A class of multifunctional fluorescent dyes and their applications

Abstract

A class of multifunctional fluorescent dyes and their applications, belonging to the field of functional dye technology. This class of multifunctional dyes, as dual-color ratiometric pH fluorescent probes, introduces different fluorophores at the distal and proximal amino positions of amino acids to form a class of ratiometric fluorescent probes with FRET phenomenon, and is finally anchored to the target position through the active ester connected to the carboxyl group on the amino acid. This dual-color ratiometric pH fluorescent probe has the ability to quantitatively monitor pH and can monitor the dynamic changes and metabolic processes of the target object in situ. It can be used as a convenient strategy that is easy to use and does not require cell modification, which is conducive to use in a variety of scenarios and is expected to become a pH fluorescent sensing tool with application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of functional dyes, and in particular relates to a class of multifunctional fluorescent dyes and applications. Background Art

[0002] A cell is a highly active and unified complex organism, and its microenvironment changes all the time with the cell state. The intracellular microenvironment (such as pH, viscosity, temperature, membrane potential, etc.) is an important indicator of its functional state. At present, most functional fluorescent dyes realize their labeling function through environmental aggregation, specific binding, etc., and use changes in fluorescence intensity to display microenvironment information. When the microenvironment changes, unstable labeling methods and signal transduction interfered by the environment will inevitably cause inaccurate labeling positions and inaccurate test microenvironment information. Using cell microenvironment indicators to realize the study of normal physiological and pathological states of cells requires not only fluorescent dyes to provide fluorescent signals, but also groups that can locate target cells or subcellular organelles and can accurately and stably monitor signals.

[0003] In the biological field, the visualization of intracellular pH is of great significance for cell activities. Various strategies have been introduced to develop pH sensors. Among them, FRET-based pH sensors are favored by researchers because of their single excitation and dual emission component ratio signals that are not affected by concentration and luminescence intensity. However, most FRET probes generally lack fixation of the target position, so long-term in situ tracking in complex biological environments is greatly limited. This is because it is difficult to connect energy donors and acceptors while retaining the targeting group in molecular design. At present, a bioorthogonal connection method has been developed to achieve targeted fixation of FRET probes. However, a convenient strategy that is easy to use and does not require cell modification and can be used in a variety of scenarios using a universal labeling strategy has not been well developed. There is an urgent need to rationally design and provide dual-color FRET-type pH fluorescent probes with stable targeting groups for studying in situ dynamic changes and metabolic processes in organisms. Summary of the invention

[0004] The present invention studies multifunctional fluorescent dyes and their applications, and develops a class of multifunctional dyes that have the ability to fix labeled proteins and accurately indicate the in-situ behavior of the labeled position. It can be used as a FRET ratio pH fluorescent dye to dynamically track the pH changes and distribution of proteins and bacteria in living cells in situ. Compared with traditional pH fluorescent dyes, due to the use of FRET-type two-color fluorophores and stable covalently linked labels, it can achieve long-term and accurate pH monitoring in situ in organisms, and is used for more potential multifunctional fluorescent sensing applications and biological research.

[0005] The present invention first provides a multifunctional fluorescent dye having the following structure of general formula I or II:

[0006]

[0007] Among them, R 0 Each independently selected from H, n=an integer of 0-18, m=an integer of 0-18, specifically, n=an integer of 0-10, m=an integer of 0-10, X - is an anion selected from BF4 - , Cl - Br - ,I - 、NO 3- 、SO 4 2- 、C1O 4 - , CH 3 COO - , CH 3 SO 3 - CF 3 SO 3 - ;

[0008] Said The total number of positive charges is equal to the total number of negative charges carried by X-;

[0009] Y is selected from

[0010] R 1 Selected from

[0011] R 2 Selected from the following structures:

[0012]

[0013] Among them, R 3 Each independently selected from H, methyl, R 4 Each independently selected from H, methyl or ethyl, R 5 Each independently selected from F, Cl, Br, I, R 6 Each is independently selected from H or Br.

[0014] The multifunctional fluorescent dye has good application prospects in pH fluorescence sensing.

[0015] The multifunctional fluorescent dye is used in the preparation of materials capable of fixing and tracking proteins. Specifically, the multifunctional fluorescent dye is used as a functional dye for long-range tracking and monitoring of the local pH of the marked target to prepare materials capable of fixing and tracking proteins.

