WO2023011656A1 - 一种荧光染料及其制备方法和用途 - Google Patents

一种荧光染料及其制备方法和用途 Download PDF

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WO2023011656A1
WO2023011656A1 PCT/CN2022/110699 CN2022110699W WO2023011656A1 WO 2023011656 A1 WO2023011656 A1 WO 2023011656A1 CN 2022110699 W CN2022110699 W CN 2022110699W WO 2023011656 A1 WO2023011656 A1 WO 2023011656A1
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group
alkyl
optionally
fluorescent dye
compound
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French (fr)
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张大生
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纳莹(上海)生物科技有限公司
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Priority to EP22852367.6A priority Critical patent/EP4382526A1/en
Priority to JP2024506818A priority patent/JP2024528240A/ja
Publication of WO2023011656A1 publication Critical patent/WO2023011656A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates to the technical field of fluorescent dyes, in particular to a fluorescent dye with low background fluorescence, good water solubility, high viscosity sensitivity and adjustable molecular structure, and a preparation method and application thereof.
  • Cell viscosity is one of the important parameters affecting the diffusion rate of substances. By affecting the mobility of substances, it has a great impact on the transportation, signal transduction, recognition and interaction of biomacromolecules. At the same time, the change of intracellular viscosity has a certain relationship with the occurrence of atherosclerosis, diabetes, Alzheimer's disease and tumor, and it is a potential indicator of these diseases. Therefore, it is of great significance to detect the viscosity in cells. Due to its unique advantages of sensitivity, real-time, in-situ, and visualization, the fluorescence method is an important means of measuring intracellular microscopic viscosity.
  • the measurement of intracellular viscosity generally uses fluorescent probes based on molecular rotors.
  • Molecular rotors are a special class of fluorescent molecules.
  • the energy lost in the non-radiative transition of the excited state changes with the change of viscosity.
  • the increase in viscosity or surrounding rigidity will hinder the twisting of the molecular rotor, and the energy lost in the non-radiative transition of the molecular rotor will decrease. , specifically manifested as an increase in the fluorescence intensity of the molecular rotor. Therefore, the fluorescence intensity of the molecular rotor can directly reflect the viscosity of the intracellular microenvironment.
  • Viscosity-responsive molecular rotor Fluorescent molecules are often used in the design of fluorescence-activated light-up probes (fluorogenic probes) in addition to detecting the micro-viscosity in cells. This is mainly because when the fluorescent molecule of the molecular rotor binds to protein, nucleic acid or other biomolecules, its twist rotation is limited (equivalent to an increase in viscosity), the fluorescence is lit, and the unbound molecules are in a twisted state and do not emit light. Fluorescence achieves low background for biomolecules and highly specific fluorescence lighting. Fluorescence imaging of low-abundance biomolecules in living organisms requires a lower background while ensuring a high signal-to-noise ratio.
  • the widely used thiazole orange dye has a good signal-to-noise ratio when labeling macromolecular proteins and tRNA, but it also shows relatively strong background fluorescence, which is severely limited when labeling some small biomolecules with low abundance. . Therefore, it is extremely important to develop fluorescent dye molecules with low background, sensitive viscosity response, and high signal-to-noise ratio, and will have wider applications in more fields.
  • the permanent bright fluorescent probes have a strong fluorescent background due to the problem that their own fluorescence cannot be quenched, such as traditional fluorescent dyes such as coumarin, fluorescein, and rhodamine , they themselves emit bright fluorescence in water. When combined with target substances, they must be washed before imaging, but often cause strong background fluorescence due to inability to wash thoroughly.
  • the second is the background fluorescence caused by incomplete quenching of quenched fluorescent probes.
  • rhodamine uses typical quenching groups to quench fluorescence with PET and FRET, and imaging RNA results in strong background fluorescence due to incomplete quenching. .
  • the third is the background fluorescence caused by lipophilicity of environment-responsive fluorescent probes due to their poor water solubility.
  • the environment-responsive fluorescent molecules themselves do not fluoresce in water. When combined with the target, the fluorescence is illuminated. However, if the water solubility of the fluorescent probe is too poor, it will bind to the cell membrane structure due to the principle of similarity and compatibility, so that it can also be activated. Some fluorescence, causing background problems. Therefore, as a viscosity-responsive fluorescent probe, to solve the problem of background fluorescence, improving the water solubility of the probe is a key factor.
  • fluorescent dyes as fluorescent probes over fluorescent proteins
  • the structure of small-molecule fluorescent dyes can be adjusted, so that the wavelength can be adjusted to achieve different colors of biomolecules.
  • the structures of small-molecule fluorescent probes are adjustable, many of them require many steps in organic synthesis, and the yields of many molecular synthesis are not high, so a relatively high price has been paid.
  • long wavelength has obvious advantages, but in the structure of existing dyes, long-wavelength molecules generally have relatively large conjugation, and large conjugation generally means that the water solubility of the compound is relatively poor, then Can cause problems with background fluorescence.
  • the wavelength can be significantly changed, and the wavelength can be shifted to a longer wavelength, then the fluorescent molecular structure can be kept small and the wavelength can be lengthened, which can ensure that the dye molecule is water soluble. It will be of great significance for biological imaging to realize the long wavelength of dye molecules at the same time.
  • the object of the present invention is to provide a fluorescent dye with low background fluorescence, good water solubility, sensitive viscosity response and adjustable wavelength.
  • the present invention provides a kind of fluorescent dye, comprises electron donor part D, conjugated system E and electron acceptor part A, and described fluorescent dye is as formula (I)
  • the electron donor moiety-D is (X 1 )(X 2 )N-, X 1 and X 2 are independently selected from hydrogen, alkyl, or modified alkyl, X 1 and X 2 are optionally connected to each other, and N Atoms together form an aliphatic ring;
  • the conjugated system E is formed by at least one conjugation connection selected from an aromatic subcycle and an aromatic heterocyclic ring, wherein each hydrogen atom contained in the conjugated system E is optionally independently substituted by an alkyl group;
  • X 1 and X 2 in the electron donor part D- are optionally together with the N atom to form an aliphatic heterocyclic ring with the conjugated system E;
  • the electron acceptor moiety optionally forms a ring structure of the following formula (I-1):
  • R 1 is independently selected from -O-, -S-, and -(NR a )-, wherein R a is selected from hydrogen, or alkyl;
  • X is independently selected from hydrogen, alkyl or modified alkyl
  • alkyl is C 1 -C 30 straight chain or branched chain alkyl; preferably, C 1 -C 10 straight chain or branched chain alkyl; preferably, C 1 -C 7 Straight chain or branched chain alkyl; preferably, C 1 -C 5 straight chain or branched chain alkyl; preferably, selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl Base, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3 -Dimethylbuty
  • the carbon atom is replaced, which means that the carbon atom or the carbon atom and the hydrogen atom on it are replaced by the corresponding group;
  • the "aliphatic heterocyclic ring” is a saturated or unsaturated 4-10 membered monocyclic or polycyclic aliphatic heterocyclic ring containing at least one heteroatom selected from N, O, or S on the ring, and the aliphatic heterocyclic ring When it contains an S atom, it is -S-, -SO- or -SO 2 -; the aliphatic ring is optionally substituted by an alkyl group;
  • arylene ring is a 6-13-membered monocyclic or fused bicyclic or fused polycyclic arylene group
  • heteromatic ring is a 5-12 membered monocyclic or fused bicyclic or fused polycyclic heteroaromatic group containing at least one heteroatom selected from N, O, S or Si on the ring ;
  • halogen atoms are each independently selected from F, Cl, Br, I;
  • the "primary amino group” is RNH 2 group
  • the "secondary amino group" is an RNHR' group
  • the "tertiary amino group” is an RNR'R" group
  • Each R, R', R" is independently a single bond, an alkyl group, an alkylene group, a modified alkyl group or a modified alkylene group;
  • alkylene is C 1 -C 10 straight chain or branched chain alkylene, optionally C 1 -C 6 branched or branched chain alkylene;
  • each of the modified alkyl groups or modified alkylene groups is independently selected from -OH, -O-, -NH 2 , ethylene glycol units (-(CH 2 CH 2 O)n-) , C 1 ⁇ C 8 alkyl, C 1 ⁇ C 8 haloalkyl (preferably C 2 ⁇ C 6 haloalkyl), C 1 ⁇ C 8 alkylcarboxy (preferably C 2 ⁇ C 6 alkylcarboxy) -SO 2 - A group of at least one of NH-, halogen atom, cyano group and nitro group, wherein n is 1-30, more preferably 1-10; more preferably 1-2;
  • the C 1 -C 8 alkyl group is methyl, ethyl, propyl, isopropyl
  • the C 1 -C 8 alkoxy group is methoxy, ethoxy, propoxy, Isopropoxy
  • the C 1 -C 8 acyloxy is acetoxy, ethyl, propyl, isopropyl
  • the C 1 -C 8 haloalkyl is trifluoromethyl, chloromethyl, bromine methyl;
  • the aliphatic heterocycle is selected from morpholine, thiomorpholine, tetrahydropyridine;
  • each of X1 and X2 is independently substituted by one or more C1-10 straight-chain or branched-chain alkyl groups selected from hydroxyl, cyano, halogen, and carboxyl;
  • conjugated system E is selected from structures in the following formulas (I-2-1)-(I-2-20):
  • conjugated system E and (X 1 )(X 2 )N- form an aliphatic ring as shown in (I-3-1)-(I-3-4):
  • the electron acceptor part A is one selected from the following formulas (I-4-1)-(I-4-43):
  • the fluorescent dye formula (I) is selected from compounds of the following formulas:
  • the second aspect of the present invention is to provide the method for preparing above-mentioned fluorescent dye, it is characterized in that, comprise the synthetic reaction step of formula (a) compound and formula (b) compound reaction generation fluorescent dye shown in formula (I):
  • the third aspect of the present invention is to provide the use of the above-mentioned fluorescent dyes in viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection.
