (s, 1H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.75 (t, 2H, J=5.6 Hz), 3.55 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 26

A fluorescence-activated covalently labeling fluorescent probe 26 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 26

This probe was obtained by following the general procedure for probe 1, and the yield was 58%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.98 (brs, 1H), 8.22 (s, 1H), 8.09 (d, 1H, J=8.0 Hz), 8.02 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.43 (s, 1H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 27

A fluorescence-activated covalently labeling fluorescent probe 27 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 27

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.02 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.43 (s, 1H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.49 (s, 9H).

Example 28

A fluorescence-activated covalently labeling fluorescent probe 28 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 28

This probe was obtained by following the general procedure for probe 1, and the yield was 63%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.02 (s, 1H), 7.93 (d, 1H, J=5.6), 7.77 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.30 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.43 (s, 1H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.75 (t, 2H, J=5.6 Hz), 3.55 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 29

A fluorescence-activated covalently labeling fluorescent probe 29 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 29

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.09 (d, 1H, J=8.0 Hz), 8.02 (s, 1H), 7.96 (d, 1H, J=5.6), 7.90 (d, 1H, J=8.0 Hz), 7.75 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.43 (s, 1H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 30

A fluorescence-activated covalently labeling fluorescent probe 30 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 26

This compound was obtained by following the general procedure for compound 21, and the yield was 87%. ¹H-NMR (400 MHz, CDCl₃): δ=8.02 (s, 1H), 7.66 (d, 1H, J=8.4 Hz), 7.44-7.48 (m, 1H), 7.41 (m, 1H), 7.29 (m, 1H), 3.60 (t, 2H, J=5.6 Hz), 3.34 (t, J=8.0 Hz, 3H), 3.10 (s, 3H).

Compound 27

This compound was obtained by following the general procedure for compound 22, and the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=9.92 (s, 1H), 7.81 (s, 1H), 7.68 (d, J=9.0 Hz, 1H), 6.92 (d, J=2.0 Hz, 1H), 6.82 (d, J=9.1, 2.3 Hz, 1H), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.10 (s, 3H).

Compound 28

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.22 (s, 1H), 8.02 (s, 1H), 6.43 (s, 1H), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.11 (s, 3H), 1.48 (s, 9H).

Probe 30

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.20 (s, 1H), 7.81 (s, 2H), 7.68 (d, J=9.0 Hz, 1H), 7.40 (m, 4H), 6.92 (d, J=2.0 Hz, 1H), 6.82 (d, J=9.1, 2.3 Hz, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.11 (s, 3H), 1.51 (s, 9H).

Example 31

A fluorescence-activated covalently labeling fluorescent probe 31 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 29

This compound was obtained by following the general procedure for compound 4, and the yield was 93%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.45 (s, 1H), 8.09 (d, J=8.00 Hz, 2H), 8.07 (s, 1H), 7.94 (d, J=8.00 Hz, 2H), 7.51 (m, 1H), 7.41 (m, 1H), 6.45 (s, 1H), 3.61 (t, 3H, J=8.0 Hz), 3.34 (t, J=8.0 Hz, 3H), 3.21 (s, 3H).

Probe 31

This probe was obtained by following the general procedure for probe 1, and the yield was 71%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.45 (s, 1H), 8.09 (d, J=8.00 Hz, 2H), 8.07 (s, 1H), 7.94 (d, J=8.00 Hz, 2H), 7.81 (s, 1H), 7.51 (m, 1H), 7.41 (m, 5H), 6.45 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 3H, J=8.0 Hz), 3.34 (t, J=8.0 Hz, 3H), 3.21 (s, 3H).

Example 32

A fluorescence-activated covalently labeling fluorescent probe 32 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 30

This compound was obtained by following the general procedure for compound 1, and the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.09 (d, 1H, J=8.00 Hz), 7.94 (d, 1H, J=8.00 Hz), 7.81 (s, 1H), 7.68 (d, J=9.0 Hz, 1H), 7.51 (m, 1H), 7.41 (m, 1H), 6.92 (d, J=2.0 Hz, 1H), 6.82 (d, J=9.1, 2.3 Hz, 1H), 6.45 (s, 1H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.21 (s, 3H).

Probe 32

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.09 (d, 1H, J=8.00 Hz), 7.94 (d, 1H, J=8.00 Hz), 7.81 (s, 2H), 7.68 (d, J=9.0 Hz, 1H), 7.51 (m, 1H), 7.41 (m, 5H), 6.92 (d, J=2.0 Hz, 1H), 6.82 (d, J=9.1, 2.3 Hz, 1H), 6.45 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (t, 2H, J=8.0 Hz), 3.36 (t, J=8.0 Hz, 2H), 3.21 (s, 3H).

Example 33

A fluorescence-activated covalently labeling fluorescent probe 33 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye

Probe 33

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. 1H-NMR (400 MHz, CDCl₃): δ=9.99 (brs, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.43 (s, 1H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.21 (s, 3H), 1.48 (s, 9H).

Example 34

A fluorescence-activated covalently labeling fluorescent probe 34 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 34

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=8.22 (s, 1H), 8.02 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.43 (s, 1H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 3.21 (s, 3H), 1.48 (s, 9H).

Example 35

A fluorescence-activated covalently labeling fluorescent probe 35 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 29

This compound was obtained by following the general procedure for compound 26, and the yield was 42%. ¹H-NMR (400 MHz, CDCl₃): δ=7.62 (d, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (dd, 1H, J₁=9.0 Hz, J₂=2.4 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H).

Compound 30

Compound 29 (0.965 g, 5 mmol) was dissolved in 50 ml acetonitrile in a 100 ml round-bottom flask, and potassium carbonate (1.38 g, 10 mmol) and bromohexane (1.33 g, 6 mmol) were added. The resulting mixture was heated to reflux in an oil bath under the protection of Ar. After the reaction was completed, filtering was carried out, and the solvent was removed by rotary evaporation. The residue was dissolved in 100 ml ethyl acetate, then washed with water, saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate. The solvent was completely removed by rotary evaporation. The residue was purified by gel silica gel column chromatography to give a waxy solid 1.182 g, and the yield was 71%. ¹H-NMR (400 MHz, CDCl₃): δ=7.64 (d, 1H, J=8.8 Hz), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 3.12-3.09 (t, 2H, J=7.6 Hz), 1.59-1.65 (m, 24H), 0.89 (t, 3H, J=2.0 Hz).

Compound 31

This compound was obtained by following the general procedure for compound 27, and the yield was 56%. 1H-NMR (400 MHz, CDCl3): δ=9.92 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 3.12-3.09 (t, 2H, J=7.6 Hz), 1.59-1.65 (m, 24H), 0.89 (t, 3H, J=2.0 Hz).

Compound 32

This compound was obtained by following the general procedure for compound 1, and the yield was 87%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 3.12-3.09 (t, 2H, J=7.6 Hz), 1.59-1.65 (m, 33H), 0.89 (t, 3H, J=2.0 Hz).

Probe 35

This probe was obtained by following the general procedure for probe 1, and the yield was 60%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 7.78 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 3.12-3.09 (t, 2H, J=7.6 Hz), 1.59-1.65 (m, 33H), 0.89 (t, 3H, J=2.0 Hz).

Example 36

A fluorescence-activated covalently labeling fluorescent probe 36 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 33

Compound 29 (0.580 g, 3 mmol) was dissolved in 60 ml of toluene in a 100 ml round-bottom flask, 1 ml of acrylonitrile and 1 ml of acetic acid were added, heated to reflux in an oil bath for 24 h under the protection of Ar. After the reaction was completed, the system was poured into 100 ml of water, the organic phase was separated, and the aqueous phase was extracted twice with 50 ml of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a product 0.487 g. The yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=7.62 (d, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (dd, 1H, J₁=9.0 Hz, J₂=2.4 Hz), 3.72 (t, 2H, J=6.8 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 2.57 (t, 2H, J=6.8 Hz).

Compound 34

This compound was obtained by following the general procedure for compound 27, and the yield was 43%. ¹H-NMR (400 MHz, CDCl₃): δ=9.97 (s, 1H), 7.60 (d, 1H), 7.80 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (dd, 1H, J₁=9.0 Hz, J₂=2.4 Hz), 3.72 (t, 2H, J=6.8 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 2.57 (t, 2H, J=6.8 Hz).

Compound 35

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.60 (d, 1H), 7.80 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (dd, 1H, J₁=9.0 Hz, J₂=2.4 Hz), 3.72 (t, 2H, J=6.8 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 2.57 (t, 2H, J=6.8 Hz), 1.49 (s, 9H).

Probe 36

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 7.60 (d, 1H), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.72 (t, 2H, J=6.8 Hz), 3.62 (t, J=8.0 Hz, 3H), 3.35 (t, J=8.0 Hz, 3H), 2.57 (t, 2H, J=6.8 Hz), 1.49 (s, 9H).

Example 37

A fluorescence-activated covalently labeling fluorescent probe 37 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 36

This compound was obtained by following the general procedure for compound 21, and the yield was 67%. ¹H-NMR (400 MHz, CDCl₃): δ=7.62 (d, 1H, J=8.8 Hz), 7.15 (d, 1H, J=5.6 Hz), 7.08-7.01 (m, 2H), 6.81 (d, 1H, J=2.4 Hz), 3.62 (t, 4H, J=8.0 Hz), 3.35 (t, 4H, J=8.0 Hz).

Compound 37

This compound was obtained by following the general procedure for compound 27, and the yield was 67%. ¹H-NMR (400 MHz, CDCl₃): δ=9.99 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 3.62 (t, 4H, J=8.0 Hz), 3.35 (t, 4H, J=8.0 Hz).

Compound 38

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.83 (s, 1H), 7.69 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 3.62 (t, 4H, J=8.0 Hz), 3.35 (t, 4H, J=8.0 Hz), 1.49 (s, 9H).

Probe 37

This probe was obtained by following the general procedure for probe 1, and the yield was 44%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 9.99 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.81 (s, 1H), 7.69 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (t, 4H, J=8.0 Hz), 3.35 (t, 4H, J=8.0 Hz), 1.49 (s, 9H).

Example 38

A fluorescence-activated covalently labeling fluorescent probe 38 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 39

This compound was obtained by following the general procedure for compound 10, and the yield was 45%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.83 (s, 1H), 7.69 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 3.56-3.68 (m, 24H), 3.38 (s, 3H), 1.49 (s, 9H).

Probe 38

This probe was obtained by following the general procedure for probe 1, and the yield was 67%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.81 (s, 1H), 7.69 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.56-3.68 (m, 24H), 3.38 (s, 3H), 1.49 (s, 9H).

Example 39

A fluorescence-activated covalently labeling fluorescent probe 39 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 39

This probe was obtained by following the general procedure for probe 11, and the yield was 65%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 9.99 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.81 (s, 1H), 7.69 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.81 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (t, 4H, J=8.0 Hz), 3.35 (t, 4H, J=8.0 Hz), 3.21 (t, 2H, 5.6 Hz), 2.71 (t, 2H, 5.6 Hz), 2.31 (m, 2H), 1.49 (s, 9H).