[0016] The application of the multifunctional fluorescent dye is specifically to study the in situ dynamic tracking of proteins and the behavioral distribution of different bacteria in living cells.

[0017] The multifunctional fluorescent dye can realize dynamic visualization monitoring of the in-situ pH environment at the marked position. Specifically, compound F is covalently linked to the thiol or exposed amino group of the research object of interest, and a living cell confocal microscope is used as a fluorescence collection tool to obtain dual-color fluorophore fluorescence and fluorescence intensity, thereby revealing the characteristics of the behavior distribution of the research object of interest in the environment.

[0018] Beneficial effects of the present invention: This type of multifunctional dye is used as a dual-color ratiometric pH fluorescent probe by introducing different fluorophores at the distal amino position and the proximal amino position of the amino acid to form a type of ratiometric fluorescent probe with FRET phenomenon, and finally anchored to the target position through the active ester connected to the carboxyl group on the amino acid. The dual-color ratiometric pH fluorescent probe has the ability to quantitatively monitor pH and can realize the monitoring of dynamic changes and metabolic processes of the target object in situ. It can be used as a convenient strategy that is easy to use and does not require cell modification, which is conducive to use in a variety of scenarios and is expected to become a pH fluorescent sensing tool with application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the high-resolution mass spectrum of the multifunctional dye F1.

[0020] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum characterization diagram of the multifunctional dye F1.

[0021] Figure 3 This is the carbon NMR spectrum characterization diagram of the multifunctional dye F1.

[0022] Figure 4 The UV absorption and fluorescence emission spectra of the multifunctional dye F1.

[0023] Figure 5 This is the fluorescence emission spectrum of the multifunctional dye F1 after binding to BSA protein in vitro.

[0024] Figure 6 This is the SDS gel electrophoresis diagram of the multifunctional dye F1 after binding to BSA protein in vitro.

[0025] Figure 7 This is the pH cycle diagram of the multifunctional dye F1.

[0026] Figure 8 This is the fluorescence imaging of 4T1 cells stained with the multifunctional dye F1.

[0027] Fig. 9 This is the fluorescence emission spectrum of the multifunctional dye F17.

[0028] Fig.10 This is the fluorescence emission spectrum of the multifunctional dye F17 after binding to BSA protein in vitro. DETAILED DESCRIPTION

[0029] The following non-limiting embodiments may enable a person of ordinary skill in the art to more fully understand the present invention, but do not limit the present invention in any way. Any person familiar with the present technical field may make equivalent replacements or changes according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which shall fall within the protection scope of the present invention.

[0030] Specifically, the multifunctional fluorescent dye containing Y is selected from the following structures:

[0031]

[0032] In the specific structures of some multifunctional fluorescent dyes, R 2 Selected from the following structures:

[0033]

[0034] Among them, R 5 Each is independently selected from F, Cl, Br, and I.

[0035] In the specific structures of some multifunctional fluorescent dyes, R 2 Selected from the following structures:

[0036]

[0037] The multifunctional fluorescent dye described in general formula I selects amino acids as the connecting bridge of the dye, connecting the energy donor fluorophore BDP part on the one hand and the energy acceptor sulfonate rhodamine part on the other hand to achieve appropriate intramolecular energy transfer and spectral separation. In addition, it is anchored to the target protein of interest through the active ester connected to the amino acid. The multifunctional fluorescent dye responds to pH through the switch ring of the rhodamine structure, and can be used as a pH sensor to achieve accurate perception of the in situ environment of the organism at the molecular level. The multifunctional dye mainly has the following three characteristics: (1) Using amino acids of different structures as bridges, a one-step esterification reaction covalently connects the fluorophore (fluoroborane pyrrole) as an energy donor, a one-step amidation reaction connects the sulfonate rhodamine as an energy acceptor, and the residual carboxyl group is used as the target effect, so that the dye has multiple functions. (2) Adopting Or the fluorescence resonance energy transfer (FRET) quantitative measurement method has built-in correction for environmental effects; (3) Multifunctional amino acids are used as connecting bridges to ensure that the dual dyes form FRET, while using the amino acid carboxyl group to provide labeling sites. This greatly reduces the difficulty of molecular design and synthesis. The dye molecules have suitable energy transfer and bacterial / cellular protein targeting capabilities, and can monitor the local pH dynamics of bacteria / cells on site.