  • the fourth aspect of the present invention is to provide the use of the above-mentioned fluorescent dyes in the preparation of reagents for viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection.
  • the fifth aspect of the present invention provides a fluorescence-activated light-up probe, which is characterized by comprising the above-mentioned fluorescent dye.
  • the sixth aspect of the present invention provides the use of the above-mentioned fluorescence-activated light-up probe in protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection.
  • the seventh aspect of the present invention provides the use of the above-mentioned fluorescence-activated light-up probes in the preparation of protein fluorescent labels, nucleic acid fluorescent labels, protein quantification or detection, or nucleic acid quantification or detection reagents.
  • the fluorescent dye obtained in the present invention has long-wavelength emission (the longest emission wavelength reaches 800nm). Its fluorescence intensity increases with the increase of environmental viscosity, the logarithm of fluorescence intensity and the logarithm of solvent viscosity have a good linear relationship, the relationship between fluorescence intensity and viscosity conforms to the Hoffman equation, and has a high slope, It is sensitive to viscosity, has a high activation factor and is insensitive to polarity changes.
  • the fluorescent dye obtained in the present invention can be used to measure the viscosity of a sample, for example, it is applicable to the test of microscopic viscosity.
  • the obtained fluorescent dye can specifically bind to the corresponding antibody, aptamer or amyloid, or bind to a protein label or enzyme through a ligand or inhibitor to obtain a series of fluorescent activation points Bright-type probes for fluorescent labeling, quantification, or monitoring of proteins, enzymes, or nucleic acids.
  • Figure 1 is a diagram of the fluorescence emission intensity of dye dye7 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Fig. 2 is a linear relationship diagram between viscosity conditions and fluorescence intensity of dye dye7 (1 ⁇ 10 -5 M);
  • Fig. 3 is a graph of fluorescence emission intensity of dye dye9 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Fig. 4 is a linear relationship diagram between viscosity conditions and fluorescence intensity of dye dye9 (1 ⁇ 10 -5 M);
  • Fig. 5 is a diagram of fluorescence emission intensity of dye dye12 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 6 is a linear relationship diagram between the viscosity condition and the fluorescence intensity of dye dye12 (1 ⁇ 10 -5 M);
  • Fig. 7 is a graph of fluorescence emission intensity of dye dye25 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 8 is a linear relationship diagram between the viscosity condition and the fluorescence intensity of dye dye25 (1 ⁇ 10 -5 M);
  • Fig. 9 is a graph of fluorescence emission intensity of dye dye40 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 10 is a linear relationship diagram between the viscosity condition and the fluorescence intensity of dye dye40 (1 ⁇ 10 -5 M);
  • Fig. 11 is a diagram of fluorescence emission intensity of dye dye7 (1 ⁇ 10 -5 M) under different polarity conditions;
  • Fig. 12 is a diagram of fluorescence emission intensity of dye dye9 (1 ⁇ 10 -5 M) under different polarity conditions
  • Fig. 13 is a diagram of fluorescence emission intensity of dye dye12 (1 ⁇ 10 -5 M) under different polarity conditions
  • Fig. 14 is a diagram of fluorescence emission intensity of dye dye25 (1 ⁇ 10 -5 M) under different polarity conditions;
  • Fig. 15 is a diagram of fluorescence emission intensity of dye dye40 (1 ⁇ 10 -5 M) under different polarity conditions;
  • Figure 16 is a comparison diagram of emission spectra of dye dye5 and dye6;
  • Figure 17 is a comparison diagram of emission spectra of dyes dye11, dye12, and dye14;
  • Figure 18 is a comparison diagram of emission spectra of dye dye12 and dye13;
  • Figure 19 is the emission spectrum of dye dye39
  • Figure 20 is the emission spectrum of dye dye42
  • Figure 21 is a comparison of background fluorescence of dye dye1 and dye48 in PBS solution
  • Figure 22 is a comparison of background fluorescence of dyes dye22 and dye49 in PBS solution
  • Fig. 23 is a comparison chart of background fluorescence of dye dye40 and dye50 in PBS solution
  • Figure 24 is a comparison of background fluorescence of dye dye9, dye10 and dye47 in PBS solution
  • Figure 25 is a diagram of dyes dye1, dye12, dye25, dye41 used to label aptamers in cells;
  • Figure 26 is the flow cytogram of dye dye3 binding RNA aptamer
  • Figure 27 is the flow cytogram of dye dye13 binding RNA aptamer
  • Figure 28 is the flow cytogram of dye dye21 binding RNA aptamer
  • Figure 29 is a flow cytogram of dye dye34 binding to RNA aptamers
  • Figure 30 is the flow cytogram of dye dye50 binding RNA aptamer
  • the synthesis steps of dye2 refer to the synthesis method of dye1, and the yield is 76%.
  • the synthesis steps of dye3 refer to the synthesis method of dye1, and the yield is 82%.
  • the synthesis method of dye4 refers to the synthesis steps of dye1, and the yield is 70%.
  • the synthesis method of compound 4 refers to the synthesis method of compound 3, and the yield is 56%.
  • 1 H NMR (400MHz,DMSO-d 6 ) ⁇ 10.11(s,1H)6.98–6.94(m,1H),6.94–6.88(m,1H),6.57(td,J 7.5,1.2Hz,1H) ,3.57–3.42(m,2H),3.13–3.00(m,2H),2.87(s,3H).
  • Dye5 was synthesized according to the synthesis method of dye1, and the yield was 65.8%.
  • Dye6 refers to the synthetic method of dye1, and the yield is 75.8%.
  • 1 H NMR (400MHz,DMSO-d 6 ) ⁇ 13.15(s,1H),7.42(s,1H),7.38–7.15(m,2H),6.82(d,J 8.7Hz,1H),3.76– 3.57(m,2H),3.13–3.05(m,2H),3.03(s,3H).
  • Dye refers to the synthetic method of dye1, and the yield is 84.2%.
  • Dye8 refers to the synthetic method of dye1, and the yield is 80.3%.
  • Dye9 refers to the synthetic method of dye1, and the yield is 88.6%.
  • Dye10 refers to the synthetic method of dye1, and the yield is 82.4%.
  • Dye11 refers to the synthetic method of dye1, and the yield is 78.4%.
  • Dye12 was synthesized according to dye1, and the yield was 78.3%.
  • Dye13 was synthesized according to dye1, and the yield was 87.1%.
  • Dye14 was synthesized according to the synthesis method of dye1, and the yield was 87.1%.
  • Dye15 was synthesized according to dye1, and the yield was 84.7%.
  • Dye17 was synthesized according to dye1, and the yield was 82.7%.
  • Dye refers to the synthetic method of dye1, and the yield is 52.3%.
  • 1H-NMR (400MHz, CDCl3): ⁇ 8.02(s, 1H), 7.57(s, 1H), 7.92(s, 1H), 3.95(m, 4H), 3.45(m, 8H), 3.25(s, 3H), 3.10(s, 3H).
  • dye19 was synthesized according to the synthesis method of dye1, and the yield was 88.9%.
  • 1 H-NMR (400MHz, CDCl 3 ): ⁇ 8.23(s, 1H), 7.57(s, 1H), 7.92(s, 1H), 3.25(s, 3H), 3.10(s, 3H) 3.08(m ,2H), 1.58-1.43(m,18H).
  • Dye20 refers to the synthetic method of dye1, and the yield is 81.2%.
  • Dye21 was synthesized according to the synthesis method of dye1, and the yield was 82.5%.
  • Dye22 was synthesized according to the synthesis method of dye1, and the yield was 90.1%.
  • Dye23 refers to the synthetic method of dye1, and the yield is 79.5%.
  • Dye24 was synthesized according to the synthesis method of dye1, and the yield was 68.5%.
  • Dye25 refers to the synthesis method of dye1, and the yield is 70.0%.
  • dye26 refers to the synthetic method of dye17, and the yield is 70.0%.
  • Dye27 was synthesized according to the synthesis method of dye1, and the yield was 65.9%.
  • Dye28 was synthesized according to the synthesis method of dye1, and the yield was 65.9%.
  • Dye29 was synthesized according to the synthesis method of dye1, and the yield was 55.1%.
  • dye30 refers to the synthetic method of compound dye1, and the yield is 45.2%.
  • Dye31 refers to the synthetic method of compound dye1, and the yield is 80.2%.
  • the synthesis method of dye33 refers to compound dye1, and the yield is 60.2%.
  • dye36 refers to the synthetic method of compound dye1, and the yield is 49.7%.
  • Dye37 refers to the synthetic method of compound dye1, and the yield is 59.6%.
  • the synthetic method of dye39 refers to compound dye1, and the yield is 58.2%.
  • the synthetic method of dye40 refers to compound dye1, and the yield is 69.2%.
  • the synthesis method of dye41 refers to compound dye1, and the yield is 56.6%.
  • the synthetic method of dye42 refers to compound dye1, and the yield is 38.2%.
  • Dye43 refers to the synthetic method of compound dye1, and the yield is 78.2%.
  • dye44 was synthesized according to the synthetic method of compound dye1.
  • dye45 was synthesized according to the synthesis method of dye1.
  • dye46 was synthesized according to the synthesis method of dye1.