Example 40

A fluorescence-activated covalently labeling fluorescent probe 40 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 41

This compound was obtained by following the general procedure for compound 27, and the yield was 68%. ¹H-NMR (400 MHz, CDCl₃): δ=7.62 (d, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0 Hz), 6.82 (dd, 1H, J₁=9.0 Hz, J₂=2.4 Hz), 3.55 (t, 2H, J=7.6 Hz), 3.35 (t, 2H, J=6.8 Hz), 3.13 (s, 3H), 1.24 (m, 2H).

Probe 40

This probe was obtained by following the general procedure for probe 13, and the yield was 88%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 7.81 (s, 1H), 7.82 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0 Hz), 6.82 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.55 (t, 2H, J=7.6 Hz), 3.35 (t, 2H, J=6.8 Hz), 3.13 (s, 3H), 1.24 (m, 2H).

Example 41

A fluorescence-activated covalently labeling fluorescent probe 41 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 44

This compound was obtained by following the general procedure for compound 14, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.22 (s, 1H), 8.02 (s, 1H), 6.43 (s, 1H), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.11 (s, 3H), 3.00 (s, 3H), 2.15 (t, 3H, J=2.4 Hz), 1.48 (s, 9H).

Probe 41

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 6.43 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.11 (s, 3H), 3.00 (s, 3H), 2.15 (t, 3H, J=2.4 Hz), 1.48 (s, 9H).

Example 42

A fluorescence-activated covalently labeling fluorescent probe 42 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 45

This compound was obtained by following the general procedure for compound 1, and the yield was 93%. ¹H-NMR (400 MHz, CDCl₃): δ=8.22 (s, 1H), 8.02 (s, 1H), 7.63-7.68 (m, 5H), 6.43 (s, 1H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.15 (s, 3H), 3.11 (s, 3H).

Probe 42

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.63-7.68 (m, 5H), 6.43 (s, 1H), 7.40 (m, 4H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, J=8.0 Hz, 3H), 3.34 (t, J=8.0 Hz, 3H), 3.15 (s, 3H), 3.11 (s, 3H).

Example 43

A fluorescence-activated covalently labeling fluorescent probe 43 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 46

This compound was obtained by following the general procedure for compound 21, and the yield was 78%. ¹H-NMR (400 MHz, CDCl₃): δ=8.07 (s, 1H), 7.77 (d, 1H, J=1.6 Hz), 7.66 (d, 1H, J=8.4 Hz), 7.50 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Compound 47

This compound was obtained by following the general procedure for compound 22, and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=9.99 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Compound 48

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H), 1.50 (s, 9H).

Probe 43

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (m, 5H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H), 1.50 (s, 9H).

Example 44

A fluorescence-activated covalently labeling fluorescent probe 44 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 44

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=9.99 (brs, 1H), 8.01 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (m, 1H), 7.33 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H), 1.50 (s, 9H).

Example 45

A fluorescence-activated covalently labeling fluorescent probe 45 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 45

This probe was obtained by following the general procedure for probe 1, and the yield was 67%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.89 (d, 1H, J=5.6), 7.75 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H), 1.50 (s, 9H).

Example 46

A fluorescence-activated covalently labeling fluorescent probe 46 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 49

This compound was obtained by following the general procedure for compound 1, and the yield was 97%. ¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.36-7.42 (m, 3H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Probe 46

This probe was obtained by following the general procedure for probe 1, and the yield was 65%. ¹H-NMR (400 MHz, CDCl₃): δ=12.32 (s, 1H), 10.01 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.32-7.37 (m, 3H), 7.40 (m, 4H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Example 47

A fluorescence-activated covalently labeling fluorescent probe 47 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 50

This compound was obtained by following the general procedure for compound 1, and the yield was 97%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 7.90 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Probe 47

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.04 (d, 1H, J=8.0 Hz), 7.90 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.81 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (m, 5H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Example 48

A fluorescence-activated covalently labeling fluorescent probe 48 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 48

This probe was obtained by following the general procedure for probe 1, and the yield was 97%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 7.93 (d, 1H, J=5.6), 7.90 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.75 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Example 49

A fluorescence-activated covalently labeling fluorescent probe 49 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 51

This compound was obtained by following the general procedure for compound 1, and the yield was 87%. ¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Probe 49

This probe was obtained by following the general procedure for probe 1, and the yield was 31%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.32 (s, 1H), 10.05 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.61 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.4 Hz), 7.40 (m, 5H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.11 (s, 3H).

Example 50

A fluorescence-activated covalently labeling fluorescent probe 50 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 52

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=7.98 (s, 1H), 7.64-7.48 (m, 7H), 7.40 (dd, 1H, J₁=8.4 Hz, J₂=1.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.13 (s, 3H), 3.11 (s, 3H).

Probe 50

This probe was obtained by following the general procedure for probe 1, and the yield was 67%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 7.98 (s, 1H), 7.81 (s, 1H), 7.64-7.48 (m, 7H), 7.40 (m, 5H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.13 (s, 3H), 3.11 (s, 3H).

Example 51

A fluorescence-activated covalently labeling fluorescent probe 51 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 53

This compound was obtained by following the general procedure for compound 21, and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=8.67 (s, 1H), 7.95 (d, 1H, J=10.0 Hz), 7.15 (d, 1H, J=2.4 Hz), 7.00 (dd, 1H, J₁=8.8 Hz, J₂=2.4 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Compound 54

This compound was obtained by following the general procedure for compound 22, the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=10.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.07-7.04 (m, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Compound 55

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.07-7.04 (m, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H), 1.50 (s, 9H).

Probe 51

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.07-7.04 (m, 2H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H), 1.50 (s, 9H).

Example 52

A fluorescence-activated covalently labeling fluorescent probe 52 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 52

This probe was obtained by following the general procedure for probe 1, and the yield was 69%. ¹H-NMR (400 MHz, CDCl₃): δ=9.99 (brs, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.19 (s, 2H), 7.07-7.04 (m, 2H), 6.10 (s, 1H), 5.26 (s, 2H), 4.36 (s, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H), 1.50 (s, 9H).

Example 53

A fluorescence-activated covalently labeling fluorescent probe 52 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 53

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.90 (d, 1H, J=5.6), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 7.07-7.04 (m, 2H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H), 1.50 (s, 9H).

Example 54

A fluorescence-activated covalently labeling fluorescent probe 54 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 56

This compound was obtained by following the general procedure for compound 1, and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.07-7.04 (m, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Probe 54

This probe was obtained by following the general procedure for probe 1, and the yield was 65%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.40 (m, 4H), 7.36-7.42 (m, 2H), 7.07-7.04 (m, 2H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Example 55

A fluorescence-activated covalently labeling fluorescent probe 55 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 57

This compound was obtained by following the general procedure for compound 1, and the yield was 97%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 8.00 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.07-7.04 (m, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Probe 55

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 8.00 (d, 1H, J=10.0 Hz), 7.95 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.07-7.04 (m, 2H), 3.61 (t, 2H, J=8.0 Hz), 3.34 (t, 2H, J=8.0 Hz), 3.10 (s, 3H).

Example 56

A fluorescence-activated covalently labeling fluorescent probe 56 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 58

N-methyl-N-hydroxyethylaniline (1.88 g, 12.5 mmol) and NaHCO₃ (1.57 g, 18.7 mmol) were dissolved in 48 ml of dichloromethane and 36 ml of water, cooled to 0° C., and I₂ (3.0 g, 11.8 mmol) was added slowly, then, the system was gradually warmed to room temperature, stirred for 30 min, the system was diluted with 300 ml of dichloromethane and 40 ml of water; the organic phase was separated, and the organic phase was washed with water, sodium thiosulfate solution and brine, respectively, and dried over anhydrous sodium sulfate; the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a pure compound 22 (2.46 g, 92%). ¹H-NMR (400 MHz, CDCl₃): δ=7.46 (d, 1H, J=7.60 Hz), 6.56 (d, 1H, J=7.60 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 2.94 (s, 3H).

Compound 59

Compound 58 (5.54 g, 20 mmol), Pd(PPh₃)₂Cl₂ (213 mg, 0.3 mmol), cuprous iodide (38 mg, 0.2 mmol) were dissolved in 20 ml of trimethylamine. After stirred at room temperature for 30 min under the protection of Ar, propargyl alcohol (1.53 ml, 26 mmol) was added, and the mixture was stirred at room temperature for 24 hrs. After the reaction was completed, the mixture was filtered, the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a brown solid of 3.20 g, and the yield was 78%. ¹H-NMR (400 MHz, CDCl₃): δ=7.30 (d, 2H, J=8.8 Hz), 6.60 (d, 2H, J=8.8 Hz), 4.45 (d, 2H, J=4.0 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 60

Compound 59 (2.05 g, 10 mmol) was dissolved in 50 ml of tetrahydrofuran, 10 g of active manganese dioxide was added, and stirred under the protection of Ar for 24 h at room temperature. When the reaction was completed, the solution was filtered, and the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a brown solid of 1.42 g, and the yield was 70%. ¹H-NMR (400 MHz, CDCl₃): δ=9.37 (s, 1H), 7.49 (d, 2H, J=8.8 Hz), 6.74 (d, 2H, J=8.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 61

This compound was obtained by following the general procedure for compound 61, and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.47 (d, 2H, J=8.8 Hz), 6.76 (d, 2H, J=8.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.50 (s, 9H).

Probe 56

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.47 (d, 2H, J=8.8 Hz), 7.40 (m, 4H), 6.76 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.50 (s, 9H).

Example 57

A fluorescence-activated covalently labeling fluorescent probe 57 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 57

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.47 (d, 2H, J=8.8 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.76 (d, 2H, J=8.8 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.50 (s, 9H).

Example 58

A fluorescence-activated covalently labeling fluorescent probe 58 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 62

This compound was obtained by following the general procedure for compound 1, and the yield was 90%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.47 (d, 2H, J=8.8 Hz), 7.36-7.42 (m, 2H), 6.76 (d, 2H, J=8.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 58

This probe was obtained by following the general procedure for probe 1, and the yield was 50%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.47 (d, 2H, J=8.8 Hz), 7.40 (m, 4H), 7.36-7.42 (m, 2H), 6.76 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 59

A fluorescence-activated covalently labeling fluorescent probe 59 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 63

This compound was obtained by following the general procedure for compound 1, and the yield was 90%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 8.01 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.47 (d, 2H, J=8.8 Hz), 7.45 (t, 1H, J=8.0 Hz), 6.76 (d, 2H, J=8.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 59

This probe was obtained by following the general procedure for probe 1, and the yield was 49%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.04 (d, 1H, J=8.0 Hz), 8.01 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.81 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.47 (d, 2H, J=8.8 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (m, 4H), 6.76 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 60

A fluorescence-activated covalently labeling fluorescent probe 60 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 64

The synthesis was carried out by the method disclosed in WO2009152165 (A2), Dec. 17, 2009. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.84 (s, 1H), 7.37 (s, 1H), 7.24 (s, 1H).