[0038] The present invention also provides a method for preparing the multifunctional fluorescent dye described above, which is synthesized by referring to related substances reported in existing literature, using rhodamine sulfonate and BDP as raw materials, and is prepared by condensation reaction, specifically comprising the following steps: 1) dehydration condensation of compound A and amino acid-Boc compound (compound Z) to obtain compound C;

[0039]

[0040] 2) Compound C reacts with fluoroborylpyrrole isothiocyanate or fluorescein isothiocyanate to obtain compound E;

[0041]

[0042] Wherein, compound D is selected from the following structures:

[0043]

[0044] Among them, R 3 , R 4 , R 5 , R 6 The definitions of are the same as those in the above general structural formula.

[0045] 3) Compound E and The target compound F is generated by esterification reaction;

[0046]

[0047] Among them, R 0 , R 1 , R 2 , The definitions of Y are the same as those in the general structural formula.

[0048] Specifically, the preparation method of the multifunctional fluorescent dye can be carried out by the following steps:

[0049] 1) Compound A is dissolved in an organic solvent, phosphorus oxychloride is added, and the mixture is heated under reflux for more than 5 hours. After the color of the mixture deepens, the mixture is cooled, spin-dried, and redissolved in dry acetonitrile. The mixture is added dropwise to a mixed system of an amino acid-Boc compound (compound Z), acetonitrile or N,N-dimethylformamide, and triethylamine at 0° C., and stirred at room temperature for more than 12 hours. The mixture is cooled, filtered, washed, dried, and subjected to column chromatography to obtain compound B. Compound C is obtained by removing Boc from compound B;

[0050] The molar ratio of the compound A to the amino acid-Boc compound (compound z) is 1:1-6.

[0051] 2) In an inert gas atmosphere, compound C was dissolved in an organic solvent, and then compound D (isothiocyanate of BDP or FITC) and 10% by mass of NaHCO were added. 3 , stirring at room temperature for more than 20 hours under the protection of inert gas, washing with water, drying, filtering and purification to obtain compound E, compound C and compound D are reacted in a molar ratio of 1:1.

[0052] 3) In an inert gas atmosphere, compound E is dissolved in an organic solvent, and then EDCI, Stir at room temperature for more than 3 hours under the protection of inert gas; remove most of the solvent, pour the residue into ether to precipitate solid, filter and dry to obtain compound F. Compound E and EDCI react at a molar ratio of 1:1-2.

[0053] The molar ratio of compound A to amino acid-Boc compound (compound Z) in step 1 is preferably 1:3.

[0054] The heating reflux described in step 1 is preferably refluxed for more than 8 hours.

[0055] The organic solvent in step 3 is selected from DMF, acetonitrile, tetrahydrofuran or a mixture thereof. The molar ratio of compound E to EDCI is more preferably 1:1.2.

[0056] In the following examples, unless otherwise specified, all reagents used can be prepared by conventional methods or purchased from commercial sources.

[0057] Example 1

[0058] Synthesis route of dual-color ratiometric pH fluorescent probe 4SR-L-BDP (F1):

[0059]

[0060] (1) Synthesis of intermediate C1

[0061] In a nitrogen atmosphere, A1 (1.23 g, 2.36 mmol) was dissolved in 25 ml of 1,2-dichloroethane. POCl was added dropwise under ice bath. 3 (0.362 g, 2.36 mmol), then heated to 90 ° C and refluxed for 24 h, cooled to room temperature, cooled, dried by rotation, redissolved in dry acetonitrile, and dripped into a mixed system of acetonitrile / triethylamine containing Boc-L-lysine (1.74 g, 7.08 mmol) at 0 ° C, and kept at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was poured into water, extracted with DCM 3 times, and the organic phase was washed 3 times with brine, dried over anhydrous sodium sulfate, dried by rotation, and column chromatography to obtain compound B 1 The obtained intermediate B1 was dissolved in 20 ml of a mixed solvent of TFA / DCM = 1:1 (v:v), stirred at room temperature, and monitored by TLC. After the reaction was completed, it was poured into water, extracted with DCM three times, and the organic phase was washed with brine three times, dried over anhydrous sodium sulfate, and spin-dried to obtain compound C1.