  • the emission wavelengths of the above-mentioned molecular rotors are 620nm, 576nm, 650nm, 670nm, and 720nm, respectively.
  • the results showed that the fluorescence intensity of the fluorescent dyes with the same concentration increased with the increase of the environmental viscosity.
  • the logarithm of the fluorescence intensity and the logarithm of the solvent viscosity conform to the Huffman equation, have a good linear relationship, and have a high slope, as shown in Figures 2, 4, 6, 8, and 10, which prove that the above-mentioned dyes have a significant effect on the viscosity Responsive, can be used for viscosity testing of unknown samples.
  • the fluorescent dye of the present invention has good specificity to the viscous environment, and can reduce the non-specific fluorescence caused by the polar response.
  • Dyes dye1 and dye48; dye22 and dye49; dye40 and dye50; dye9, dye10 and dye47 were added to PBS to prepare a solution with a final concentration of 1 ⁇ 10 -5 M, and the corresponding maximum excitation wavelength was used to excite the dyes to detect their concentration in PBS.
  • the fluorescence intensity in each group of samples is normalized with the maximum fluorescence intensity as 100, as shown in Figures 21, 22, 23, and 24 respectively. The results show that compared with the fluorescent dyes reported in the literature, the fluorescent dyes of the present application With a special electron-withdrawing group structure part, the fluorescent dye of this application has lower background fluorescence.
  • the dye Before the binding, the dye basically does not emit fluorescence, but after the binding, the fluorescence is significantly activated, and the compound can also bind to the protein in the cell.
  • the cells expressing the aptamer emit Bright fluorescence, and no fluorescence in cells that do not express the aptamer, which indicates that the dye of the present invention has good cell membrane permeability and can be used for labeling the aptamer in living cells.
  • the detection results can be seen as follows As shown in the flow diagrams 26-29, dye3, dye13, dye21, and dye34 have very low dye background before binding, and the fluorescence is significantly activated after binding, and the dye can also combine with RNA aptamers to emit bright fluorescence in cells, basically There is no non-specific fluorescence activation, which shows that the fluorescent dye of the present invention has very low background fluorescence and very low non-specific fluorescence in cells, and can be used for labeling low-abundance biological macromolecules such as nucleic acids, while dye50 has obvious background fluorescence , as shown in Figure 30, is difficult to label low-abundance biomacromolecules such as nucleic acids.

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Abstract

一种式(I)所示的荧光染料及其制备方法和用途，所述荧光染料包括电子供体部分D、共轭体系E和电子受体部分A。其具有长波长发射的能力具有种背景荧光低、水溶性好，粘度响应敏感、波长可调的荧光染料。

Description

一种荧光染料及其制备方法和用途 技术领域
本发明涉及荧光染料技术领域，具体涉及一种背景荧光低、水溶性好，粘性敏感性高、分子结构可调的荧光染料及其制备方法和用途。
背景技术
细胞粘度是影响物质扩散速率的重要参数之一，通过影响物质的移动性，对物质的运输、信号传导、识别及生物大分子的相互作用有着极大的影响。同时细胞内粘度的改变与粥样硬化、糖尿病、阿尔茨海默病及肿瘤的发生有一定的关系，是这些疾病潜在的指示指标。因此，检测细胞内的粘度具有非常重要的意义。荧光方法由于具有灵敏、即时、原位、可视等独特优势是测试细胞内微观粘度的一种重要手段。不同于宏观大体积粘稠流体的粘度的测定可以使用转子粘度计，落球粘度计等仪器，细胞内粘度的测定一般采用基于分子转子的荧光探针。分子转子是一类特殊的荧光分子，其在激发态非辐射跃迁损失的能量随粘度的改变而变化，粘度或周围刚性的增加会阻碍分子转子的扭曲，分子转子非辐射跃迁损失的能量会减少，具体的表现为分子转子的荧光强度升高。所以分子转子的荧光强度就可以直接反映细胞内微环境的粘度。
粘度响应的分子转子荧光分子除了检测细胞内的微粘度之外，他还常常被用于荧光激活点亮型探针(fluorogenicprobe)的设计。这主要是因为当分子转子荧光分子和蛋白、核酸或其他的生物分子结合后限制了它的扭曲转动(等同于黏性的增加)，荧光被点亮，而没有结合的分子处于扭曲状态不发荧光，则实现了对生物分子的低背景，高特异性的荧光点亮。对于生物体中低丰度的生物分子的荧光造影在保证高的信噪比的同时要求更低的背景。如应用广泛的噻唑橙染料在标记大分子蛋白和tRNA时有好的信噪比，但也表现出了比较强的背景荧光，在标记一些丰度低的小的生物分子时受到比较严重的限制。所以发展低背景，粘度响应敏感，信噪比高的荧光染料分子极其重要，将会在更多的领域里有更加广泛的应用。
各类荧光探针背景高原因主要有：一是常亮型的荧光探针因为本身荧光无法淬灭的问题导致强的荧光背景，如香豆素，荧光素，罗丹明这类传统的荧光染料，他们本身在水里发出明亮的荧光，当和靶标物质结合后必须要进行清洗才能成像，但往往因为没法彻底清洗的原因造成强的背景荧光。二是淬灭型的荧光探针因为淬灭不彻底导致的背景荧光，如罗丹明用典型的淬灭基团利用PET、FRET淬灭荧光后成像RNA因为淬灭不彻底导致了强的背景荧光。三是环境响应型荧光探针因为水溶性差而亲脂导致的背景荧光。环境响应型的荧光分子本身在水里不发荧光，当与靶标结合后荧光被点亮，但荧光探针如果水溶性太差因为相似相容的原理就会和细胞膜结构结合，从而也能激活一些荧光，造成背景的问题。所以作为粘度响应型的荧光探针，要解决背景荧光的问题，改善探针的水溶性是一个很关键的因素。
荧光染料作为荧光探针之于荧光蛋白而言最大的优势在于小分子荧光染料结构可调，从而波长可调以实现对生物分子不同颜色的标记。虽说小分子荧光探针的结构可调，但很多在有机合成上需要很多步骤，很多的分子合成产率也不高，所以付出了比较高的代价。再者是作为荧光染料，长波长具有明显的优势，但现有的染料的结构中，长波长的分子一般具有比较大的共轭，大的共轭一般意味着化合物的水溶性比较差，则会导致背景荧光的问题。如果只改变染料分子上的一个化学元素就可以使波长发生明显的改变，并且可以使波长向长波长移动，那么就可以保证荧光分子结构小的同时使波长变长，这就可以保证染料分子水溶性的同时实现染料分子的长波长化，这对于生物成像将有十分重大的意义。
发明内容
本发明的目的在于提供一种具有种背景荧光低、水溶性好，粘度响应敏感、波长可调的荧光染料。
本发明一方面，提供一种荧光染料，包括电子供体部分D、共轭体系E和电子受体部分A，所述荧光染料如式(I)
Figure PCTCN2022110699-appb-000001
电子供体部分-D为(X 1)(X 2)N-，X 1、X 2独立地选自氢、烷基、或改性烷基，X 1，X 2任选相互连接，与N原子一起形成脂杂环；
共轭体系E为选自亚芳香环、亚芳香杂环中的至少一种共轭连接而形成，其中共轭体系E所含的各氢原子任选独立地被烷基取代；
电子供体部分D-中的X 1、X 2任选独立地和N原子一起与共轭体系E形成脂杂环；
电子受体部分任选形成下式(I-1)环状结构：
Figure PCTCN2022110699-appb-000002
其中，
R 1独立地选自-O-、-S-、和-(NR a)-，其中R a选自氢、或烷基；
R 2独立地选自＝O、＝S、和＝NH；
R 3独立地选自＝O、＝S、＝NH；
X独立地选自氢、烷基或改性烷基；
其中，所述“烷基”为C 1-C 30的直链或支链的烷基；优选地，为C 1-C 10直链或支链烷基；优选地，为C 1-C 7直链或支链烷基；优选地，为C 1-C 5直链或支链烷基；优选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
所述“改性烷基”为烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-SO 2-、-(S＝O)-、伯氨基、仲氨基、叔氨基、中的至少一种基团置换所得的基团，所述改性烷基具有1～ 30个碳原子(可选地，所述改性烷基具有1～12个碳原子；可选地，所述改性烷基具有1～10个碳原子；可选地，所述改性烷基具有1～8个碳原子；可选地，所述改性烷基具有1～5个碳原子；可选地，所述改性烷基具有1～2个碳原子)，