Compound 65

This compound was obtained by following the general procedure for compound 21, and the yield was 85%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.84 (s, 1H), 7.37 (s, 1H), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 66

This compound was obtained by following the general procedure for compound 22, and the yield was 56%. 1H-NMR (400 MHz, DMSO-d₆): δ=10.04 (s, 1H), 7.84 (s, 1H), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 67

This compound was obtained by following the general procedure for compound 1, and the yield was 88%. 1H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.84 (s, 1H), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.50 (s, 9H).

Probe 60

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.84 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.24 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.50 (s, 9H).

Example 61

A fluorescence-activated covalently labeling fluorescent probe 61 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 68

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.01 (s, 1H), 7.84 (s, 1H), 7.74 (1H, d, J=4.0 Hz), 7.55 (1H, d, J=4.0 Hz), 7.36-7.42 (2H, m), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 61

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.84 (s, 1H), 7.81 (s, 1H), 7.74 (1H, d, J=4.0 Hz), 7.55 (1H, d, J=4.0 Hz), 7.36-7.42 (6H, m), 7.24 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 62

A fluorescence-activated covalently labeling fluorescent probe 62 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 68

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.07 (1H, d, J=8.0 Hz), 8.01 (s, 1H), 7.90 (1H, d, J=8.0 Hz), 7.84 (s, 1H), 7.53 (1H, t, J=8.0 Hz), 7.45 (1H, t, J=8.0 Hz), 7.24 (s, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 62

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.07 (1H, d, J=8.0 Hz), 8.01 (s, 1H), 7.90 (1H, d, J=8.0 Hz), 7.84 (s, 1H), 7.81 (s, 1H), 7.53 (1H, t, J=8.0 Hz), 7.45 (1H, t, J=8.0 Hz), 7.40 (m, 4H), 7.24 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 63

A fluorescence-activated covalently labeling fluorescent probe 63 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 70

6-Bromo-1-benzothiophene (0.43 g, 2 mmol) was dissolved in 50 ml of dry dihalomethane, potassium acetate (0.4 g, 4 mmol) was added, and bromine (0.32 g, 2 mmol) was added in an ice bath. The system was slowly risen to room temperature. When the reaction was completed, 100 ml of saturated sodium thiosulfate solution was added. The organic phase was separated, and the aqueous phase was extracted three times with dichloromethane, and the organic phase was combined and dried by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a yellow product 0.64 g. and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=7.81 (s, 1H). 7.68 (d, J=9.0 Hz, 1H), 6.92 (d, J=2.0 Hz, 1H).

Synthesis of Compound 71

Compound 70 (1.27 g, 3.43 mmol) was dissolved in 50 ml of dry triethylamine, and dichloroditetra(triphenylphosphine) palladium (120.2 mg, 0.171 mmol), cuprous iodide (65.2 mg, 0.343 mmol) and trimethylsilylacetylene (344 mg, 3.43 mmol) were added, heated in an oil bath for 24 h under the protection of Ar. After completion of the reaction, 5 ml of water was added to quench the reaction. The solvent was completely removed by rotary evaporation. The residue was dissolved in diethyl ether, and filtered. Rotary evaporation was carried out to give a residue, which was used in the next step without purification.

The crude product was dissolved in 30 ml of NMP, added with sodium sulfide nonahydrate (0.87 g, 3.63 mmol). The mixture was heated in an oil bath at 190° C. for 12 h under the protection of Ar, cooled at room temperature, and added with 20 ml of saturated ammonium chloride solution, and the resulting was extracted with dichloromethane three times, and the organic phase was combined and dried over anhydrous Na₂SO₄, and Na₂SO₄ was removed by filtration. The solvent was removed by rotary evaporation to give a residue which was purified by gel silica gel column chromatography to give a yellow solid 0.85 g, and the yield was 49%. ¹H-NMR (400 MHz, CDCl3): δ=7.87 (m, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.41 (m, 1H), 7.32 (d, J=5.4 Hz, 1H).

Compound 72

This compound was obtained by following the general procedure for compound 21, and the yield was 75%. ¹H-NMR (400 MHz, CDCl₃): δ=7.86 (m, 1H), 7.73 (m, 1H), 7.54 (d, J=5.4 Hz, 1H), 7.41 (m, 1H), 7.32 (d, J=5.4 Hz, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 73

This compound was obtained by following the general procedure for compound 22, and the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=9.71 (s, 1H), 7.87 (m, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.32 (d, J=5.4 Hz, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Compound 74

This compound was obtained by following the general procedure for compound 1, and the yield was 93%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.87 (m, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.32 (d, J=5.4 Hz, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.49 (s, 9H).

Probe 63

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 7.81 (s, 1H), 8.01 (s, 1H), 7.87 (m, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.40 (m, 4H), 7.32 (d, J=5.4 Hz, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H), 1.49 (s, 9H).

Example 64

A fluorescence-activated covalently labeling fluorescent probe 64 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 75

This compound was obtained by following the general procedure for compound 75, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.87 (m, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.32 (d, J=5.4 Hz, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 64

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.87 (m, 1H), 7.81 (s, 1H), 7.71 (m, 1H), 7.51 (d, J=5.4 Hz, 1H), 7.40 (m, 4H), 7.32 (d, J=5.4 Hz, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 65

A fluorescence-activated covalently labeling fluorescent probe 65 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 76

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.87 (m, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.71 (m, 1H)), 7.55 (d, 1H, J=4.0 Hz), 7.51 (d, J=5.4 Hz, 1H), 7.36-7.42 (m, 2H), 7.32 (d, J=5.4 Hz, 1H), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Probe 65

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.93 (d, 1H, J=5.6), 7.87 (m, 1H), 7.77 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.71 (m, 1H)), 7.55 (d, 1H, J=4.0 Hz), 7.51 (d, J=5.4 Hz, 1H), 7.36-7.42 (m, 4H), 7.32 (d, J=5.4 Hz, 1H), 7.19 (d, 2H, J=8.0 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.78 (t, 2H, J=4.80 Hz), 3.44 (t, 2H, J=4.80 Hz), 3.02 (s, 3H).

Example 66

A fluorescence-activated covalently labeling fluorescent probe 66 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 77

Compound 58 (0.554 g, 2 mmol) and 5-aldehyde-2-thiopheneboronic acid (0.374 g, 2.4 mmol) were dissolved in 10 ml of toluene, 10 ml of ethanol, and 2 ml of 2 N K₂CO₃ solution was added. After heating in an oil bath at 85° C. for 5 hrs under the protection of Ar, the reaction was completed, and the solution was cooled at room temperature, and then the reaction was quenched with water (10 ml). The organic phase was separated and the aqueous phase was extracted with dichloromethane. The organic phase was combined, washed with brine, dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to give a residue which was purified by gel silica gel column chromatography to give a pure product 0.339 g, and the yield was 65%. ¹H-NMR (400 MHz, CDCl₃): δ=9.81 (s, 1H), 7.68 (s, 1H), 7.55 (d, 1H, J=8.00 Hz), 7.25 (d, 2H, J=8.00 Hz), 6.78 (d, 2H, J=8.00 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H).

Compound 78

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=8.01 (s, 1H), 7.68 (s, 1H), 7.55 (d, 1H, J=8.00 Hz), 7.25 (d, 2H, J=8.00 Hz), 6.78 (d, 2H, J=8.00 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 1.50 (s, 9H).

Probe 66

This probe was obtained by following the general procedure for probe 1, and the yield was 54%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.68 (s, 1H), 7.55 (d, 1H, J=8.00 Hz), 7.40 (m, 4H), 7.25 (d, 2H, J=8.00 Hz), 6.78 (d, 2H, J=8.00 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 1.50 (s, 9H).

Example 67

A fluorescence-activated covalently labeling fluorescent probe 67 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 79

Cyanoacetic acid (1.0 g, 10 mmol) was added to a 25 ml round bottom flask, and 2-methoxyethylamine was added and stirred at room temperature under the protection of Ar. After completion of the reaction, 10 ml of anhydrous diethyl ether was added, and the mixture was separated by ultrasonication, filtered, and dried in vacuo to give a white solid. ¹H-NMR (400 MHz, CDCl₃): δ=6.5 (s, 1H), 3.48-3.52 (m, 4H), 3.38 (s, 3H).

Compound 80

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.31 (s, 1H), 8.22 (bt, 1H), 7.82 (d, 1H, J=4.00 Hz), 7.58 (d, 2H, J=8.80 Hz), 7.50 (d, 2H, J=4.00 Hz), 6.77 (d, 2H, J=8.80 Hz), 4.74 (bt, 1H), 3.57 (t, 2H, J=5.20 Hz), 3.41-3.48 (m, 4H), 3.38 (t, 2H, J=5.20 Hz), 3.27 (s, 3H), 3.01 (s, 3H).

Probe 67

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.31 (s, 1H), 8.22 (bt, 1H), 7.82 (m, 2H), 7.58 (d, 2H, J=8.80 Hz), 7.50 (d, 2H, J=4.00 Hz), 7.40 (m, 4H), 6.77 (d, 2H, J=8.80 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.74 (bt, 1H), 4.40 (d, 2H, J=4.8 Hz), 3.57 (t, 2H, J=5.20 Hz), 3.41-3.48 (m, 4H), 3.38 (t, 2H, J=5.20 Hz), 3.27 (s, 3H), 3.01 (s, 3H).

Example 68

A fluorescence-activated covalently labeling fluorescent probe 68 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 68

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.99 (s, 1H), 8.31 (s, 1H), 8.22 (bt, 1H), 7.82 (d, 1H, J=4.00 Hz), 7.58 (d, 2H, J=8.80 Hz), 7.50 (d, 2H, J=4.00 Hz), 7.39 (d, 2H, J=8.4 Hz), 7.26 (d, 2H, J=8.4 Hz), 7.09 (s, 2H), 6.77 (d, 2H, J=8.80 Hz), 6.10 (s, 1H), 5.26 (s, 2H), 4.74 (bt, 1H), 4.36 (s, 2H), 3.57 (t, 2H, J=5.20 Hz), 3.41-3.48 (m, 4H), 3.38 (t, 2H, J=5.20 Hz), 3.27 (s, 3H), 3.01 (s, 3H).

Example 69

A fluorescence-activated covalently labeling fluorescent probe 69 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 69

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.31 (s, 1H), 8.22 (bt, 1H), 7.93 (d, 1H, J=5.6), 7.82 (d, 1H, J=4.00 Hz), 7.75 (s, 1H), 7.58 (d, 2H, J=8.80 Hz), 7.50 (d, 2H, J=4.00 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.77 (d, 2H, J=8.80 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.74 (bt, 1H), 4.45 (d, 2H, J=5.6 Hz), 3.57 (t, 2H, J=5.20 Hz), 3.41-3.48 (m, 4H), 3.38 (t, 2H, J=5.20 Hz), 3.27 (s, 3H), 3.01 (s, 3H).