[0062] (2) Synthesis of intermediate E1

[0063] Under nitrogen atmosphere, compound C1 (0.551 g, 1 mmol) was dissolved in 15 mL of acetonitrile and 10% NaHCO 3 The mixture was stirred at room temperature for 20 h under the protection of inert gas, and the solvent was distilled off under reduced pressure from the reaction system. A small amount of methanol or DMF was added to dissolve the reaction product, which was precipitated in a large amount of ether and filtered. The filter cake was purified by column chromatography (methanol / dichloroethane system = 1 / 10) to obtain compound E1.

[0064] (3) Synthesis of intermediate F1

[0065] At room temperature, compound E1 (0.4 g, 0.429 mmol) was dissolved in 5 ml of anhydrous DMF solvent, and EDCI (0.08 g, 0.515 mmol) and sodium salt of N-hydroxysuccinimide sulfonate (0.13 g, 0.515 mmol) were added in sequence, and stirred at room temperature for more than 3 hours. Most of the solvent was removed by vacuum distillation of the reaction system, and the compound F1 was precipitated in a large amount of ether and filtered to obtain the compound F1. The mass spectrum and NMR data of F1 are shown in Figure 1-3 shown.

[0066] Example 2

[0067]

[0068] The synthesis method is as in Example 1. replace Synthesis of compound E2.

[0069] Example 3

[0070]

[0071] (1) Synthesis of intermediate C3

[0072] In a nitrogen atmosphere, A1 (1.23 g, 2.36 mmol) was dissolved in 25 ml of 1,2-dichloroethane. POCl was added dropwise under ice bath. 3 (0.362g, 2.36mmol), then heated to 90℃ and refluxed for 24h, cooled, spin-dried, redissolved in dry acetonitrile, and dripped into 10ml DMF / triethylamine (v:v=10:1) mixed system containing Boc-tryptophan (2.17g, 7.08mmol) at 0℃, and kept at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was poured into water, extracted with DCM 3 times, the organic phase was washed with brine 3 times, dried over anhydrous sodium sulfate, spin-dried, and column chromatography to obtain compound B3. B3 was added to 20ml TFA / DCM=1:1 (v:v) mixed solvent and stirred at room temperature for 24h. After the reaction was completed, the solvent was removed under reduced pressure to obtain the crude product of compound C3.

[0073] (2) Synthesis of intermediate E3

[0074] In a nitrogen atmosphere, compound C3 (0.612 g, 1 mmol) was dissolved in a mixed solvent of 15 mL of anhydrous DMF and triethylamine (v:v=10:1), and then compound D1 (0.382 g, 1 mmol) was added thereto, and the mixture was stirred uniformly at room temperature. After the reaction was completed, the solvent was removed by distillation under reduced pressure, a small amount of methanol or DMF was added to dissolve the reaction product, precipitated in a large amount of ether, filtered, and the filter cake was purified by column chromatography (methanol / dichloroethane system=1 / 10) to obtain compound E3.

[0075] Example 4

[0076]

[0077] The synthesis method is as in Example 3. replace Synthesis of compound E4.

[0078] Example 5

[0079]

[0080] (1) Synthesis of intermediate C5

[0081] In a nitrogen atmosphere, A1 (1.23 g, 2.36 mmol) was dissolved in 25 ml of 1,2-dichloroethane. POCl was added dropwise under ice bath. 3(0.362g, 2.36mmol), then heated to 90℃ and refluxed for 24h, cooled, spin-dried, redissolved in dry acetonitrile, dripped into 10ml acetonitrile / triethylamine (v:v=10:1) mixed system containing glycine (0.532g, 7.08mmol) at 0℃, left overnight at room temperature, and monitored the reaction by TLC. Poured into water, extracted with DCM 3 times, washed with brine 3 times, dried over anhydrous sodium sulfate, and spin-dried. The obtained intermediate and Boc-tyrosine (1.99g, 7.07mmol) were dissolved in 5ml anhydrous DMF solvent, and then EDCI (0.439g, 2.83mmol) and 4-dimethylaminopyridine (0.029g, 0.236mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was poured into water, extracted with DCM 3 times, washed with brine 3 times, dried over anhydrous sodium sulfate, spin-dried, and column chromatography was performed to obtain intermediate B5. B5 was added to 20 ml of a TFA / DCM=1:1 (v:v) mixed solvent and stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product of compound C5.