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“脂杂环”为环上含有选自N、O、或S中的至少一种杂原子的饱和或不饱和的4～10元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被烷基取代；
所述“亚芳香环”为6～13元单环或稠合双环或稠合多环的亚芳香基团；
所述“亚芳香杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的5～12元单环或稠合双环或稠合多环的亚杂芳香基团；
所述“卤原子”各自独立地选自F、Cl、Br、I；
所述“伯胺基”为RNH 2基团；
所述“仲胺基”为RNHR'基团；
所述“叔胺基”为RNR'R”基团；
各个R、R'、R”各自独立地为单键、烷基、亚烷基、改性烷基或改性亚烷基；
所述“亚烷基”为C 1-C 10直链或支链亚烷基，可选为C 1-C 6支链或支链亚烷基；
所述“改性亚烷基”为C 1-C 10(可选为C 1-C 6)烷基或亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中一种基团置换所得的基团；所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
可选地，所述改性烷基或改性亚烷基各自独立地为含有选自-OH、-O-、-NH 2、乙二醇单元(-(CH 2CH 2O)n-)、C 1～C 8烷基、C 1～C 8卤代烷基(优选C 2～C 6卤代烷基)、C 1～C 8烷基羧基(优选C 2～C 6烷基羧基)-SO 2-NH-、卤原子、氰基、硝基中至少一种基团的基团，其中，n为1～30，更优选为1～10；更优选为1～2；
可选地，所述C 1～C 8烷基为甲基、乙基、丙基、异丙基，所述C 1～C 8烷氧基为甲氧基、乙氧基、丙氧基、异丙氧基，所述C 1～C 8酰基氧基为乙酰氧基、乙基、丙基、异丙基，所述C 1～C 8卤代烷基为三氟甲基、氯甲基、溴甲基；
可选地，所述脂杂环选自吗啉、硫代吗啉，四氢吡啶；
可选地，X 1和X 2各自独立地被1个或多个选自羟基、氰基、卤原子、羧基的基团取代的C 1-10直链或支链烷基；
可选地，所述共轭体系E选自下式(I-2-1)-(I-2-20)中的结构：
Figure PCTCN2022110699-appb-000003
Figure PCTCN2022110699-appb-000004
或者，所述共轭体系E与(X 1)(X 2)N-形成如下(I-3-1)-(I-3-4)所示的脂杂环：
Figure PCTCN2022110699-appb-000005
可选地，所述电子受体A部分为选自下式(I-4-1)-(I-4-43)中的一种：
Figure PCTCN2022110699-appb-000006
Figure PCTCN2022110699-appb-000007
可选地，所述荧光染料式(I)选自下式化合物：
Figure PCTCN2022110699-appb-000008
Figure PCTCN2022110699-appb-000009
本发明的第二个方面是提供制备上述的荧光染料的方法，其特征在于，包括将式(a)化合物与式(b)化合物反应生成式(I)所示荧光染料的合成反应步骤：
Figure PCTCN2022110699-appb-000010
本发明的第三个方面是提供上述的荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途。
本发明的第四个方面是提供上述的荧光染料在制备粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测试剂中的用途。
本发明的第五个方面提供一种荧光激活点亮型探针，其特征在于，包括上述荧光染料。
本发明的第六个方面提供上述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途。
本发明的第七个方面提供上述的荧光激活点亮型探针在制备蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测试剂中的用途。
本发明所得荧光染料具有长波长发射(最长发射波长达800nm)。它的荧光强度随环境粘度的增大而增强，荧光强度的对数和溶剂粘度的对数关系具有很好的线性关系，荧光强度与粘度的关系符合霍夫曼方程，并且具有较高斜率，其对粘度反应灵敏，激活倍数高并且对极性变化反应不灵敏。
本发明所得荧光染料可用于测定样品的粘度，例如适用于微观粘度的测试。根据另一方面的具体实施方案，所得荧光染料可与对应的抗体、适配体或淀粉样蛋白等特异性结合，或者通过配体或抑制剂与蛋白标签或酶键合，获得系列荧光激活点亮型探针，用于蛋白、酶或核酸的荧光标记、定量或监测。
附图说明：
图1为染料dye7(1×10 -5M)在不同粘度条件下荧光发射强度图；
图2为染料dye7(1×10 -5M)粘度条件与荧光强度的线性关系图；
图3为染料dye9(1×10 -5M)在不同粘度条件下荧光发射强度图；
图4为染料dye9(1×10 -5M)粘度条件与荧光强度的线性关系图；
图5为染料dye12(1×10 -5M)在不同粘度条件下荧光发射强度图；
图6为染料dye12(1×10 -5M)粘度条件与荧光强度的线性关系图；
图7为染料dye25(1×10 -5M)在不同粘度条件下荧光发射强度图；
图8为染料dye25(1×10 -5M)粘度条件与荧光强度的线性关系图；
图9为染料dye40(1×10 -5M)在不同粘度条件下荧光发射强度图；
图10为染料dye40(1×10 -5M)粘度条件与荧光强度的线性关系图；
图11为染料dye7(1×10 -5M)在不同极性条件下荧光发射强度图；
图12为染料dye9(1×10 -5M)在不同极性条件下荧光发射强度图；
图13为染料dye12(1×10 -5M)在不同极性条件下荧光发射强度图；
图14为染料dye25(1×10 -5M)在不同极性条件下荧光发射强度图；
图15为染料dye40(1×10 -5M)在不同极性条件下荧光发射强度图；
图16为染料dye5，dye6的发射光谱对比图；
图17为染料dye11，dye12，dye14的发射光谱对比图；
图18为染料dye12，dye13的发射光谱对比图；
图19为染料dye39的发射光谱；
图20为染料dye42的发射光谱；
图21为染料dye1和dye48在PBS溶液中的本底荧光对比图；
图22为染料dye22和dye49在PBS溶液中的本底荧光对比图；
图23为染料dye40和dye50在PBS溶液中的本底荧光对比图；
图24为染料dye9，dye10和dye47在PBS溶液中的本底荧光对比图；
图25为染料dye1，dye12，dye25，dye41用于细胞中标记适配体的图；
图26为染料dye3结合RNA适配体的流式细胞图；
图27为染料dye13结合RNA适配体的流式细胞图；
图28为染料dye21结合RNA适配体的流式细胞图；
图29为染料dye34结合RNA适配体的流式细胞图；
图30为染料dye50结合RNA适配体的流式细胞图；
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于对本发明进行示例性说明，并不用于限制本发明。
Figure PCTCN2022110699-appb-000011
化合物1
5-溴噻吩-2甲醛(0.5g.2.6mmol)和2-甲氨基乙醇(0.78g,10.4mmol)溶于10ml水中，Ar保护条件下油浴加热100℃，反应过夜，反应结束，冷却至室温，二氯甲烷萃取3遍，饱和食盐水洗，无水硫酸钠干燥，旋干有机相，残余物进行柱层析，得到化合物10.4g，产率84％。 1H NMR(400MHz,DMSO-d 6)δ9.40(s,1H),7.65(d,J＝4.5Hz,1H),6.12(d,J＝4.5Hz,1H),3.62(q,J＝5.5Hz,2H),3.47(t,J＝5.6Hz,2H),3.09(s,3H).
dye1
Figure PCTCN2022110699-appb-000012
化合物1(0.2g，1.08mmol)和3-甲基噻唑烷-2，4-二酮(0.13g，1.30mmol)溶于50ml无水乙醇中，加入催化量哌啶，Ar保护条件下油浴加热90℃，反应3h，反应结束，冷却至室温，体系有大量固体析出，过滤，滤饼用冷乙醇洗两次，真空烘干，即得黄色化合物dye10.2g，产率71.4％。 1H NMR(400MHz,DMSO-d 6)δ7.71(s,1H),7.43(d,J＝4.4Hz,1H),6.15(d,J＝4.4Hz,1H),3.62(q,J＝5.5Hz,2H),3.46(t,J＝5.6Hz,2H),3.20(s，3),3.09(s,3H).
化合物2
Figure PCTCN2022110699-appb-000013
化合物2的合成步骤参照化合物1的合成方法，产率为50％。 1H NMR(400MHz,DMSO-d 6)δ9.70(s,1H),6.82(s,1H),3.62(q,J＝5.5Hz,2H),3.47(t,J＝5.6Hz,2H),3.09(s,3H).
dye2
Figure PCTCN2022110699-appb-000014
dye2的合成步骤参照dye1的合成方法，产率76％。 1H NMR(400MHz,DMSO-d 6)δ9.70(s,1H),6.82(s,1H),4.10(q,J＝7.0Hz4H),3.62(q,J＝5.5Hz,2H),3.47(t,J＝5.6Hz,2H),3.09(s,3H),1.2(t,J＝7.1Hz3H).
化合物3
Figure PCTCN2022110699-appb-000015
苯并吗啉(0.30g，2mmol)溶于20mLDMF，加入碳酸铯(0.78g，2.4mmol)，碘甲烷(0.31g，2.2mmol)，Ar保护条件下65℃油浴加热，反应4h,反应完毕，冷却至室温，体系倒入50mL水中，二氯甲烷萃取3次×50mL，合并有机相，旋转蒸干溶剂，柱色谱分离得产物0.290g，产率90.6％。 1H NMR(400MHz,DMSO-d 6)δ6.98–6.94(m,1H),6.94–6.88(m,1H),6.67(dd,J＝8.1,1.2Hz,1H),6.57(td,J＝7.5,1.2Hz,1H),3.38–3.32(m,2H),3.05–2.80(m,2H),2.69(s,3H).
三口烧瓶中加入10mlDMF置于冰浴中冷却5min，滴加0.2ml三氯氧磷，冰浴条件下搅拌1h,上述化合物溶于DMF中，滴加到体系中，Ar保护和冰浴条件下搅拌0.5h，体系缓慢升至室温，继续搅拌5h，反应完毕，加入饱和碳酸钠溶液调pH＝10.0，室温条件下搅拌过夜，次日分出有机相，水相用二氯甲烷50ml萃取三次，合并有机相，饱和食盐水洗涤两次，有机相用无水硫酸钠干燥，旋转蒸干溶剂，残余物柱色谱分离得黄色固体0.25g，产率74％。 1H NMR(400MHz,DMSO-d 6)δ10.26(s,1H)6.98–6.94(m,1H),6.94–6.88(m,1H),6.57(td,J＝7.5,1.2Hz,1H),3.38–3.32(m,2H),3.05–2.80(m,2H),2.69(s,3H).
dye3
Figure PCTCN2022110699-appb-000016
dye3
dye3的合成步骤参照dye1的合成方法，产率82％。 1H NMR(400MHz,DMSO-d 6)δ11.71(s,1H),11.56(s,1H),6.98–6.94(m,1H),6.94–6.88(m,1H),6.65(s,1H),6.57(td,J＝7.5,1.2Hz,1H),3.38–3.32(m,2H),3.05–2.80(m,2H),2.69(s,3H).
dye4
Figure PCTCN2022110699-appb-000017
dye4的合成方法参照dye1的合成步骤，产率70％。 1H NMR(400MHz,DMSO-d 6)δ13.81(s,1H),6.98–6.94(m,1H),6.94–6.88(m,1H),6.65(s,1H),6.57(td,J＝7.5,1.2Hz,1H),3.38–3.32(m,2H),3.05–2.80(m,2H),2.69(s,3H).