Example 70

A fluorescence-activated covalently labeling fluorescent probe 70 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 81

The synthesis was carried out by the method disclosed in the reference (WO2013142841 (A1) Sep. 26, 2013). ¹H-NMR (400 MHz, CDCl₃): δ=7.87 (s, 2H), 7.54 (s, 1H), 7.42 (d, J=5.6 Hz, 1H), 7.39 (d, J=5.6 Hz, 1H).

Compound 82

This compound was obtained by following the general procedure for compound 21, and the yield was 76%. ¹H-NMR (400 MHz, CDCl₃): δ=7.87 (s, 2H), 7.54 (s, 1H), 7.42 (d, J=5.6 Hz, 1H), 7.39 (d, J=5.6 Hz, 1H), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H).

Compound 83

This compound was obtained by following the general procedure for compound 22, and the yield was 62%. ¹H-NMR (400 MHz, CDCl3): δ=9.99 (s, 1H), 7.89 (s, 2H), 7.59 (s, 1H), 7.27 (s, 1H), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H).

Compound 84

This compound was obtained by following the general procedure for compound 1, and the yield was 88%. ¹H-NMR (400 MHz, CDCl₃): δ=7.89 (s, 2H), 7.59 (s, 1H), 7.27 (s, 1H), 7.05 (s, 1H), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H).

Probe 70

This probe was obtained by following the general procedure for probe 1, and the yield was 54%. ¹H-NMR (400 MHz, CDCl₃): δ=12.22 (s, 1H), 10.11 (s, 1H), 7.89 (s, 2H), 7.81 (s, 1H), 7.59 (s, 1H), 7.40 (m, 4H), 7.27 (s, 1H), 7.05 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H).

Example 71

A fluorescence-activated covalently labeling fluorescent probe 71 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 85

This compound was obtained by following the general procedure for compound 1, and the yield was 95%. ¹H-NMR (400 MHz, CDCl₃): δ=7.98 (s, 1H), 7.89 (s, 2H), 7.59 (s, 1H), 7.27 (s, 1H), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.48-3.50 (m, 4H), 3.38 (s, 3H), 3.06 (s, 3H).

Probe 71

This probe was obtained by following the general procedure for probe 1, and the yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=11.92 (s, 1H), 10.21 (s, 1H), 7.98 (s, 1H), 7.89 (s, 2H), 7.81 (s, 1H), 7.59 (s, 1H), 7.40 (m, 4H), 7.27 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.48-3.50 (m, 4H), 3.38 (s, 3H), 3.06 (s, 3H).

Example 72

A fluorescence-activated covalently labeling fluorescent probe 72 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 72

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, CDCl₃): δ=7.98 (s, 1H), 7.93 (d, 1H, J=5.6), 7.89 (s, 2H), 7.75 (s, 1H), 7.59 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.27 (s, 1H), 7.19 (d, 2H, J=8.0 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.48-3.50 (m, 4H), 3.38 (s, 3H), 3.06 (s, 3H).

Example 73

A fluorescence-activated covalently labeling fluorescent probe 73 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 87

The synthesis was carried out by the method disclosed in the literature (Takuya M. et al. RSC Adv. 2015.5.55406-55410). ¹H-NMR (400 MHz, CDCl₃): δ=7.20 (d, 2H, J=4.8 Hz), 7.07 (d, 2H, J=4.8 Hz), 0.41 (s, 6H).

Compound 88

Compound 87 (0.4 g, 1.8 mmol) was dissolved in 100 ml of anhydrous tetrahydrofuran, cooled to −30° C., added with N-bromodibutimide, stirred under the protection of Ar for 2 hrs, and 5 ml of water was added to quench the reaction. The system was returned to room temperature, and the solvent was completely removed by rotary evaporation, and the residue was dissolved in 100 ml of dichloromethane and washed with water three times. The organic phase was dried over anhydrous Na₂SO₄, and Na₂SO₄ was removed by filtration. The solvent was removed by rotary evaporation to give a residue which was purified by gel silica gel column chromatography to give a white solid 0.31 g, and the yield was 57%. ¹H-NMR (400 MHz, CDCl₃): δ=7.73 (s, 1H), 7.42 (d, 1H, J=4.8 Hz), 7.15 (d, 1H, J=4.8 Hz), 0.46 (s, 6H).

Compound 89

This compound was obtained by following the general procedure for compound 21, and the yield was 76%. ¹H-NMR (400 MHz, CDCl₃): δ=7.73 (s, 1H), 7.42 (d, 1H, J=4.8 Hz), 7.15 (d, 1H, J=4.8 Hz), 3.86 (t, 2H, J=4.80 Hz), 3.56 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Compound 90

This compound was obtained by following the general procedure for compound 22, and the yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=9.87 (s, 1H), 7.83 (s, 1H), 7.10 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Compound 91

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.83 (s, 1H), 7.11 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Probe 73

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.82 (s, 1H), 10.00 (s, 1H), 7.83 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.11 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Example 74

A fluorescence-activated covalently labeling fluorescent probe 74 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 92

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.83 (s, 1H), 7.11 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 1.50 (s, 9H), 0.42 (s, 6H).

Probe 74

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ=10.92 (s, 1H), 9.84 (s, 1H), 7.83 (s, 1H), 7.71 (s, 1H), 7.40 (m, 4H), 7.11 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 1.50 (s, 9H), 0.46 (s, 6H).

Example 75

A fluorescence-activated covalently labeling fluorescent probe 75 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 93

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=7.83 (s, 1H), 7.74 (1H, d, J=4.0 Hz), 7.55 (1H, d, J=4.0 Hz), 7.36-7.42 (2H, m), 7.11 (s, 1H), 4.12 (2H, s), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.42 (s, 6H).

Probe 75

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, CDCl₃): δ=11.22 (s, 1H), 10.01 (s, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.74 (1H, d, J=4.0 Hz), 7.70 (1H, d, J=4.0 Hz), 7.63-7.48 (m, 5H), 7.46 (m, 4H), 7.36-7.42 (2H, m), 7.11 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 4.12 (2H, s), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.13 (s, 3H), 3.06 (s, 3H), 0.46 (s, 6H).

Example 76

A fluorescence-activated covalently labeling fluorescent probe 76 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 94

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H), 7.11 (s, 1H), 6.50 (s, 1H), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Probe 76

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, CDCl₃): δ=12.42 (s, 1H), 10.01 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.4 (m, 4H), 7.11 (s, 1H), 6.50 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.06 (s, 3H), 0.46 (s, 6H).

Example 77

A fluorescence-activated covalently labeling fluorescent probe 77 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 95

The synthesis was carried out by the method disclosed in the literature (Martinez M. et al. Org. Biomol. Chem. 2012, 10.3892-3898). ¹H-NMR (400 MHz, CDCl₃): δ=7.24 (dd, J₁=5.2 Hz, J₂=1.2 Hz, 1H), 7.13 (dd, 1H, J₁=3.6 Hz, J₂=1.2 Hz), 7.03 (dd, 1H, J₁=5.2 Hz, J₂=1.2 Hz), 6.99 (d, 1H, J=3.8 Hz), 6.93 (d, 1H, J=3.6 Hz).

Compound 96

This compound was obtained by following the general procedure for compound 21, and the yield was 78%. ¹H-NMR (400 MHz, CDCl₃): δ=7.25 (dd, J₁=5.2 Hz, J₂=1.2 Hz, 1H), 7.13 (dd, 1H, J₁=3.6 Hz, J₂=1.2 Hz), 7.03 (dd, 1H, J₁=5.2 Hz, J₂=1.2 Hz), 6.99 (d, 1H, J=3.8 Hz), 6.93 (d, 1H, J=3.6 Hz), 3.85 (t, 2H, J=4.80 Hz), 3.46 (t, 2H, J=4.80 Hz), 3.10 (s, 3H).

Compound 97

This compound was obtained by following the general procedure for compound 22, and the yield was 65%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.75 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 5.81 (d, 1H, J=4.00 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.27 (s, 3H), 3.13 (s, 3H).

Compound 98

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 5.81 (d, 1H, J=4.00 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H), 1.50 (s, 9H).

Probe 77

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.52 (s, 1H), 10.01 (s, 1H), 8.00 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.00 Hz), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.27 (s, 3H), 3.13 (s, 3H), 1.50 (s, 9H).

Example 78

A fluorescence-activated covalently labeling fluorescent probe 78 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 99

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.40 (m, 4H), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.00 Hz), 5.46 (s, 2H), 4.40 (d, 2 H, J=4.8 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.48-3.52 (m, 4H), 3.38 (s, 3H), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Probe 78

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 5.81 (d, 1H, J=4.00 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.48-3.52 (m, 4H), 3.38 (s, 3H), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Example 79

A fluorescence-activated covalently labeling fluorescent probe 79 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 100

This compound was obtained by following the general procedure for compound 1, and the yield was 97%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 7.94 (d, 1H, J=8.0 Hz), 7.89 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 5.81 (d, 1H, J=4.00 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Probe 79

This probe was obtained by following the general procedure for probe 1, and the yield was 48%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.82 (s, 1H), 10.21 (s, 1H), 8.04 (d, 1H, J=8.0 Hz), 7.94 (d, 1H, J=8.0 Hz), 7.89 (s, 1H), 7.81 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (m, 4H), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.00 Hz), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Example 80

A fluorescence-activated covalently labeling fluorescent probe 80 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 80

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.81 (d, 1H, J=4.00 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H), 1.50 (s, 9H).

Example 81

A fluorescence-activated covalently labeling fluorescent probe 81 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 99

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.57 (d, 1H, J=4.00 Hz), 7.51 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 5.81 (d, 1H, J=4.00 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Probe 81

This probe was obtained by following the general procedure for probe 1, and the yield was 58%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.00 (s, 1H), 7.93 (d, 1H, J=5.6), 7.79 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.57 (d, 1H, J=4.00 Hz), 7.51 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 4H), 7.19 (d, 2H, J=8.0 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.81 (d, 1H, J=4.00 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Example 82

A fluorescence-activated covalently labeling fluorescent probe 82 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 82

This probe was obtained by following the general procedure for probe 1, and the yield was 57%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 7.94 (d, 2H, J=8.0 Hz), 7.89 (s, 1H), 7.75 (s, 1H), 7.57 (d, 1H, J=4.00 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 7.13 (d, 1H, J=4.00 Hz), 6.95 (d, 1H, J=4.00 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.81 (d, 1H, J=4.00 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.67 (t, 2H, J=5.60 Hz), 3.35 (t, 2H, J=5.60 Hz), 3.13 (s, 3H).

Example 83

A fluorescence-activated covalently labeling fluorescent probe 83 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 101

The synthesis was carried out by the method disclosed in the literature (WO2002020499 (A1). Apr. 14, 2002). ¹H-NMR (400 MHz, DMSO-d₆): δ=6.91 (m, 2H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H) 4.71 (t, 1H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H).

Compound 102

This compound was obtained by following the general procedure for compound 22, and the yield was 57%. ¹H-NMR (400 MHz, DMSO-d₆): δ=9.89 (s, 1H), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H) 4.71 (t, 1H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H).

Compound 103

This compound was obtained by following the general procedure for compound 1, and the yield was 88%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (s, 1H), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H) 4.71 (t, 1H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H), 1.49 (s, 9H).