[0082] (2) Synthesis of intermediate E5

[0083] The synthesis method was as in step 2 of Example 1. Compound C5 (0.4 g, 0.622 mmol) was dissolved in 10 mL of acetonitrile and 10% NaHCO in a nitrogen atmosphere. 3 The mixture was stirred evenly and stirred at room temperature for more than 20 h under the protection of inert gas. The reaction system was distilled under reduced pressure to remove the solvent. A small amount of methanol or DMF was added to dissolve the reaction product, which was precipitated in a large amount of ether and filtered. The filter cake was purified by column chromatography (methanol / dichloroethane system = 1 / 10) to obtain compound E5.

[0084] Example 6

[0085]

[0086] The synthesis method is as in Example 5. replace Synthesis of compound E6.

[0087] Example 7

[0088]

[0089] The synthesis method is as in Example 5. replace Synthesis of compound E7.

[0090] Example 8

[0091]

[0092] (1) Synthesis of Compound F2

[0093] At room temperature, compound E2 (0.931 g, 1 mmol) was dissolved in 5 ml of anhydrous DMF solvent, and then EDCI (0.186 g, 1.2 mmol) and sodium salt of N-hydroxysuccinimide sulfonate (0.304 g, 1.2 mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the large solvent, precipitated in a large amount of ether, and filtered in a Buchner funnel to obtain compound F2. The product structure was identified by HRMS.

[0094] Example 8

[0095]

[0096] The synthesis method was similar to Example 7 to synthesize compound F3. The structure of the product was identified by HRMS.

[0097] Example 9

[0098]

[0099] The synthesis method was similar to Example 7 to synthesize compound F4. The structure of the product was identified by HRMS.

[0100] Example 10

[0101]

[0102] The synthesis method was similar to Example 7 to synthesize compound F5. The structure of the product was identified by HRMS.

[0103] Embodiment 11

[0104]

[0105] At room temperature, compound E6 (1.01 g, 1 mmol) was dissolved in 5 ml of anhydrous DMF solvent, and then EDCI (0.186 g, 1.2 mmol) and sodium salt of N-hydroxysuccinimide sulfonate (0.304 g, 1.2 mmol) were added in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the solvent, precipitated in a large amount of ether, and filtered in a Buchner funnel to obtain compound F6. The product structure was identified by HRMS.

[0106] Example 12

[0107]

[0108] The synthesis method was similar to Example 11 to synthesize compound F7. The structure of the product was identified by HRMS.

[0109] Embodiment 13

[0110]

[0111] (1) Synthesis of Compound F8

[0112] At room temperature, compound E7 (0.935 g, 1 mmol) was dissolved in 5 ml of anhydrous dichloromethane solvent, and then EDCI (0.186 g, 1.2 mmol) and N-hydroxysuccinimide (0.138 g, 1.2 mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the solvent, precipitated in a large amount of n-hexane, and filtered in a Buchner funnel to obtain compound F8. The product structure was identified by HRMS.

[0113] Embodiment 14

[0114]

[0115] The synthesis method was similar to Example 13 to synthesize compound F9. The structure of the product was identified by HRMS.

[0116] Embodiment 15

[0117]

[0118] The synthesis method was similar to Example 13 to synthesize compound F10. The structure of the product was identified by HRMS.

[0119] Example 16

[0120]

[0121] The synthesis method was similar to Example 13 to synthesize compound F11. The structure of the product was identified by HRMS.

[0122] Embodiment 17

[0123]

[0124] At room temperature, compound E6 (1.01 g, 1 mmol) was dissolved in 5 ml of anhydrous dichloromethane solvent, and then EDCI (0.186 g, 1.2 mmol) and N-hydroxysuccinimide (0.138 g, 1.2 mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the solvent, precipitated in a large amount of n-hexane, and filtered in a Buchner funnel to obtain compound F12. The product structure was identified by HRMS.

[0125] Embodiment 18

[0126]

[0127] The synthesis method was similar to Example 13 to synthesize compound F13. The structure of the product was identified by HRMS.