化合物4
Figure PCTCN2022110699-appb-000018
化合物4的合成方法参照化合物3的合成方法，产率56％。 1H NMR(400MHz,DMSO-d 6)δ10.11(s,1H)6.98–6.94(m,1H),6.94–6.88(m,1H),6.57(td,J＝7.5,1.2Hz,1H),3.57–3.42(m,2H),3.13–3.00(m,2H),2.87(s,3H).
dye5
Figure PCTCN2022110699-appb-000019
dye5参照dye1的合成方法,产率65.8％。 1H NMR(400MHz,DMSO-d 6)δ11.75(s,1H),7.42(s,1H),7.38–7.15(m,2H),6.82(d,J＝8.7Hz,1H),3.76–3.57(m,2H),3.13–3.05(m,2H),3.03(s,3H).
dye6
Figure PCTCN2022110699-appb-000020
dye6参照dye1的合成方法,产率75.8％。 1H NMR(400MHz,DMSO-d 6)δ13.15(s,1H),7.42(s,1H),7.38–7.15(m,2H),6.82(d,J＝8.7Hz,1H),3.76–3.57(m,2H),3.13–3.05(m,2H),3.03(s,3H).
Figure PCTCN2022110699-appb-000021
化合物5参照化合物3的合成方法，产率65.7％。 1H NMR(400MHz,DMSO-d 6)δ10.21(s,1H)，7.33(dd,J＝8.8,2.2Hz,1H),7.15(d,J＝2.2Hz,1H),6.63(d,J＝8.8Hz,1H),5.47(d,J＝1.5Hz,1H),2.87(s,3H),1.96(d,J＝1.4Hz,3H),1.34(s,6H).
Figure PCTCN2022110699-appb-000022
dye参照dye1的合成方法,产率84.2％。 1H NMR(400MHz,DMSO-d 6)δ13.86(s,1H),7.41(s,1H),7.33(dd,J＝8.8,2.2Hz,1H),7.15(d,J＝2.2Hz,1H),6.63(d,J＝8.8Hz,1H),5.47(d,J＝1.5Hz,1H),2.87(s,3H),1.96(d,J＝1.4Hz,3H),1.34(s,6H).
dye8
Figure PCTCN2022110699-appb-000023
dye8参照dye1的合成方法,产率80.3％。 1H NMR(400MHz,DMSO-d 6)δ7.41(s,1H),7.33(dd,J＝8.8,2.2Hz,1H),7.15(d,J＝2.2Hz,1H),6.63(d,J＝8.8Hz,1H),5.47(d,J＝1.5Hz,1H),4.40(m,2H),2.87(s,3H),2.61(m,2H)1.96(d,J＝1.4Hz,3H),1.34(s,6H).
化合物6
Figure PCTCN2022110699-appb-000024
化合物6参照化合物3的合成方法，产率60.7％。 1H NMR(400MHz,DMSO-d 6)δ10.25(s,1H),6.95(s,2H),3.25(dd,J＝6.6,4.9Hz,4H),2.68(t,J＝6.3Hz,4H),2.05–1.57(m,4H).
dye9
Figure PCTCN2022110699-appb-000025
dye9参照dye1的合成方法,产率88.6％。 1H NMR(400MHz,DMSO-d 6)δ12.21(s,1H),7.31(s,1H),6.95(s,2H),3.25(dd,J＝6.6,4.9Hz,4H),2.68(t,J＝6.3Hz,4H),2.05–1.57(m,4H).
dye10
Figure PCTCN2022110699-appb-000026
dye10参照dye1的合成方法,产率82.4％。 1H NMR(400MHz,DMSO-d 6)δ19.21(s,1H),7.31(s,1H),6.95(s,2H),3.25(dd,J＝6.6,4.9Hz,4H),2.68(t,J＝6.3Hz,4H),2.05–1.57(m,4H).
化合物7、8
Figure PCTCN2022110699-appb-000027
化合物7：
2-溴并二噻吩(0.438g，2mmol)溶于15mLN-甲基-N-羟乙基胺，加入铜粉(6.4mg，0.01mmol)，碘化亚铜(19mg，0.01mmol)，磷酸三钾(0.850g，4mmol)，Ar保护条件下80℃油浴加热过夜，反应完毕，冷却至室温，体系倒入50mL水中，二氯甲烷萃取3次×50mL，合并有机相，旋转蒸干溶剂，柱色谱分离得黄色产物0.362g，产率85％。 1H-NMR(400MHz，CDCl 3)：δ＝7.92(s，1H)，7.63(d，1H，J＝5.2Hz)，7.31(d，1H，J＝5.2Hz)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
化合物8：
化合物8(0.426g，2mmol)溶于50ml无水二氯甲烷中，加入1ml三乙胺，冰浴条件 下缓慢滴加醋酸酐(0.3ml，3mmol)，滴加完毕，体系缓慢升至室温，搅拌3h，反应完毕，加水100ml，分出有机相，水相用二氯甲烷50ml二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，旋转蒸干溶剂，残余物无需进一步纯化，直接用于下一步。
上述残余物溶于50ml二氯甲烷中，加入二甲基甲酰胺5ml，冰浴条件下加入三氯氧磷2ml，Ar保护条件下搅拌0.5h，体系缓慢升至室温，继续搅拌5h，反应完毕，加入饱和碳酸钠溶液调pH＝10.0，室温条件下搅拌过夜，次日分出有机相，水相用二氯甲烷50ml萃取三次，合并有机相，饱和食盐水洗涤两次，有机相用无水硫酸钠干燥，旋转蒸干溶剂，残余物柱色谱分离得黄色固体0.285g，产率59％。 1H-NMR(400MHz，CDCl 3)：δ＝10.01(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
dye11
Figure PCTCN2022110699-appb-000028
dye11参照dye1的合成方法,产率78.4％。 1H-NMR(400MHz，CDCl 3)：δ＝12.01(s，1H),7.57(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
Figure PCTCN2022110699-appb-000029
dye12参照dye1的合成方法,产率78.3％。 1H-NMR(400MHz，CDCl 3)：δ＝13.25(s，1H),7.57(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
dye13
Figure PCTCN2022110699-appb-000030
dye13参照dye1的合成方法,产率87.1％。 1H-NMR(400MHz，CDCl 3)：δ＝11.35(s，1H),11.25(s，1H),7.87(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
dye14
Figure PCTCN2022110699-appb-000031
dye14参照dye1的合成方法,产率87.1％。 1H-NMR(400MHz，CDCl 3)：δ＝18.35(s，1H),6.87(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)。
dye15
Figure PCTCN2022110699-appb-000032
dye15参照dye1的合成方法,产率84.7％。 1H-NMR(400MHz，CDCl 3)：δ＝7.42(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.34(s,3H),3.10(s，3H)。
dye16
Figure PCTCN2022110699-appb-000033
dye16参照dye1的合成方法,产率82.1％。 1H-NMR(400MHz，CDCl 3)：δ＝6.72(s，1H)，7.92(s，1H)，7.63(s，1H)3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.52(s,3H),3.10(s，3H)。
dye17
Figure PCTCN2022110699-appb-000034
dye17参照dye1的合成方法,产率82.7％。 1H-NMR(400MHz，CDCl 3)：δ＝12.03(s,1H),11.41(s,1H)6.72(s，1H)，7.92(s，1H)，7.63(s，1H),4.40(m,2H),3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.52(s,3H),3.10(s，3H),2.62(m,2H).
dye18
Figure PCTCN2022110699-appb-000035
化合物9
化合物9参照献(Organic&Biomolecular Chemistry,7(1),142-154；2009)的合成方法合成。1H-NMR(400MHz，CDCl3)：δ＝10.05(s，1H),7.57(s，1H)，7.92(s，1H)，3.95(m，4H)，3.45(m，8H)，3.10(s，3H)。
dye参照dye1的合成方法,产率52.3％。1H-NMR(400MHz，CDCl3)：δ＝8.02(s,1H),7.57(s，1H)，7.92(s，1H)，3.95(m，4H)，3.45(m，8H)，3.25(s,3H),3.10(s，3H)。
dye19
Figure PCTCN2022110699-appb-000036
化合物10
化合物10参照文献(Journal of Organic Chemistry,83(15),8533-8542；2018)的方法合成，产率52.1％。1H-NMR(400MHz，CDCl3)：δ＝10.15(s，1H),7.57(s，1H)，7.92(s，1H)，3.10(s，3H)3.08(m,2H),1.58-1.43(m,18H)。
dye19参照dye1的合成的方法合成，产率88.9％。 1H-NMR(400MHz，CDCl 3)：δ＝8.23(s，1H),7.57(s，1H)，7.92(s，1H)，3.25(s,3H),3.10(s，3H)3.08(m,2H),1.58-1.43(m,18H)。
化合物11、12、13
Figure PCTCN2022110699-appb-000037
化合物11
噻吩并[3,2-b]噻吩(10g,71.31mmol)加入到250ml三口烧瓶中，加入120ml无水四氢呋喃溶解,置于-78℃下冷却，1.6M的正丁基锂49ml滴加到体系中，保持-78℃2h,关闭制冷，缓慢回到室温，搅拌过夜，反应完后，冰浴下加入40ml水淬灭反应，体系倒入200ml二氯甲烷中，萃取，水相用二氯甲烷萃取三次，有机相用无水硫酸钠干燥，旋干溶剂，残余物柱 色谱分离得化合物10.7g。产率90％。 1H NMR(400MHz,Chloroform-d)δ9.97(s,1H),7.94(s,1H),7.70(d,J＝5.3Hz,1H),7.33(d,J＝5.3Hz,1H).