Probe 83

This probe was obtained by following the general procedure for probe 1, and the yield was 68%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.42 (s, 1H), 10.01 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H), 1.49 (s, 9H).

Example 84

A fluorescence-activated covalently labeling fluorescent probe 84 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 84

This probe was obtained by following the general procedure for probe 1, and the yield was 48%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.21 (s, 1H), 7.19 (d, 2H, J=8.0 Hz), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H), 1.49 (s, 9H).

Example 85

A fluorescence-activated covalently labeling fluorescent probe 85 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 104

This compound was obtained by following the general procedure for compound 1, and the yield was 88%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H) 4.71 (t, 1H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H).

Probe 85

This probe was obtained by following the general procedure for probe 1, and the yield was 49%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.82 (s, 1H), 10.51 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.47 (m, 4H), 7.36-7.42 (m, 2H), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H).

Example 86

A fluorescence-activated covalently labeling fluorescent probe 86 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 86

This probe was obtained by following the general procedure for probe 1, and the yield was 91%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 8.02 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.21 (s, 1H), 6.71 (d, 1H, J=8.0 Hz), 6.50 (m, 1H) 4.71 (t, 1H, J=5.6 Hz), 3.62 (m, 2H), 3.56 (m, 2H), 3.35 (m, 2H), 3.00 (m, 2H).

Example 87

A fluorescence-activated covalently labeling fluorescent probe 87 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 106

The synthesis was carried out by the method disclosed in Hao Y et al. Tetrahedron 2012. 68.552-558. ¹H-NMR (400 MHz, CDCl₃): δ=7.06 (d, 1H, J=7.6 Hz), 6.94 (t, 1H, J=7.6H), 6.58 (t, 1H, J=7.2 Hz), 6.4 (d, 1H, 7.8 Hz), 5.19 (s, 1H), 2.35 (s, 3H), 1.36 (s, 6H).

Compound 107

Compound 106 (1.73 g, 10 mmol) was added to a 250 ml round bottom flask, potassium carbonate (2.76 g, 20 mol), bromoethanol (2.48 g, 20 mmol) was added, and 120 ml of acetonitrile was added. The mixture was heated to reflux in an oil bath for 48 h under the protection of Ar. After completion of the reaction, the system was filtered, and the solvent was completely removed by rotary evaporation. The residue was dissolved in 100 ml of dichloromethane, washed three times with 50 ml of water, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a brown product 1.76 g, and the yield was 81%. ¹H-NMR (400 MHz, CDCl₃): δ=7.06 (d, 1H, J=7.6 Hz), 6.94 (t, 1H, J=7.6H), 6.58 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Compound 108

This compound was obtained by following the general procedure for compound 22, and the yield was 65%. ¹H-NMR (400 MHz, CDCl₃): δ=9.89 (s, 1H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Compound 109

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (s, 1H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.50 (s, 9H), 1.36 (s, 6H).

Probe 87

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, CDCl₃): δ=11.82 (s, 1H), 9.73 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.50 (s, 9H), 1.36 (s, 6H).

Example 88

A fluorescence-activated covalently labeling fluorescent probe 88 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 110

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.30 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Probe 88

This probe was obtained by following the general procedure for probe 1, and the yield was 56%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.72 (s, 1H), 9.56 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.49 (m, 4H), 7.36-7.42 (m, 2H), 7.30 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Example 89

A fluorescence-activated covalently labeling fluorescent probe 89 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 111

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Probe 89

This probe was obtained by following the general procedure for probe 1, and the yield was 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.76 (s, 1H), 9.86 (s, 1H), 8.04 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.90 (d, 1H, J=8.0 Hz), 7.81 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.40 (m, 4H), 7.45 (t, 1H, J=8.0 Hz), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Example 90

A fluorescence-activated covalently labeling fluorescent probe 90 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 90

This probe was obtained by following the general procedure for probe 1, and the yield was 52%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.03 (s, 1H), 7.93 (d, 1H, J=5.6), 7.75 (s, 1H), 7.38 (d, 1H, J=7.6 Hz), 7.31 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.50 (s, 9H), 1.36 (s, 6H).

Example 91

A fluorescence-activated covalently labeling fluorescent probe 91 for CLIP protein tags was constructed using a molecular rotor as viscosity-sensitive fluorescent dye.

Probe 91

This probe was obtained by following the general procedure for probe 1, and the yield was 59%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 7.99 (s, 1H), 7.93 (d, 1H, J=5.6), 7.90 (d, 1H, J=8.0 Hz), 7.75 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.36 (d, 1H, J=7.6 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.19 (d, 2H, J=8.0 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.16 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Example 92

A fluorescence-activated covalently labeling fluorescent probe 92 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 112

The synthesis was carried out by the method disclosed in the literature (Ping Yan. et al. J. Org. Chem. 2008, 73, 6587-6594). ¹H-NMR (400 MHz, CDCl₃): δ=7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 1.50 (s, 6H).

Compound 113

This compound was obtained by following the general procedure for compound 21, and the yield was 76%. ¹H-NMR (400 MHz, CDCl₃): δ=7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz) 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Compound 114

This compound was obtained by following the general procedure for compound 22, and the yield was 66%. ¹H-NMR (400 MHz, CDCl₃): δ=9.89 (s, 1H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Compound 115

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.89 (s, 1H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (m, 15H).

Probe 92

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, 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=5.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (m, 15H).

Example 93

A fluorescence-activated covalently labeling fluorescent probe 93 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 116

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.89 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 4.12 (s, 2H), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Probe 93

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.42 (s, 1H), 10.01 (s, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 6H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 4.12 (s, 2H), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Example 94

A fluorescence-activated covalently labeling fluorescent probe 94 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 117

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=8.04 (d, 1H, J=8.0 Hz), 7.93 (d, 1H, J=8.0 Hz), 7.89 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 4.24 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Probe 94

This probe was obtained by following the general procedure for probe 1, and the yield was 45%. ¹H-NMR (400 MHz, DMSO-d₆): 12.42 (s, 1H), 10.01 (s, 1H), δ=8.04 (d, 1H, J=8.0 Hz), 7.93 (d, 1H, J=8.0 Hz), 7.89 (s, 1H), 7.81 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.40 (m, 4H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 4.24 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Example 95

A fluorescence-activated covalently labeling fluorescent probe 95 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 95

This probe was obtained by following the general procedure for probe 1, and the yield was 58%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.93 (d, 1H, J=7.2 Hz), 7.89 (s, 1H), 7.75 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.23 (d, 2H, J=8.0 Hz), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.15 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (m, 15H).

Example 96

A fluorescence-activated covalently labeling fluorescent probe 96 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 96

This probe was obtained by following the general procedure for probe 1, and the yield was 55%. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.93 (d, 1H, J=7.2 Hz), 7.89 (s, 1H), 7.79 (s, 1H), 7.74 (d, 1H, J=4.0 Hz), 7.55 (d, 1H, J=4.0 Hz), 7.36-7.42 (m, 2H), 7.31 (d, 2H, J=8.0 Hz), 7.18 (m, 3H), 6.96 (d, 2H, J=5.6 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.15 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 4.12 (s, 2H), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Example 97

A fluorescence-activated covalently labeling fluorescent probe 97 for CLIP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Probe 97

This probe was obtained by following the general procedure for probe 1, and the yield was 98%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.04 (d, 1H, J=8.0 Hz), 7.93 (m, 2H), 7.89 (s, 1H), 7.75 (s, 1H), 7.53 (t, 1H, J=8.0 Hz), 7.45 (t, 1H, J=8.0 Hz), 7.33 (d, 2H, J=8.0 Hz), 7.23 (d, 2H, J=8.0 Hz), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.06 (d, 1H, J=5.6 Hz), 5.27 (s, 2H), 5.15 (s, 2H), 4.45 (d, 2H, J=5.6 Hz), 4.24 (s, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H), 1.50 (s, 6H).

Example 98

A fluorescence-activated covalently labeling fluorescent probe 98 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 118

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.89 (s, 1H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 98

This probe was obtained by following the general procedure for probe 1, and the yield was 33%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.12 (s, 1H), 10.05 (s, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.4 (m, 4H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 99

A fluorescence-activated covalently labeling fluorescent reference probe 99 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 99

Compound 112 (0.375 g, 1 mmol), Compound 2 (0.297 g, 1.1 mmol) and benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (0.625 g, 1.2 mmol) were dissolved in 15 ml of anhydrous dimethylformamide, and 0.2 ml of triethylamine was added, and the mixture was stirred at room temperature for 2 hrs under the protection of Ar. After the completion of the reaction, the solvent was completely removed by rotary evaporation, and the residue was purified by gel silica gel column chromatography to give a yellow solid 0.458, and the yield was 73%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.12 (s, 1H), 10.05 (s, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.4 (m, 4H), 7.18 (s, 1H), 6.96 (d, 2H, J=5.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 100

A fluorescence-activated covalently labeling fluorescent reference probe 100 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 119

The synthesis was carried out by the method disclosed in (Shirisha Gurrapu et al. ACS Med. Chem. Lett. 2015. 6.558-561). ¹H-NMR (400 MHz, DMSO-d₆): δ=8.11 (s, 1H), 7.97 (d, 2H, J=9.0 Hz), 6.69 (d, 2H, J=9.6 Hz), 3.20 (s, 6H).

Comparative Probe 100

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 81%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.52 (s, 1H), 10.01 (s, 1H), 8.11 (s, 1H), 7.97 (d, 2H, J=9.0 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 6.69 (d, 2H, J=9.6 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.20 (s, 6H).

Example 101

A fluorescence-activated covalently labeling fluorescent probe 101 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 120

This compound was obtained by following the general procedure for compound 1, and the yield was 95%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.07 (s, 1H), 7.93 (d, 2H, J=9.2 Hz), 6.85 (d, 2H, J=9.2 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H).

Probe 101

This probe was obtained by following the general procedure for probe 1, and the yield was 35%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.22 (s, 1H), 10.01 (s, 1H), 8.07 (s, 1H), 7.93 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.4 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H).

Comparative Example 102

A fluorescence-activated covalently labeling fluorescent reference probe 102 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 102

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 65%. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.22 (s, 1H), 10.01 (s, 1H), 8.07 (s, 1H), 7.93 (d, 2H, J=9.2 Hz), 7.81 (s, 1H), 7.4 (m, 4H), 6.85 (d, 2H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.55-3.59 (m, 4H), 3.08 (s, 3H).