[0128] Embodiment 19

[0129]

[0130] (1) Synthesis of Compound E8

[0131] A1 (1.23 g, 2.36 mmol) was dissolved in 25 ml of 1,2-dichloroethane. POCl was added dropwise under ice bath. 3 (0.362g, 2.36mmol), then heated to 90℃ and refluxed for 24h, cooled to room temperature, cooled, spin-dried, redissolved in dry acetonitrile, and dripped into 10ml acetonitrile / triethylamine (v:v=10:1) mixed system containing Boc-L-lysine (1.74g, 7.08mmol) at 0℃, and kept at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was poured into water, extracted with DCM 3 times, the organic phase was washed with brine 3 times, dried with anhydrous sodium sulfate, spin-dried, and column chromatography to obtain compound B1. The obtained intermediate B1 was dissolved in 20ml TFA / DCM=1:1 (v:v) mixed solvent, stirred at room temperature, and monitored by TLC. After the reaction was completed, it was poured into water, extracted with DCM 3 times, the organic phase was washed with brine 3 times, dried with anhydrous sodium sulfate, and spin-dried to obtain compound C1. Under nitrogen atmosphere, C1 (0.552 g, 1 mmol) was dissolved in 15 mL of anhydrous acetonitrile and 10% NaHCO 3 The mixture was stirred evenly and heated under reflux for 24 h. After the reaction system was cooled to room temperature, the solvent was distilled off under reduced pressure, a small amount of methanol or DMF was added to dissolve the reaction product, and the product was precipitated in a large amount of ether, and filtered in a Buchner funnel to obtain a filter cake, which was purified by column chromatography (methanol / dichloroethane system = 1 / 10) to purify compound E8.

[0132] (2) Synthesis of Compound F14

[0133] At room temperature, compound E8 (0.88 g, 1 mmol) was dissolved in 5 ml of anhydrous dichloromethane solvent, and then EDCI (0.187 g, 1.2 mmol) and N-hydroxysuccinimide (0.139 g, 1.2 mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the solvent, precipitated in a large amount of n-hexane, and filtered in a Buchner funnel to obtain compound F14. The product structure was identified by HRMS.

[0134] Embodiment 20

[0135]

[0136] The synthesis method was similar to Example 19 to synthesize compound F15. The structure of the product was identified by HRMS.

[0137] Embodiment 21

[0138]

[0139] Synthesis of compound E10

[0140] A1 (1.23 g, 2.36 mmol) was dissolved in 25 ml of 1,2-dichloroethane. POCl was added dropwise under ice bath. 3 (0.362g, 2.36mmol), then heated to 90℃ and refluxed for 24h, cooled to room temperature, cooled, spin-dried, redissolved in dry acetonitrile, and dripped into 10ml acetonitrile / triethylamine (v:v=10:1) mixed system containing Boc-L-lysine (1.74g, 7.08mmol) at 0℃, and kept at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction solution was poured into water, extracted with DCM 3 times, the organic phase was washed with brine 3 times, dried with anhydrous sodium sulfate, spin-dried, and column chromatography to obtain compound B1. The obtained intermediate B1 was dissolved in 20ml TFA / DCM=1:1 (v:v) mixed solvent, stirred at room temperature, and monitored by TLC. After the reaction was completed, it was poured into water, extracted with DCM 3 times, the organic phase was washed with brine 3 times, dried with anhydrous sodium sulfate, and spin-dried to obtain compound C1. Under nitrogen atmosphere, C1 (0.552 g, 1 mmol) was dissolved in 15 mL of anhydrous acetonitrile and 10% NaHCO 3 The mixture was stirred evenly and heated under reflux for 24 h. After the reaction system was cooled to room temperature, the solvent was distilled off under reduced pressure, a small amount of methanol or DMF was added to dissolve the reaction product, and the product was precipitated in a large amount of ether, and filtered in a Buchner funnel to obtain a filter cake, which was purified by column chromatography (methanol / dichloroethane system = 1 / 10).

[0141] Synthesis of Compound F16 At room temperature, compound E10 (0.94 g, 1 mmol) was dissolved in 5 ml of anhydrous dichloromethane solvent, and then EDCI (0.187 g, 1.2 mmol) and N-hydroxysuccinimide (0.139 g, 1.2 mmol) were added thereto in sequence, and stirred at room temperature overnight. The reaction system was distilled under reduced pressure to remove the solvent, precipitated in a large amount of n-hexane, and filtered in a Buchner funnel to obtain compound F16. The product structure was identified by HRMS.