化合物12
化合物11(10g.59.5mmol)溶于120ml干燥的DMF和乙酸(1:1)的混合溶剂中，加入NBS(11.66g,65.5mmol),Ar保护下油浴加热120℃回流过夜，反应完成后，乙酸乙酯和水萃取三遍，有机相合并干燥，旋干溶剂，剩余物柱层析得到11.2g产物，产率78％。 1H NMR(400MHz,Chloroform-d)δ10.01(s,1H),7.70(d,J＝5.3Hz,1H),7.33(d,J＝5.3Hz,1H).
化合物13
参照化合1的合成方法，产率85％。 1H NMR(400MHz,DMSO-d 6)δ10.15(s,1H),7.87(s,1H),6.39(s,1H),3.85(t，4H，J＝5.6Hz)，3.60(t，4H，J＝5.6Hz).
dye20
Figure PCTCN2022110699-appb-000038
dye20参照dye1的合成方法,产率81.2％。 1H NMR(400MHz,DMSO-d 6)δ11.15(s,1H),7.64(s,1H),7.57(s,1H),6.59(s,1H),3.85(t，4H，J＝5.6Hz)，3.60(t，4H，J＝5.6Hz).
dye21
Figure PCTCN2022110699-appb-000039
dye21参照dye1的合成方法,产率82.5％。 1H NMR(400MHz,DMSO-d 6)δ11.35(s,1H),7.64(s,1H),7.57(s,1H),6.59(s,1H),3.85(t，4H，J＝5.6Hz)，3.60(t，4H，J＝5.6Hz).
化合物12、13
Figure PCTCN2022110699-appb-000040
化合物14、15
化合物14、15，参照化合物7、8的合成方法，产率50.4％。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)。
Figure PCTCN2022110699-appb-000041
dye22参照dye1的合成方法,产率90.1％。 1H-NMR(400MHz，CDCl 3)：δ＝12.31(s,1H),7.86(s，1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)。
dye23
Figure PCTCN2022110699-appb-000042
化合物16
化合物16参照文献(Journal of the American Chemical Society,137(43),13768-13771；2015)的合成方法合成。1H-NMR(400MHz，CDCl3)：δ＝10.31(s,1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)，2.91(s,3H)。
dye23参照dye1的合成方法,产率79.5％。1H-NMR(400MHz，CDCl3)：δ＝8.02(s,1H),7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)，2.91(s,3H)。
dye24
Figure PCTCN2022110699-appb-000043
化合物17
化合物17参照文献(Journal of the American Chemical Society,131(5),1766-1774；2009)的合成方法合成。1H-NMR(400MHz，CDCl3)：δ＝10.31(s,1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)，2.21(s,6H)。
dye24参照dye1的合成方法,产率68.5％。1H-NMR(400MHz，CDCl3)：δ＝12.28(s,1H)，8.31(s，1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)，2.21(s,6H)。
化合物18
Figure PCTCN2022110699-appb-000044
化合物18
化合物18参照化合物15的合成方法。1H-NMR(400MHz，CDCl3)：δ＝10.58(s,1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H),2.68(s，3H)。
dye25
Figure PCTCN2022110699-appb-000045
dye25参照dye1的合成方法，产率70.0％。1H-NMR(400MHz，CDCl3)：δ＝8.58(s,1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H),3.21(s,3H),2.68(s，3H)。
dye26
Figure PCTCN2022110699-appb-000046
化合物19
化合物19参照文献(Organic Letters,14(24),6366-6369；2012)的合成方法合成。1H-NMR(400MHz，CDCl3)：δ＝10.05(s,1H),7.98(s,1H)，7.81(d，J＝2.0Hz，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H),3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H),3.21(s,3H)。
dye26参照dye17的合成方法，产率70.0％。1H-NMR(400MHz，CDCl3)：δ＝8.55(s,1H),7.98(s,1H)，7.81(d，J＝2.0Hz，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H),3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H),3.21(s,3H),3.10(s,1H)。
Figure PCTCN2022110699-appb-000047
化合物20
化合物20，参照化合物8的合成方法，产率30.4％。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.75(d，J＝9.0Hz，1H)，6.52(d，J＝2.0Hz，1H)，6.42(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H)。
dye27
Figure PCTCN2022110699-appb-000048
dye27参照dye1的合成方法,产率65.9％。 1H-NMR(400MHz，CDCl 3)：δ＝11.28(s,1H),7.84(s，1H)，7.75(d，J＝9.0Hz，1H)，6.52(d，J＝2.0Hz，1H)，6.42(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H)。
Figure PCTCN2022110699-appb-000049
化合物21
化合物21，参照化合物8的合成方法，产率30.4％。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.21(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，2H)，3.34(t，J＝8.0Hz，2H)，3.21(s，3H)。
dye28
Figure PCTCN2022110699-appb-000050
dye28参照dye1的合成方法,产率65.9％。 1H-NMR(400MHz，CDCl 3)：δ＝7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.60(q,J＝5.5Hz,2H),3.34(t，J＝8.0Hz，3H)，3.21(s，3H),1.2(t,J＝7.1Hz3H).
Figure PCTCN2022110699-appb-000051
化合物22
化合物22，参照化合物8的合成方法，产率30.4％。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.75(d，J＝9.0Hz，1H)，6.52(d，J＝2.0Hz，1H)，6.42(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H)。
dye29
Figure PCTCN2022110699-appb-000052
dye29参照dye1的合成方法,产率55.1％。 1H-NMR(400MHz，CDCl 3)：δ＝7.75(d，J ＝9.0Hz，1H)，6.52(d，J＝2.0Hz，1H)，6.42(d，J＝9.1，2.3Hz，1H)，3.61(t，J＝8.0Hz，3H)，3.34(t，J＝8.0Hz，3H)，3.21(s，3H),3.10(s，3H).
化合物23
Figure PCTCN2022110699-appb-000053
化合物23
化合物23，参照化合物11的合成方法，产率54.2％。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.21(s,6H).
dye30
Figure PCTCN2022110699-appb-000054
dye30参照化合物dye1的合成方法，产率45.2％。 1H-NMR(400MHz，CDCl 3)：δ＝8.01(s,1H),7.81(s，1H)，7.68(d，J＝9.0Hz，1H)，6.92(d，J＝2.0Hz，1H)，6.82(d，J＝9.1，2.3Hz，1H)，3.21(s,6H).
化合物24
Figure PCTCN2022110699-appb-000055
化合物24
化合物24，参照化合物11的合成方法，产率58.2％。 1H-NMR(400MHz，CDCl 3)：δ＝9.91(s，1H)，7.80(s，1H)，7.65(d，J＝9.1Hz，1H)，6.98(d，J＝2.1Hz，1H)，6.85(d，J＝9.0，2.3Hz，1H)，3.21(s,6H).
dye31
Figure PCTCN2022110699-appb-000056
dye31参照化合物dye1的合成方法，产率80.2％。 1H-NMR(400MHz，CDCl 3)：δ＝8.03(s,1H),7.80(s，1H)，7.65(d，J＝9.0Hz，1H)，6.98(d，J＝2.0Hz，1H)，6.85(d，J＝9.1，2.3Hz，1H)，4.44(m,2H)，3.21(s,6H)2.62(m,2H).
dye32
Figure PCTCN2022110699-appb-000057
参照化合物dye1的合成方法，产率50.2％。 1H-NMR(400MHz，CDCl 3)：δ＝7.83(s,1H),7.80(s，1H)，7.65(d，J＝9.0Hz，1H)，6.98(d，J＝2.0Hz，1H)，6.85(d，J＝9.1，2.3Hz，1H)，3.21(s,6H).