Example 103

A fluorescence-activated covalently labeling fluorescent probe 103 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 122

This compound was obtained by following the general procedure for compound 1, and the yield was 89%. ¹H-NMR (400 MHz, CDCl₃): δ=8.09 (s, 1H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Probe 103

This probe was obtained by following the general procedure for probe 1, and the yield was 89%. ¹H-NMR (400 MHz, CDCl₃): δ=11.22 (s, 1H), 9.87 (s, 1H), 8.09 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Comparative Example 104

A fluorescence-activated covalently labeling fluorescent reference probe 104 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 104

This probe was obtained by following the general procedure for probe 1, and the yield was 89%. ¹H-NMR (400 MHz, CDCl₃): δ=11.22 (s, 1H), 9.87 (s, 1H), 8.09 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.46 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Example 105

A fluorescence-activated covalently labeling fluorescent probe 105 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 123

This compound was obtained by following the general procedure for compound 1, and the yield was 99%. ¹H-NMR (400 MHz, CDCl₃): δ=8.03 (s, 1H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 3.11 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Probe 105

This probe was obtained by following the general procedure for probe 99, and the yield was 95%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.72 (s, 1H), 9.89 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.11 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Comparative Example 106

A fluorescence-activated covalently labeling fluorescent probe 106 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 106

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 95%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.72 (s, 1H), 9.89 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 7.36 (d, 1H, J=7.6 Hz), 6.78 (t, 1H, J=7.2 Hz), 6.49 (d, 1H, 7.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.11 (m, 4H), 2.35 (s, 3H), 1.36 (s, 6H).

Example 107

A fluorescence-activated covalently labeling fluorescent probe 107 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 124

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.22 (s, 1H), 8.02 (s, 1H), 6.43 (s, 1H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 107

This probe was obtained by following the general procedure for probe 1, and the yield was 39%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.72 (s, 1H), 9.79 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 6.43 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2 H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 108

A fluorescence-activated covalently labeling fluorescent reference probe 6 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 108

This probe was obtained by following the general procedure for probe 92, and the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.72 (s, 1H), 9.79 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.40 (m, 4H), 6.43 (s, 1H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 109

A fluorescence-activated covalently labeling fluorescent probe 109 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 125

This compound was obtained by following the general procedure for compound 1, and the yield was 89%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.49 (d, 2H, J=8.8 Hz), 6.74 (d, 2H, J=8.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 109

This probe was obtained by following the general procedure for probe 99, and the yield was 93%. ¹H-NMR (400 MHz, CDCl₃): δ=11.51 (s, 1H), 9.72 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.49 (d, 2H, J=8.8 Hz), 7.40 (m, 4H), 6.74 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 110

A fluorescence-activated covalently labeling fluorescent reference probe 110 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 110

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 93%. ¹H-NMR (400 MHz, CDCl₃): δ=11.51 (s, 1H), 9.72 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.49 (d, 2H, J=8.8 Hz), 7.40 (m, 4H), 6.74 (d, 2H, J=8.8 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 111

A fluorescence-activated covalently labeling fluorescent probe 111 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 127

This compound was obtained by following the general procedure for compound 1, and the yield was 98%. ¹H-NMR (400 MHz, CDCl₃): δ=7.95 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=9.0 Hz), 6.92 (d, 1H, J=2.0), 6.82 (d, 1H, J=9.2 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H)

Probe 111

This probe was obtained by following the general procedure for probe 99, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=12.12 (s, 1H), 10.09 (s, 1H), 7.95 (s, 1H), 7.81 (m, 2H), 7.68 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0), 6.82 (d, 1H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 112

A fluorescence-activated covalently labeling fluorescent reference probe 112 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 112

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=12.12 (s, 1H), 10.09 (s, 1H), 7.95 (s, 1H), 7.81 (m, 2H), 7.68 (d, 1H, J=9.0 Hz), 7.40 (m, 4H), 6.92 (d, 1H, J=2.0), 6.82 (d, 1H, J=9.2 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 113

A fluorescence-activated covalently labeling fluorescent reference probe 113 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 128

This compound was synthesized according to the method disclosed in the literature (Masahiro Ono et al. Bioorg. Med. Chem. 2009, 17, 7002-7007). ¹H-NMR (400 MHz, CDCl₃): δ=10.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.07-7.04 (m, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Compound 129

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, CDCl₃): δ=8.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.07-7.04 (m, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Probe 113

This probe was obtained by following the general procedure for the reference probe 99, and the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.42 (s, 1H), 9.89 (s, 1H), 8.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 7.07-7.04 (m, 2H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 114

A fluorescence-activated covalently labeling fluorescent probe 114 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 130

This compound was obtained by following the general procedure for compound 1, and the yield was 96%. ¹H-NMR (400 MHz, CDCl₃): δ=8.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.07-7.04 (m, 2H), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 114

This probe was obtained by following the general procedure for probe 92, and the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.42 (s, 1H), 9.89 (s, 1H), 8.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 7.07-7.04 (m, 2H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 115

A fluorescence-activated covalently labeling fluorescent reference probe 115 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 115

This probe was obtained by following the general procedure for probe 99, the yield was 89%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.42 (s, 1H), 9.89 (s, 1H), 8.06 (s, 1H), 8.03 (d, 1H, J=10.0 Hz), 7.81 (s, 1H), 7.40 (m, 4H), 7.07-7.04 (m, 2H), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 116

A fluorescence-activated covalently labeling fluorescent reference probe 116 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 131

The synthesis was carried out by the method disclosed in the literature (Marian. Z. J. et. al. Tetrahedron., 2008, 64, 10605-10618). ¹H-NMR (400 MHz, CDCl₃): δ=9.98 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 3.03 (s, 6H).

Compound 132

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 3.03 (s, 6H).

Comparative Probe 116

This probe was obtained by following the general procedure for probe 92, and the yield was 97%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.02 (s, 1H), 9.56 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.40 (m, 4H), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.03 (s, 6H).

Example 117

A fluorescence-activated covalently labeling fluorescent probe 117 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 133

This compound was obtained by following the general procedure for compound 1, and the yield was 91%. ¹H-NMR (400 MHz, CDCl₃): δ=8.00 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 117

This probe was obtained by following the general procedure for probe 99, and the yield was 97%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.02 (s, 1H), 9.56 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.40 (m, 4H), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 118

A fluorescence-activated covalently labeling fluorescent reference probe 118 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Comparative Probe 118

This probe was obtained by following the general procedure for probe 92, and the yield was 97%. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.02 (s, 1H), 9.56 (s, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.68 (d, 1H, J=4.0 Hz), 7.56 (d, 2H, J=9.0 Hz), 7.40 (m, 4H), 7.24 (d, 1H, J=4.0 Hz), 6.72 (d, 2H, J=9.0 Hz), 6.29 (s, 2H), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 119

A fluorescence-activated covalently labeling fluorescent reference probe 119 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 134

The synthesis was carried out by the method disclosed in the literature (US 20090042227 (A1). 2009 Dec. 12). ¹H-NMR (400 MHz, CDCl₃): δ=9.75 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 5.81 (d, 1H, J=4.0 Hz), 3.00 (s, 6H).

Compound 135

This compound was obtained by following the general procedure for compound 1, and the yield was 94%. ¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 5.81 (d, 1H, J=4.0 Hz), 3.00 (s, 6H).

Reference Probe 119

This probe was obtained by following the general procedure for the reference probe 99. ¹H-NMR (400 MHz, CDCl₃): δ=11.32 (s, 1H), 9.75 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.40 (m, 4H), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.0 Hz), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.00 (s, 6H).

Example 120

A fluorescence-activated covalently labeling fluorescent probe 120 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Compound 136

This compound was obtained by following the general procedure for compound 1, and the yield was 94%. ¹H-NMR (400 MHz, CDCl₃): δ=7.99 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 5.81 (d, 1H, J=4.0 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Probe 120

This probe was obtained by following the general procedure for probe 1. ¹H-NMR (400 MHz, CDCl₃): δ=11.32 (s, 1H), 9.75 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.40 (m, 4H), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.0 Hz), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Comparative Example 121

A fluorescence-activated covalently labeling fluorescent reference probe 121 for SNAP protein tags was constructed using a molecular rotor as a viscosity-sensitive fluorescent dye.

Reference Probe 121

This probe was obtained by following the general procedure for probe 99. ¹H-NMR (400 MHz, CDCl₃): δ=11.32 (s, 1H), 9.75 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.57 (d, 1H, J=4.0 Hz), 7.40 (m, 4H), 7.13 (d, 1H, J=4.0 Hz), 6.95 (d, 1H, J=4.0 Hz), 6.29 (s, 2H), 5.81 (d, 1H, J=4.0 Hz), 5.46 (s, 2H), 4.40 (d, 2H, J=4.8 Hz), 3.85 (t, 2H, J=5.6 Hz), 3.60 (t, 2H, J=5.6 Hz), 3.10 (s, 3H).

Example 122

Reference probes BG-CCVJ and BG-gly-CCVJ were prepared by the method reported in literature (T. Y. Wang et al. Chem Sci. 2016, 7, 301-307).

Probes 1 to 98, 101, 103, 105, 107, 109, 111, 114, 117, and 120, and the reference probes 99, 100, 102, 104, 106, 108, 110, 112, 113, 115, 116, 118, 119, 121, BG-CCVJ and BG-Gly-CCVJ were dissolved in dimethyl sulfoxide, respectively, to prepare mother liquors having a concentration of 1×10⁻² M, to each of which glycerol and methanol were added and uniformly mixed, to prepare solutions finally having a concentration of 1×10⁻⁵ M. Depending on the probes, fluorescence emission spectra of the probes were detected successively with the maximum excitation wavelength of each probe under the same conditions, and the results were shown in Table 1.

As can be seen from Table 1, the probes of the Examples have fluorescence emission wavelength in a wide range, and have fluorescence intensity in glycerol significantly different from that in methanol. That is, these probes are sensitive to viscosity changes and have viscosity responsiveness.

Example 123

Probes were mixed with corresponding protein tags to obtain mixed samples, in which the final probe concentration was 5 μM, and the final protein tag concentration was 10 μM. The mixed samples were incubated 37° C. for 1 hr, fluorescence spectrophotometer was used to detect the change in fluorescence intensity, and the results are shown in Table 1.

It can be seen from the quantum yield of free probe in Table 1 that the probes of the Examples and the reference probes had extremely low fluorescence prior to binding to a protein tag, close to the background fluorescence level of a PBS buffer, which indicates that prior to binding to the protein tag, the fluorescence of the viscosity-sensitive fluorescent probes has not been activated. From the quantum yield of probe bound to protein tag, after reacting with the protein tags, the probes of the Examples could be detected to have significant signal enhancement in corresponding excitation transmission channels, the fluorescence activation multiple reached several hundreds to a thousand or more, and the brightness was high, which illustrates that after the probes of the Examples are bound to the protein tags, fluorescence of the probes can be activated, and the probes have good fluorescent molecular switching properties. In contrast, though the fluorescence of the reference probes could also be activated, the quantum yield of activated fluorescence was very low, and the brightness was very poor. In particular, in view of comparison results between probe 98 and reference probe 99, probe 101 and reference probe 102, probe 103 and reference probe 104, probe 105 and reference probe 106, probe 107 and reference probe 108, probe 109 and reference probe 110, probe 111 and reference probe 112, probe 114 and reference probe 115, probe 117 and reference probe 118, probe 120 and reference probe 121 in Table 1, for the same fluorescent dye, when the ligand is connected to the electron donor part of the fluorescent dye, the fluorescence activation brightness of the probe is much higher than the reference probe in which ligand is connected to the electron acceptor part of the fluorescent dye.