[0142] Embodiment 22

[0143]

[0144] The synthesis method was similar to that of Example 21, except that fluoroborylpyrrole was replaced with fluorescein isothiocyanate to synthesize compound F17. The product structure was identified by HRMS.

[0145] Embodiment 23

[0146]

[0147] The synthesis method was similar to Example 19 to synthesize compound F18. The structure of the product was identified by HRMS.

[0148] Embodiment 24

[0149]

[0150] The synthesis method was similar to Example 19 to synthesize compound F19. The structure of the product was identified by HRMS.

[0151] Embodiment 25

[0152] The multifunctional dye F1 was dissolved in DMSO to obtain a 5 mM solution. The solution was further diluted with deionized water containing 20% ​​ethanol to a 5 μM test solution. A dilute hydrochloric acid solution or sodium hydroxide solution was added to the test solution to adjust the test pH. The absorption spectrum was tested by an Agilent 8453 UV-visible spectrophotometer, and the fluorescence spectrum was tested by an Agilent 8453 spectrometer. Figure 4 The ultraviolet absorption and fluorescence emission spectra of the multifunctional dye F1 synthesized in Example 1 show that from pH 2.03 to 12.03, the maximum absorption peak of rhodamine at 566 nm gradually decreases, and the maximum absorption peak of BDP at 500 nm gradually increases; at the same time, due to the FRET effect, two emission peaks appear in the fluorescence spectrum at 488 nm excitation light, and there is a corresponding change trend.

[0153] Labeling function: 5 mM DMSO solution containing F1 was diluted with deionized water containing 20% ​​ethanol to a 5 μM solution. It was further incubated with 100 eq (molar ratio) of BSA aqueous solution at 37°C for 1 hour. The pH was adjusted by dilute hydrochloric acid solution or sodium hydroxide solution for fluorescence spectrum test, and the fluorescence spectrum was tested by Agilent 8453 spectrometer. Figure 5 This is the fluorescence emission spectrum of the multifunctional dye F1 synthesized in Example 1 after binding to BSA protein in vitro. It can be seen from the figure that after incubation of F1 with BSA, the pH ratio maintains a good linear change in the range of 4.0-8.0, which indicates that F1 still has stable and accurate pH indication properties after labeling.

[0154] The DMSO solution of F1 was incubated with a 1% BSA protein solution in PBS at 37°C. The proteins in the solution after the reaction were then analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the fluorescence of the gel was imaged using a UV converter with an ethidium bromide filter (GBOX-HR, SYNGENE). Figure 6 As shown, corresponding to the marker in the left figure, there is a corresponding fluorescence band at 66.43KD in the right figure, which matches the molecular weight of BSA protein. Coomassie brilliant blue staining confirmed that the fluorescence came from the F17-BSA conjugate (middle figure), which shows that the probe can be stably fixed on the protein.

[0155] pH cycle curve determination: F1 was dissolved in DMSO for testing:

[0156] Spectral test: F1 was dissolved in DMSO to obtain a 5 mM solution, and the solution was further diluted with deionized water containing 20% ​​ethanol to a 5 μM test solution. Dilute hydrochloric acid solution or sodium hydroxide solution was added to the above system to adjust the test pH to 4 or 8 for fluorescence spectrum under reciprocating cycles. The fluorescence spectrum was tested using an Agilent 8453 spectrometer. Figure 7 The pH cycle curves of the multifunctional dye F1 synthesized in Example 1 at pH = 4 and pH = 8. The ratio value of F1 remained stable after 6 acid-base cycles, which proved that F1 had good acid-base stability and could achieve accurate pH monitoring in a changing microenvironment.