化合物25
Figure PCTCN2022110699-appb-000058
化合物25
化合物25，参照化合物11的合成方法，产率45.2％。 1H-NMR(400MHz，CDCl 3)：δ＝7.88(s，1H)，7.65(d，J＝9.1Hz，1H)，6.98(d，J＝2.1Hz，1H)，6.85(d，J＝9.0，2.3Hz，1H)，3.21(s,6H).
dye33
Figure PCTCN2022110699-appb-000059
dye33参照化合物dye1的合成方法，产率60.2％。 1H-NMR(400MHz，CDCl 3)：δ＝8.01(s，1H)，7.65(d，J＝9.1Hz，1H)，6.98(d，J＝2.1Hz，1H)，6.85(d，J＝9.0，2.3Hz，1H)， 3.21(s,6H).
dye34
Figure PCTCN2022110699-appb-000060
化合物26：
参照文献(Chemistry-A European Journal,22(30),10627-10637；2016)的方法合成。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.82(s，1H)，7.71(m，1H)，7.60(m，2H)，6.78(s，2H)，3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.42(s，6H)
dye34参照化合物dye1的合成方法，产率50.8％。 1H-NMR(400MHz，CDCl 3)：δ＝11.21(s,1H),7.92(s，1H)，7.82(s，1H)，7.71(m，1H)，7.60(m，2H)，6.78(s，2H)，3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.42(s，6H)
dye35
Figure PCTCN2022110699-appb-000061
dye35参照化合物dye1的合成方法，产率47.8％。 1H-NMR(400MHz，CDCl 3)：δ＝11.28(s,1H),7.84(s，1H)，7.82(s，1H)，7.71(m，1H)，7.60(m，2H)，6.78(s，2H)，3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.42(s，6H)
dye36
Figure PCTCN2022110699-appb-000062
化合物27
参照化合物26的合成的方法。 1H NMR(400MHz,CDCl 3):10.0(s,1H),8.0(s,1H),7.96–7.98(t,1H),7.89–7.90(t,1H),7.47–7.49(m,1H),3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，
dye36参照化合物dye1的合成方法，产率49.7％。 1H NMR(400MHz,CDCl 3):11.28(s,1H),8.04(s，1H),8.0(s,1H),7.96–7.98(t,1H),7.89–7.90(t,1H),7.47–7.49(m,1H),3.82(t，2H， J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，
dye37
Figure PCTCN2022110699-appb-000063
化合物28
参照化合物26的合成的方法。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.82(s，1H)，7.71(s，1H)，3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H)
dye37参照化合物dye1的合成方法，产率59.6％。 1H-NMR(400MHz，CDCl 3)：δ＝10.28(s,1H),8.05(s，1H),7.82(s，1H)，7.71(s，1H)，3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H).
dye38
Figure PCTCN2022110699-appb-000064
化合物29
参照化合物26的合成的方法。 1H-NMR(400MHz，CDCl 3)：δ＝9.92(s，1H)，7.82(s，1H)，7.71(s，1H)，7.67(s，1H),7.50(s，1H),3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H)
dye38参照化合物dye1的合成方法，产率49.6％。 1H-NMR(400MHz，CDCl 3)：δ＝11.01(s，1H)，8.04(s,1H),7.82(s，1H)，7.71(s，1H)，7.67(s，1H),7.50(s，1H),3.82(t，2H，J＝5.6Hz)，3.54(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H)
dye39
Figure PCTCN2022110699-appb-000065
化合物30
参照文献(Chemistry of Materials,31(21),8810-8819；2019)的合成方法得到。 1H-NMR(400MHz，DMSO-d 6)：δ＝10.18(s，1H)，7.48(s,1H),7.41(s,1H),7.32(m,2H)，3.69(t， J＝4.9Hz，2H)，3.12(t，J＝4.9Hz，2H)，3.10(s，3H),1.48(s,6H)
dye39参照化合物dye1的合成方法，产率58.2％。 1H-NMR(400MHz，DMSO-d 6)：δ＝14.28(s，1H)，7.95(s,1H),7.48(s,1H),7.41(s,1H),7.32(m,2H)，3.69(t，J＝4.9Hz，2H)，3.12(t，J＝4.9Hz，2H)，3.10(s，3H),1.48(s,6H)
dye40
Figure PCTCN2022110699-appb-000066
dye40参照化合物dye1的合成方法，产率69.2％。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.95(s,1H),7.48(s,1H),7.41(s,1H),7.32(m,2H)，3.69(t，J＝4.9Hz，2H)，3.12(t，J＝4.9Hz，2H)，3.21(s，3H),3.10(s，3H),1.48(s,6H)
dye41
Figure PCTCN2022110699-appb-000067
化合物32
参照文献(Journal of Organic Chemistry,73(17),6587-6594；2008)的方法合成。 1H-NMR(400MHz，DMSO-d 6)：δ＝10.18(s，1H)，7.89(s，1H)，7.18(s，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H).
dye41参照化合物dye1的合成方法，产率56.6％。 1H-NMR(400MHz，DMSO-d 6)：δ＝12.58(s，1H)，8.01(s,1H),7.89(s，1H)，7.18(s，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H).
dye42
Figure PCTCN2022110699-appb-000068
dye42参照化合物dye1的合成方法，产率38.2％。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.01(s,1H),7.89(s，1H)，7.18(s，1H),3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)，3.10(s，3H)，1.50(s，6H).
dye43
Figure PCTCN2022110699-appb-000069
化合物33
化合物33参照化合物26的合成方法合成。 1H-NMR(400MHz，DMSO-d 6)：δ＝10.18(s，1H)，7.89(s，1H)，7.36(s,2H)，7.18(s，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)3.10(s,1H).
dye43参照化合物dye1的合成方法，产率78.2％。1H-NMR(400MHz，DMSO-d6)：δ＝12.09(s,1H)，8.28(s，1H)，7.89(s，1H)，7.36(s,2H)，7.18(s，1H)，3.85(t，2H，J＝5.6Hz)，3.60(t，2H，J＝5.6Hz)3.10(s,1H).
dye44
Figure PCTCN2022110699-appb-000070
化合物34：
化合物34参照Zhu,Linyong et al,PCT Int.Appl.,2019218876,21 Nov 2019.的合成方法合成。 1H-NMR(400MHz,CDCl3):δ＝9.74(s,1H),7.76(d,1H),7.60(s,1H),7.03(d,1H),3.85(t,2H,J＝5.6Hz),3.60(t,2H,J＝5.6Hz),3.10(s,3H),1.42(s,6H)。
dye44参照化合物dye1的合成方法合成。 1H-NMR(400MHz,CDCl3):δ＝11.24(s,1H),8.02(s,1H),7.76(d,1H),7.60(s,1H),7.03(d,1H),3.85(t,2H,J＝5.6Hz),3.60(t,2H,J＝5.6Hz),3.10(s,3H),1.42(s,6H)。
dye45
Figure PCTCN2022110699-appb-000071
dye45参照dye1的合成方法合成。 1H-NMR(400MHz,CDCl 3):δ＝13.08(s,1H),8.05(s,1H),7.76(d,1H),7.60(s,1H),7.03(d,1H),3.85(t,2H,J＝5.6Hz),3.60(t,2H,J＝5.6Hz),3.10(s,3H),1.42(s,6H)。
dye46
Figure PCTCN2022110699-appb-000072
dye46参照dye1的合成方法合成。 1H-NMR(400MHz,CDCl3):δ＝15.56(s,1H),8.08(s,1H),7.76(d,1H),7.60(s,1H),7.03(d,1H),3.85(t,2H,J＝5.6Hz),3.60(t,2H,J＝5.6Hz),3.10(s,3H),1.42(s,6H)。
对比实施例
dye47
Figure PCTCN2022110699-appb-000073
dye47参照文献(European Journal of Organic Chemistry,2011(25),4773-4787)的方法合成。 1H NMR(400MHz,DMSO-d 6)δ19.21(s,1H),7.31(s,1H),6.95(s,2H),3.25(dd,J＝6.6,4.9Hz,4H),2.68(t,J＝6.3Hz,4H),2.05-1.57(m,4H).
dye48
Figure PCTCN2022110699-appb-000074
dye48参照文献(J.Am.Chem.Soc.2020,142,17515-17523)的方法合成。 1H-NMR(400MHz,Chloroform-d)δ8.07(d,J＝8.9Hz,2H),7.11(s,1H),6.70(d,J＝8.8Hz,2H),4.29(s,3H),3.05(s,6H),2.31(s,3H).
dye49
Figure PCTCN2022110699-appb-000075
dye49参照文献(J.Am.Chem.Soc.2020,142,17515-17523)的方法合成。 1H-NMR(400MHz,Chloroform-d)δ8.19(d,J＝8.6Hz,2H),7.97(d,J＝15.6Hz,1H),7.48(d,J＝8.7,2H),7.12(s,1H),6.75(d,J＝8.6,2H),6.70(d,J＝8.6,2H),6.42(d,J＝15.6Hz,1H),4.45(s,2H),3.07(s,6H),3.05(s,6H),1.49(m,9H).
dye50
Figure PCTCN2022110699-appb-000076
dye50参照文献(Angew.Chem.Int.Ed.2020,59,2–10)的方法合成。 1H-NMR(400MHz,DMSO-d6)δ8.06(d,J＝8.6Hz,2H),6.79(d,J＝8.6Hz,2H),3.28(m,6H),1.81(s,6H).