Accordingly, it can be seen that the fluorescence of the probe binding protein tag of the embodiment can be activated and has good fluorescence molecular switching properties. Compared with the reference probes formed by binding a ligand to an electron acceptor of the fluorescent dyes, the probes formed by binding a ligand to an electron donor of the fluorescent dyes in the Examples have significantly improved fluorescence activation brightness.

TABLE 1
fluorescence emission spectra of different probes
		Quantum			Fluorescence
	Quantum	yield of probe	Emission	Fluorescence	ratio in
	yield of free	bound to	wavelength/	activation	glycerol to
Name	probe	protein tag	nm	multiple	methanol
probe 1	<0.001	0.35	480	360	560
probe 2	0.001	0.15	510	146	410
probe 3	0.0019	0.27	532	139	399
probe 4	<0.001	0.35	480	490	562
probe 5	<0.001	0.15	555	1174	790
probe 6	<0.001	0.37	610	989	1183
probe 7	<0.001	0.23	480	1502	1455
probe 8	0.0018	0.12	510	68	320
probe 9	<0.001	0.21	531	745	851
probe 10	<0.001	0.39	482	431	655
probe 11	0.0013	0.36	481	270	422
probe 12	0.0012	0.41	482	351	405
probe 13	<0.001	0.29	483	315	391
probe 14	<0.001	0.18	531	771	560
probe 15	<0.001	0.21	580	277	320
probe 16	<0.001	0.21	580	359	311
probe 17	<0.001	0.15	580	498	402
probe 18	0.0017	0.45	535	265	301
probe 19	0.0012	0.45	535	361	299
probe 20	<0.001	0.32	535	398	341
probe 21	<0.001	0.48	555	560	1481
probe 22	<0.001	0.36	590	610	1821
probe 23	<0.001	0.42	610	712	1235
probe 24	<0.001	0.48	555	590	1681
probe 25	<0.001	0.36	590	670	1621
probe 26	<0.001	0.42	610	910	1123
probe 27	<0.001	0.39	555	665	1134
probe 28	<0.001	0.31	590	551	1132
probe 29	<0.001	0.33	610	621	998
probe 30	<0.001	0.55	570	535	1125
probe 31	<0.001	0.41	607	421	1135
probe 32	<0.001	0.46	609	638	997
probe 33	<0.001	0.55	570	957	1781
probe 34	<0.001	0.48	570	857	1774
probe 35	0.0012	0.53	570	433	972
probe 36	0.001	0.50	570	488	1258
probe 37	0.0012	0.56	570	469	1135
probe 38	0.0011	0.56	570	503	1011
probe 39	0.0016	0.55	570	335	892
probe 40	0.001	0.41	570	421	759
probe 41	0.0015	0.31	575	212	101
probe 42	0.0013	0.38	600	298	178
probe 43	<0.001	0.37	602	982	1028
probe 44	<0.001	0.37	603	1586	1459
probe 45	<0.001	0.29	603	958	752
probe 46	<0.001	0.21	620	563	682
probe 47	<0.001	0.26	627	358	235
probe 48	<0.001	0.16	625	428	180
probe 49	<0.001	0.11	601	541	210
probe 50	<0.001	0.29	612	416	358
probe 51	<0.001	0.25	625	392	359
probe 52	<0.001	0.25	625	435	270
probe 53	<0.001	0.18	625	579	401
probe 54	<0.001	0.17	650	498	391
probe 55	<0.001	0.25	651	382	211
probe 56	<0.001	0.39	566	989	436
probe 57	<0.001	0.31	566	1026	762
probe 58	<0.001	0.27	590	519	419
probe 59	<0.001	0.39	600	498	370
probe 60	0.0011	0.81	620	751	1199
probe 61	0.0012	0.77	620	668	817
probe 62	<0.001	0.32	640	592	711
probe 63	0.0015	0.73	620	486	531
probe 64	<0.001	0.48	620	519	632
probe 65	<0.001	0.66	650	798	391
probe 66	<0.001	0.37	620	512	386
probe 67	<0.001	0.36	620	576	276
probe 68	<0.001	0.36	620	681	381
probe 69	<0.001	0.27	620	491	217
probe 70	0.0026	0.68	640	258	131
probe 71	0.0028	0.67	640	239	121
probe 72	0.001	0.47	640	486	268
probe 73	0.0031	0.76	680	248	325
probe 74	0.0025	0.78	680	311	297
probe 75	0.0021	0.44	700	211	105
probe 76	0.0011	0.37	680	351	186
probe 77	0.0014	0.38	655	268	279
probe 78	<0.001	0.28	672	369	189
probe 79	<0.001	0.31	681	419	298
probe 80	0.0011	0.38	655	349	218
probe 81	<0.001	0.29	672	391	235
probe 82	<0.001	0.21	681	321	241
probe 83	<0.001	0.18	515	209	103
probe 84	<0.001	0.16	515	431	121
probe 85	<0.001	0.15	535	391	187
probe 86	<0.001	0.17	550	413	214
probe 87	<0.001	0.45	575	515	209
probe 88	<0.001	0.29	595	621	298
probe 89	<0.001	0.32	612	421	129
probe 90	<0.001	0.28	575	541	109
probe 91	0.0013	0.24	612	192	163
probe 92	<0.001	0.65	645	923	241
probe 93	<0.001	0.56	675	753	362
probe 94	<0.001	0.47	700	569	158
probe 95	<0.001	0.42	645	553	431
probe 96	<0.001	0.31	675	435	125
probe 97	<0.001	0.23	700	812	261
probe 98	<0.001	0.78	650	893	182
Reference	<0.001	0.01	649	53	129
Probe 99
Reference	<0.001	0.011	480	43	213
Probe 100
probe 101	<0.001	0.37	481	463	362
Reference	<0.001	0.02	480	31	212
Probe 102
probe 103	<0.001	0.27	515	651	321
Reference	<0.001	0.02	515	23	351
Probe 104
probe 105	<0.001	0.41	510	549	328
Reference	<0.001	0.01	555	62	215
Probe 106
probe 107	<0.001	0.47	555	564	526
Reference	<0.001	0.02	555	30	329
Probe 108
probe 109	<0.001	0.33	566	546	341
Reference	<0.001	0.01	567	23	218
Probe 110
probe 111	<0.001	0.49	570	681	621
Reference	<0.001	0.01	570	41	211
Probe 112
Reference	<0.001	0.01	625	36	129
Probe 113
probe 114	<0.001	0.27	625	328	230
Reference	<0.001	0.01	625	38	113
Probe 115
Reference	<0.001	0.01	620	29	125
Probe 116
probe 117	<0.001	0.39	620	562	321
Reference	<0.001	0.02	621	29	158
Probe 118
Reference	<0.001	0.01	655	15	362
Probe 119
probe 120	<0.001	0.42	656	469	421
Reference	<0.001	0.01	655	39	219
Probe 121
Reference	<0.001	0.02	501	170	260
Probe
BG-CCVJ
Reference	<0.001	—	—	60	260
Probe
BG-Gly-CCVJ

Example 124

SNAP protein tags were added to 30 μM the solutions of probe 1, probe 14, probe 21, probe 30, probe 48, probe 43, probe 56, respectively, to produce mixed sample solutions in which the final concentration of the SNAP protein tags was 0.1 μM, 0.5 μM, 0.7 μM, 1.2 μM, 4.5 μM, 8.1 μM, 13.1 μM and 14.8 μM. The mixed sample solutions were placed at 37° C. to react for 1 hr. A fluorescence spectrophotometer was used to detect the change in excitation emission spectrum of the sample, and the relationship between SNAP protein tag concentration and fluorescence intensity was drawn according to the emission spectrum intensity, and the results were shown in FIGS. 2 to 11, respectively.

As can be seen from FIG. 2, SNAP protein tag concentration within the range of 0.1 um to 14.8 um has a good linear relationship with the fluorescence intensity of the probe, so protein tags can be quantitatively detected according to the standard curve.

Example 125

HeLa cells were taken as an example to exhibit the labeling effect of the compound in mammalian cells. HeLa cells with stable expression of protein tags and HeLa-WT cells (HeLa primitive cells, with no protein tag expressed) were implanted in a 14 mm glass-bottom 96-well cell culture plate and stabilized for 10 hrs. Probes 1, 15, 21, 30, 56, 60, 63, 88 were added to the media, respectively and diluted to 5 μMs. The cells were incubated in a carbon dioxide incubator at 37° C. for 2 hrs, and then a Leica TPS-8 confocal microscopy was used to image the fluorescent changes of the labeled cells. The results of group B in FIG. 12 show that no corresponding fluorescence signal was detected in HeLa-WT cells after the addition of the aforesaid probes, indicating that the fluorescence of the probes was not affected by the intracellular environment. However, for HeLa cells with protein tags expressed shown by group A in FIG. 12, strong fluorescent signals could be detected, which are nearly 300 times stronger.

The above experiments indicate that the probe can specifically label intracellular protein tags and achieve fluorescence specific lighting, and meanwhile, the fluorescence of the probe is not affected by the intracellular environment.

Example 126

To verify that probes 1, 21, 48, 60, 66 and 77 can be used to label target proteins located by different organelles, HeLa cells were taken as an example to detect the effect of probes on labeling different subcellular proteins. HeLa cells were implanted 5000 cells per well in a 96-well glass-bottom cell culture plate. 14 hrs later, the cells were transfected with the plasmids encoding different organelle localized protein tags using Lipo2000; the original medium was removed 24 hrs after transfection, washed with a phenol-free red DMEM medium twice, and incubated with a phenol-free red medium containing 0.2 μM probes for 2 hrs, and then the cell labeling effects were detected by imaging with a leica TCS-8 confocal microscopy. Results are shown in FIG. 13. The probes can clearly display a variety of subcellular organelle structures without washing, including but not limited to nucleus, mitochondria, golgi body, endoplasmic reticulum, whole cell, cytoskeleton, extracellular membrane, lysosome, and endometrium.

These results indicate that the probe can be used as a powerful tool for labeling cell subcellular organelles.

Example 127

HeLa cells were implanted 5000 cells per well in a 96-well glass-bottom cell culture plate. 14 hrs later, the cells were transfected with pcdna3.1-clip-nls (The plasmid encoding nucleus localized CLIP tag), pcdna3.1-mito-snap (The plasmid encoding mitochondrial localized SNAP tag) using Lipo2000, 0.1 μM per well. The original medium was removed 24 hrs after transfection, washed twice with a phenol-free red DMEM medium. Then, the cells were incubated respectively with a phenol-free red media containing 0.2 μM probe 1 and probe 43 for two hrs, and then the cell labeling effects were detected by imaging with a leica TCS-8 confocal microscopy. Results are shown in FIG. 13. The probe 1 and problem 43 respectively can clearly display the structures of mitochondrial and nuclear without washing, and the co-localization coefficient of the fluorescence channel of nuclear labeled by probe 43 and the fluorescence channel of mitochondria labeled by probe 1 is less than 0.1, indicating that the two fluorescence channels will not interfere with each other.