[0157] 4T1 cell distribution: 5 mM DMSO solution containing F1 was diluted to 5 μM solution with DMEM complete medium. It was further mixed with 4T1 cells at 37°C and 5% CO. 2 Incubate for 30 min. Wash three times with PBS to remove free dye. Confocal imaging was performed using an Olympus FV1000 imaging system. The excitation wavelength was 488 nm, the acquisition channel one was 510-550 nm, and the acquisition channel two was 570-650 nm. Figure 8 This is a fluorescence imaging image of 4T1 cells stained with the multifunctional dye F17. As can be seen from the figure, after incubating F1 with cells, BDP fluorescence is distributed throughout the cytoplasm except for the nucleus, and 4SR fluorescence is only obvious in some acidic locations in the cell. This indicates that F1 may react with proteins in the cytoplasm and thus be distributed throughout the cytoplasm, rather than being affected by molecular charge or polarity and only tending to mitochondria or lysosomes.

[0158] Embodiment 26

[0159] Property test of multifunctional dye F17:

[0160] Spectral test: F17 was dissolved in DMSO to obtain a 5 mM solution. The solution was further diluted with deionized water containing 20% ​​ethanol to a 5 μM test solution. Dilute hydrochloric acid solution or sodium hydroxide solution was added to the test solution to adjust the test pH. The fluorescence spectrum was tested using an Agilent 8453 spectrometer. Fig. 9 This is the fluorescence emission spectrum of the multifunctional dye F17 synthesized in Example 22. As can be seen from the figure, as the pH value increases from 3.13 to 13.18, due to the FRET effect, two emission peaks appear in the fluorescence spectrum at 488nm excitation light, the maximum emission peak intensity of rhodamine at 580nm gradually decreases, and the maximum emission peak intensity of FITC at 520nm gradually increases.

[0161] pH indication function after protein labeling: 5mM DMSO solution containing F17 was diluted with deionized water containing 20% ​​ethanol to a 5μM solution. It was further incubated with 100eq (molar ratio) of BSA aqueous solution at 37°C for 1 hour. The pH was adjusted by dilute hydrochloric acid solution or sodium hydroxide solution for fluorescence spectrum test, and the fluorescence spectrum was tested by Agilent 8453 spectrometer. Fig.10 This is the fluorescence emission spectrum of the multifunctional dye F17 synthesized in Example 22 after binding to BSA protein in vitro. It can be seen from the figure that after incubation of F17 with BSA, the pH ratio maintains a good linear change in the range of 4.0-8.0, which indicates that F17 still has stable and accurate pH indication properties after labeling.

[0162] Embodiment 27

[0163] The properties of target compounds F13, F15, and F18 are given in the following table, and the test conditions are the same as Example 25.

[0164] Table 1 Properties of compounds F13, F15 and F18

[0165]

[0166] As shown in Table 1, compounds F13, F15, and F18 all have a sensitive response to pH and can bind to proteins without affecting the response performance to pH after binding to proteins.

Claims

1. A multifunctional fluorescent dye, characterized in that: It has the following structure: Wherein, R0 is independently selected from n=an integer of 0-18, m=an integer of 0-18, X - is an anion selected from BF4 - , Cl - Br - ,I - 、NO 3- 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - CF3SO3 - ; Said The total positive charge is equal to X - The total amount of negative charge; Y is selected from R1 is each independently selected from R2 are each independently selected from the following structures: Wherein, R3 is independently selected from H, methyl, R4 is independently selected from H, methyl or ethyl, R5 is independently selected from F, Cl, Br, I, and R6 is independently selected from H or Br.

2. A multifunctional fluorescent dye according to claim 1, characterized in that: The multifunctional fluorescent dye containing Y is selected from the following structures: 。 3. A multifunctional fluorescent dye according to claim 1 or 2, characterized in that: R2 are each independently selected from the following structures: Wherein, R5 is independently selected from F, Cl, Br, and I.

4. A multifunctional fluorescent dye according to claim 1 or 2, characterized in that: R2 are each independently selected from the following structures: 。 5. A multifunctional fluorescent dye according to claim 1 or 2, characterized in that: R2 are each independently selected from the following structures: 。 6. Use of the multifunctional fluorescent dye according to claim 1 in preparing FRET ratiometric pH fluorescent materials.

7. Use of a multifunctional fluorescent dye according to claim 1 in the preparation of protein tracking materials.

8. Use of the multifunctional fluorescent dye according to claim 1 in preparing protein fixed labeling materials.

9. Use of the multifunctional fluorescent dye according to claim 1 in preparing materials for studying the in situ dynamic tracking of proteins or the behavior and distribution of bacteria in living cells.

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