试验例1
将实施例dye1-dye42制备的荧光染料(分子转子)分别溶于二甲基亚砜中，各自制得浓度为1×10 -2M的母液，将各母液分别加入甘油和甲醇中，混合均匀，各配制终浓度为1×10 -5M的溶液，根据荧光染料不同，依次用各荧光染料最大激发波长在相同条件下检测其荧光发射图谱，结果如表1所示，表明本发明荧光染料具有长波长发射荧光，并且对粘度变化响应灵敏。
表1
Figure PCTCN2022110699-appb-000077
Figure PCTCN2022110699-appb-000078
Figure PCTCN2022110699-appb-000079
试验例2
将染料dye7，dye9，Dyedye12，dye25，dye40分别加入以下粘度16.4cp(甘油的体积比值为0％)、29.8cp(甘油的体积比值为20％)、64.5cp(甘油的体积比值为40％)、143.5cp(甘油的体积比值为60％)、377.0cp(甘油的体积比值为80％)、946.0cp(甘油的体积比值为100％)的甘油和乙二醇的混合液，配制成终浓度为1×10 -5M的溶液，不同粘度条件下荧光发射光谱如图1、3、5、7、9所示，上述各分子转子发射波长分别为620nm、576nm、650nm、670nm、720nm。结果表明：相同浓度的荧光染料的荧光强度随环境粘度的增大荧光增强。荧光强度的对数和溶剂粘度的对数关系符合哈夫曼方程，具有很好的线性关系，并且具有较高斜率，如图2、4、6、8、10所示，证明上述染料对粘度反应灵敏，可以用于未知样品的粘度测试。
将染料dye7，dye9，dye12，dye25，dye40分别溶入到不同极性的溶剂(甲苯、乙酸乙酯、甲醇、乙腈、二甲基亚砜)中测试时，荧光强度均很弱且不随溶剂的极性变化而变化，如图11、12、13、14、15所示。
以上结果表明：本发明荧光染料对粘度环境具有较好的特异性，可以降低因为极性响应而引起的非特异性荧光。
试验例3
将染料dye5,dye6,dye11，dye12，dye13，dye14,dye39,dye42加入甘油中，配制成终浓度为1×10 -5M的溶液，用相对应的最大激发波长激发荧光染料，得到发射光谱，对其中一些发射光谱进行归一化。对比染料dye5，dye6的发射光谱，如图16，当拉电子部分的氧羰基变成硫羰基后波长增长大约50nm；对比染料dye11，dye12，dye14的发射光谱，如图17，说明这个增加波长的效应可以叠加；对比染料dye12和dye13的发射光谱，如图18，当拉电子部分的五元环上的硫变成氮原子后，波长减短20nm，这证明上述染料的发射波长可以很轻易的通过改变的结构上的一个原子来实现，这就可以在保证染料结构变化不大(水溶性和膜的渗透性变化不大)的情况下实现光谱可调，并且通过这种波长调节机制，可以将染料的最大发射波长达到800以上，如图19，20所示，这对于染料用于活体成像是非常有利的。
试验例4
染料dye1与dye48；dye22与dye49；dye40与dye50；dye9，dye10与dye47加入PBS中，配制成终浓度为1×10 -5M的溶液，用相对应的最大激发波长激发染料，检测他们在PBS中的荧光强度，以各组样品最大的荧光强度为100来进行归一化，分别如图21,22,23,24所示，结果表明，与文献中报道的荧光染料比较，本申请荧光染料具有特殊的拉电子基团结构部分，使本申请荧光染料具有更低的本底荧光。
试验例5
使用持续表达RNA适配体(UUGCCAUGUGUAUGUGGGUUCGCCCACAUACUCUGAUGAUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCUUCGGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGAUCAUUCAUGGCAA)的稳定细胞株(293T/17)和对照细胞(293T/17)，在常规哺乳动物细胞培养条件下(37℃，5％二氧化碳，100％相对湿度)，待其生长至细胞汇合度达90％后，将染料dye1,dye12，dye25，dye41终浓度为2uM的细胞培养基替换原有细胞培养基孵育10min(37℃，5％二氧化碳，100％相对湿度)，共聚焦显微镜使用染料对应波长进行观察，结合前染料基本不发荧光，结合后荧光被显著激活，并且化合物在细胞里也可以和蛋白质结合，如图25所示，表达适配体的细胞发出明亮的荧光，没有表达适配体的细胞则没有荧光，这说明本发明的染料具有很好的细胞膜渗透性，可用于活细胞适配体的标记。
试验例6
使用持续表达RNA适配体(UUGCCAUGUGUAUGUGGGUUCGCCCACAUACUCUGAUGAUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCGGUGUAGUAGGUCCUUCGGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGACCGGAAGAAUUGAUCUCGGCCGGAUCAUUCAUGGCAA)的稳定细胞株(293T/17)和对照细胞(293T/17)，在常规哺乳动物细胞培养条件下(37℃，5％二氧化碳，100％相对湿度)，待其生长至细胞汇合度达90％后，将细胞消化下来，800rpm离心，并使用含有1μMdye1,dye12，dye25，dye41分子的PBS重悬细胞并孵育5分钟后进行流式检测，检测结果可见如流式图26-29所示，dye3,dye13，dye21，dye34结合前染料本底很低，结合后荧光被显著激活并且染料在细胞里也可以和RNA适配体结合发出明亮的荧光，基本上没有非特异性荧光激活，这说明本发明的荧光染料在细胞里具有很低的背景荧光和很低的非特异性荧光，可用于低丰度生物大分子如核酸的标记，而dye50具有明显的背景荧光，如图30所示，难以用于低丰度生物大分子如核酸的标记。
Figure PCTCN2022110699-appb-000080

Claims (10)

  1. 一种荧光染料，包括电子供体部分D、共轭体系E和电子受体部分A，所述荧光染料如式(I)
    Figure PCTCN2022110699-appb-100001
    电子供体部分-D为(X 1)(X 2)N-，X 1、X 2独立地选自氢、烷基、或改性烷基，X 1，X 2任选相互连接，与N原子一起形成脂杂环；
    共轭体系E为选自亚芳香环、亚芳香杂环中的至少一种共轭连接而形成，其中共轭体系E所含的各氢原子任选独立地被烷基取代；
    电子供体部分D-中的X 1、X 2任选独立地和N原子一起与共轭体系E形成脂杂环；
    电子受体部分任选形成下式(I-1)环状结构：
    Figure PCTCN2022110699-appb-100002
    其中，
    R 1独立地选自-O-、-S-、和-(NR a)-，其中R a选自氢、或烷基；
    R 2独立地选自＝O、＝S、和＝NH；
    R 3独立地选自＝O、＝S、＝NH；
    X独立地选自氢、烷基或改性烷基；
    其中，所述“烷基”为C 1-C 30的直链或支链的烷基；优选地，为C 1-C 10直链或支链烷基；优选地，为C 1-C 7直链或支链烷基；优选地，为C 1-C 5直链或支链烷基；优选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
    所述“改性烷基”为烷基的任意碳原子被选自卤原子、-O-、-OH、-CO-、-SO 2-、-(S＝O)-、伯氨基、仲氨基、叔氨基、中的至少一种基团置换所得的基团，所述改性烷基具有1～30个碳原子(可选地，所述改性烷基具有1～12个碳原子；可选地，所述改性烷基具有1～10个碳原子；可选地，所述改性烷基具有1～8个碳原子；可选地，所述改性烷基具有1～5 个碳原子；可选地，所述改性烷基具有1～2个碳原子)，
    所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
    所述“脂杂环”为环上含有选自N、O、或S中的至少一种杂原子的饱和或不饱和的4～10元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被烷基取代；
    所述“亚芳香环”为6～13元单环或稠合双环或稠合多环的亚芳香基团；
    所述“亚芳香杂环”为环上含有选自N、O、S或Si中的至少一种杂原子的5～12元单环或稠合双环或稠合多环的亚杂芳香基团；
    所述“卤原子”各自独立地选自F、Cl、Br、I；
    所述“伯胺基”为RNH 2基团；
    所述“仲胺基”为RNHR'基团；
    所述“叔胺基”为RNR'R”基团；
    各个R、R'、R”各自独立地为单键、烷基、亚烷基、改性烷基或改性亚烷基；
    所述“亚烷基”为C 1-C 10直链或支链亚烷基，可选为C 1-C 6支链或支链亚烷基；
    所述“改性亚烷基”为C 1-C 10(可选为C 1-C 6)烷基或亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中一种基团置换所得的基团；所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
  2. 根据权利要求1所述的荧光染料，其特征在于，所述改性烷基或改性亚烷基各自独立地为含有选自-OH、-O-、-NH 2、乙二醇单元(-(CH 2CH 2O)n-)、C 1～C 8烷基、C 1～C 8卤代烷基(优选C 2～C 6卤代烷基)、C 1～C 8烷基羧基(优选C 2～C 6烷基羧基)-SO 2-NH-、卤原子、氰基、硝基中至少一种基团的基团，其中，n为1～30，更优选为1～10；更优选为1～2；
    可选地，所述C 1～C 8烷基为甲基、乙基、丙基、异丙基，所述C 1～C 8烷氧基为甲氧基、乙氧基、丙氧基、异丙氧基，所述C 1～C 8酰基氧基为乙酰氧基、乙基、丙基、异丙基，所述C 1～C 8卤代烷基为三氟甲基、氯甲基、溴甲基；
    可选地，所述脂杂环选自吗啉、硫代吗啉，四氢吡啶；
    可选地，X 1和X 2各自独立地被1个或多个选自羟基、氰基、卤原子、羧基的基团取代的C 1-10直链或支链烷基；
  3. 根据权利要求1或2所述的荧光染料，其特征在于，所述共轭体系E选自下式(I-2-1)-(I-2-20)中的结构：
    Figure PCTCN2022110699-appb-100003
    Figure PCTCN2022110699-appb-100004
    或者，所述共轭体系E与(X 1)(X 2)N-形成如下(I-3-1)-(I-3-4)所示的脂杂环：
    Figure PCTCN2022110699-appb-100005
    可选地，所述电子受体A部分为选自下式(I-4-1)-(I-4-43)中的一种：
    Figure PCTCN2022110699-appb-100006
    Figure PCTCN2022110699-appb-100007
  4. 根据权利要求1-3任一项所述的荧光染料，其特征在于，所述荧光染料式(I)选自下式化合物：
    Figure PCTCN2022110699-appb-100008
    Figure PCTCN2022110699-appb-100009
  5. 制备权利要求1-4任一项所述荧光染料的方法，其特征在于，包括将式(a)化合物与式(b)化合物反应生成式(I)所示荧光染料的合成反应步骤：
    Figure PCTCN2022110699-appb-100010
  6. 权利要求1-4任一项所述荧光染料荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途。
  7. 权利要求1-4任一项所述荧光染料在制备粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测试剂中的用途。
  8. 荧光激活点亮型探针，其特征在于，包括权利要求1-4任一项所述荧光染料。
  9. 权利要求8所述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途。
  10. 权利要求8所述的荧光激活点亮型探针在制备蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测试剂中的用途。
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