The above experiments indicated that the spectra of fluorescence groups of different probes would not interfere with each other, and orthogonal labeling imaging could be performed simultaneously.

Example 128

First, a SNAP protein tag expressing plasmid, pcdna3.1-SNAP (sample group), and a control plasmid without SNAP protein tag expressing, pcdna3.1-CAT (control group), were introduced into mice. This method dissolves plasmids in a large volume of solution and rapidly injects them into mice through tail vein injection. The mouse liver and kidneys absorb DNA and then express the target protein. 20 hrs after plasmid injection, 0.4 μM probe 88 dissolved in 200 μl PBS was injected into mice through tail vein injection to label the SNAP protein tag. 6 hrs later, the mice were dissected, and the fluorescence differences in the liver parts of different mouse samples were detected by KODAK multi-spectral in vivo imaging system. Results are shown in FIG. 15. The liver fluorescence of the control plasmid pcdna3.1-CAT injected with probe 88 was very low, close to the background fluorescence level of the blank liver without the probe, while the liver of SNAP plasmid pcdna3.1-SNAP injected with probe 88 had strong fluorescence, and the signal intensity was more than 20 times higher than that of the control group.

The above experiments showed that the fluorescence of the probe was not affected by the internal environment of animals, and it could be applied to live animals in vivo, and it could specifically label SNAP protein tags expressed in liver parts and generate strong fluorescence signals.

Example 129

Through the directed evolution displayed by phage, after six rounds of SNAP-tag screening, two SNAP-tag mutants were cloned, which were SNAP F33G and SNAP V164E, respectively. The two SNAP-tag mutants were labeled by probes 1, 21, 40, respectively in accordance with the method described in Example 123. As shown by the results, under the same conditions, the quantum yield after fluorescence activation of the probes was 2.1 to 1.6 times of the original yield, as compared with the sequences disclosed by a literature (Bright Mollwitz et al. Biochem 2012, 51, 986-986).

Attached is the DNA sequences of SNAP F33G and SNAP V164E:

DNA sequence of SNAP F33G (SEQ ID NO: 1):

ATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGGG
CAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCGGCC
TGGGCAAAGGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCC
GCCGTGCTGGGCGGACCAGAGCCACTGATGCAGGCCACCGCCTGGCTCAA
CGCCTACTTTCACCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCCC
TGCACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCAGGTGCTGTGG
AAACTGCTGAAAGTGGTGAAGTTCGGAGAGGTCATCAGCTACAGCCACCT
GGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGTGAAAACCGCCC
TGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAG
GGCGACCTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTG
GCTGCTGGCCCACGAGGGCCACAGACTGGGCAAGCCTGGGCTGGGTTAA

DNA sequence of SNAP V164E (SEQ ID NO: 2):

ATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGGG
CAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCC
TGGGCAAAGGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCC
GCCGTGCTGGGCGGACCAGAGCCACTGATGCAGGCCACCGCCTGGCTCAA
CGCCTACTTTCACCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCCC
TGCACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCAGGTGCTGTGG
AAACTGCTGAAAGTGGTGAAGTTCGGAGAGGTCATCAGCTACAGCCACCT
GGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGTGAAAACCGCCC
TGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAG
GGCGACCTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGAGAAAGAGTG
GCTGCTGGCCCACGAGGGCCACAGACTGGGCAAGCCTGGGCTGGGTTAA

Example 130

To verify the correlation between the fluorescent activation of the probe of the present invention and the existence of proteins, SNAP protein in mammalian cells was taken as an example, and the fluorescence changes of SNAP protein binding probes after protein degradation were detected in HeLa cells by taking AID degradation system as an example. Firstly, HeLa cells were implanted 20,000/cm² in a 20 mm glass-bottom cell culture dish, and 14 hrs later, plasmids pcdna3.1-TIR1 and pcdna3.1-SNAP-IAA17-H2B were transfected by invitrogen's lipofectmain2000 transfection reagent. 24 hrs after the cell transfection, the original cell culture medium was replaced with a phenol-free red DMEM medium containing 1 μM of probe 21 to label the cells. The cell samples were incubated in a carbon dioxide incubator at 37° C. for 1 hour. After the labeling was completed, the fluorescence signal of labeled cells was detected by imaging with a Leica SP8 laser confocal microscopy, and indoleacetic acid (IAA) was added to induce SNAP-IAA17-H2B protein to degrade, and the cell fluorescence changes were detected during the protein degradation. Results are as shown in FIG. 16, the SNAP-IAA17-H2B protein was located in the nucleus (0 min), and protein degradation was induced by indoleacetic acid. With the increase of time, the fluorescence signal of SNAP-IAA17-H2B protein was gradually decreased. When indoleacetic acid was added for 120 min, the fluorescence signal was basically invisible, and the protein degradation rate was consistent with the results reported in literature. The above experiment indicates that the fluorescent properties of probes in mammalian cells also depend on the presence of proteins. When proteins exist, the fluorescence is activated, and when proteins are degraded, the fluorescence disappears, which can be used to track and monitor the degradation process of target proteins.

Example 131

To verify that the probe of the present invention can be used to monitor the assembly and degradation process of biological macromolecules in mammalian cells in real time, the process of tracing the assembly of intercellular protein CX43 to form intercellular channels in mammalian cells by the probe was detected by taking HeLa cells as an example. The C-terminal of CX43 gene was fused with SNAP gene, and HeLa cell line with stable expression of fusion protein CX43-SNAP was constructed and obtained by lentivirus infection technology. HeLa-CX43-SNAP cell lines were implanted in a 20 mm glass-bottom cell culture dish 10 hrs before the probe labeling. To conduct labeling, a phenol-free red DMEM medium was first used to dilute the probe 21 to 2 μM to replace the original cell culture medium. Cells were incubated in a carbon dioxide incubator at 37° C. for 1 hour. The cells were then washed twice using a fresh phenol-free red DMEM medium, with unbound probe 21 removed at 2-minute intervals. Then, DMEM phenol-free red culture medium containing 1 μM probe 1 was added to label cells. Leica SP8 confocal microscopy was used to monitor for a long time the fluorescence intensity and position changes of labeled cell samples at the corresponding fluorescence channels of probes 21 and 1, as shown in FIGS. 17A and 17B, respectively. A long-columnar specific fluorescence signal between two cells can be seen in the fluorescence channel of probe 21, as shown in FIG. 17A, consistent with the results reported in the literature (Guido Gaietta et al. Science 2002, 296, 503-507). When probe 1 was just added, the fluorescence signal could not be detected in its corresponding fluorescence channel, as shown in FIG. 17B, indicating that probe 21 labeled all CX43 proteins present in the cell. With the extension of culture time, new CX43-SNAP proteins were synthesized and labeled by probe 1. After labeling for 2 hrs, a fluorescent signal of probe 1 appeared and gradually increased at the edge of intercellular channels labeled by probe 21, while the fluorescent signal of probe 21 originally at the intercellular channels gradually decreased, indicating that the CX43-SNAP protein newly synthesized in intercellular channels replaced the original CX43-SNAP protein gradually from the surrounding to the center. The superimposed channel are as shown in FIG. 17C, consistent with the results reported in literature. The above experimental results demonstrate that SNAP series of compounds can be applied to real-time monitoring of the assembly and degradation of biomacromolecules in cells.

Claims

1. A fluorescent probe, which has a structure as shown in formula (I);

2. The fluorescent probe according to claim 1, wherein the R2 is selected from the following structures, or a bicyclic fused aromatic ring or fused aromatic heterocyclic ring formed by the following structures themselves or fused with each other:

3. The fluorescent probe according to claim 1, characterized in that the fluorescent probe is selected from the following structures:

4. A process for preparing the fluorescent probe according to claim 1, comprising a step of reacting a fluorescent dye of formula (II) with a ligand and a linker:

5. A fluorescent activated protein specific labeling method, comprising the following steps: contacting the fluorescent probe according to claim 1 with a target protein of a protein tag or a fusion protein tag, labeling reaction taking place at the ligand moiety of the fluorescent probe with the protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is covalently labeled on the protein tag; optionally, a reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or an in situ medium in which the target protein of the protein tag or the fused protein tag is located; optionally, the in situ medium is intracellular media, organelle media, living tissue media, Hood or body fluids.

6. A probe kit comprising the fluorescent probe according to claim 1, optionally further comprising a biocompatible medium which is at least one selected from dimethyl sulfoxide, a buffer, and physiological saline; optionally the buffer is phosphate buffer.

7. A fluorescent activated protein specific labeling method, comprising the following steps: contacting the fluorescent probe according to claim 1 with a target protein of a protein tag or a fusion protein tag, labeling reaction taking place at the ligand moiety of the fluorescent probe with the protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is covalently labeled on the protein tag; optionally, a reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or an in situ medium in which the target protein of the protein tag or the fused protein tag is located; optionally, the in situ medium is intracellular media, organelle media, living tissue media, blood or body fluids.

8. A fluorescent activated protein specific labeling method, comprising the following steps: contacting the fluorescent probe according to claim 2 with a target protein of a protein tag or a fusion protein tag, labeling reaction taking place at the ligand moiety of the fluorescent probe with the protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is covalent's/labeled on the protein tag; optionally, a reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or an in situ medium in which the target protein of the protein tag or the fused protein tag is located; optionally, the in situ medium is intracellular media, organelle media, living tissue media, blood or body fluids.

9. A fluorescent activated protein specific labeling method, comprising the following steps: contacting the fluorescent probe according to claim 3 with a target protein of a protein tag or a fusion protein tag, labeling reaction taking place at the ligand moiety of the fluorescent probe with the protein tag, and the fluorescent probe is labeled onto the protein tag; optionally, the fluorescent probe is covalently labeled on the protein tag; optionally, a reaction medium of the labeling reaction is selected from a pure protein solution, a cell lysate or an in situ medium in which the target protein of the protein tag or the fused protein tag is located; optionally, the in situ medium is intracellular media, organelle media, living tissue media, blood or body fluids.

10. A probe kit, comprising the fluorescent probe according to claim 2, optionally, the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is at least one selected from dimethyl sulfoxide, a buffer, and physiological saline; optionally, the buffer includes phosphate buffer.

11. A probe kit, comprising the fluorescent probe according to claim 1, optionally, the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is at least one selected from dimethyl sulfoxide, a buffer, and physiological saline; optionally, the buffer includes phosphate buffer.

12. A probe kit, comprising the fluorescent probe according to claim 3, optionally, the probe kit further comprises a biocompatible medium; optionally, the biocompatible medium is at least one selected from dimethyl sulfoxide, a buffer, and physiological saline; optionally, the buffer includes phosphate buffer.

13. The fluorescent probe according to claim 1, wherein, the structure of the Formula (I-2) is selected from the group consisting of the following Formulas:

14. The fluorescent probe according to claim 1, wherein said modified alkyl group is a group containing one or more substituents from C1 to C4 alkyl groups, —O—, —O—CO—, or —NH—CO—.

15. The fluorescent probe according to claim 14, wherein the C1 to C4 alkyl groups are methyl, ethyl, propyl, isopropyl, and isobutyl.