CN116535378A

CN116535378A - Rhodamine fluorescent dye and application thereof

Info

Publication number: CN116535378A
Application number: CN202210099035.XA
Authority: CN
Inventors: 陈知行; 刘天妍; 杨中天; 张源; 陈朋; 张昊霖
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2023-08-04

Abstract

The invention provides rhodamine fluorescent dye and application thereof. The rhodamine fluorescent dye comprises rhodamine dye molecules and a triplet quencher cyclooctatetraene which is covalently coupled with the rhodamine dye molecules. Because rhodamine can be applied to all living cells, the application range of the dye is widened; in addition, the space conformation of rhodamine enables the distance between the chromophore and the triplet quencher to be further shortened, so that phototoxicity can be reduced more effectively, and therefore, the rhodamine fluorescent dye which is formed by covalently coupling the triplet quencher cyclooctatetraene to rhodamine dye molecules can realize low phototoxicity imaging of various organelles and proteins at the living cell level.

Description

Rhodamine fluorescent dye and application thereof

Technical Field

The invention relates to the technical field of biological fluorescence imaging, in particular to rhodamine fluorescent dye and application thereof.

Background

With the continuous innovation of microscope technology, the biological fluorescence imaging technology is one of the important means for researching biological problems. Common optical microscopes, fluorescence microscopes, and confocal microscopes, have increasingly required space-time resolution for imaging and information contained therein. After the super-resolution imaging technology obtains the nobel chemical prize in 2014, a plurality of unknown structures and the interrelation thereof are revealed under the ultra-high resolution of the nanometer level, and the imaging field formally meets the super-resolution era. Development of super-resolution fluorescence imaging relies on co-optimization with hardware of the microscope and fluorescent dyes. For example: random optical reconstruction microscopy (Stochastic Optical Resolution Microscopy, STORM) requires the property that fluorescent dyes can be efficiently and repeatedly converted to light within a certain period of time; stimulated emission depletion fluorescence microscopy (Stimulated Emission Depletion Microscopy, STED) requires a beam of ultra-intense light to achieve resolution degradation, which clearly places higher demands on the light stability of the dye.

Limited heretofore by the development of instrumentation and dyes, much of the imaged data comes from immobilized biological samples. The future for super-resolution imaging must be long-term, large-field imaging at the living cell, tissue level. In both STORM and STED, repeated excitation of dye molecules or the use of ultra-intense light losses not only causes damage to the dye itself, but also fatal strikes to living biological samples. During the imaging process, the resulting damage to the living cell, tissue, etc. sample is referred to as phototoxicity. For example: in observing the dynamic activity of mitochondria for a long time, the mitochondria often become swollen and rounded with the extension of the observation time, and then rupture to induce apoptosis.

Phototoxicity is a common phenomenon in which triplet oxygen molecules, mainly derived from chromophore triplet excited state highly active species, and ground states in the environment, undergo energy transfer by collisions, thereby generating singlet oxygen and other reactive oxygen species (reactive oxygen species, ROS). These reactive oxygen species react with biological macromolecules such as lipids, proteins, DNA and disrupt their normal physiological structure and function. The problem of phototoxicity clearly places a completely new dimension on the fluorescent dyes. In addition to the requirement that the fluorescent dye have photostability against bleaching, it is also required to have low phototoxicity to the sample to match the biocompatibility of the living sample. The solution to reduce the phototoxicity of fluorescent dyes is therefore to reduce the triplet excited state component of fluorescence.

The triplet state of fluorescent dyes is a key factor leading to photobleaching and phototoxicity of the dye. Thus reducing the lifetime of the triplet state, pulling the dye in the triplet state back to the ground state as far as possible is the only solution strategy, currently mainly the following techniques:

1. and (3) deoxidizing the system: in a single-molecule system, in order to increase the photostability of dye molecules, an oxygen removal mode is generally adopted to inhibit the occurrence of high-activity triplet excited dye molecules and oxygen.

2. Triplet quenchers (Triplet State Quenchers, TSQs) were added to the imaging system: triplet quenchers undergo photophysical or photochemical quenching by collisions with dye molecules in solvents, the fluorophore being protected in an intermolecular form. Among the photophysical triplet quenchers are Cyclooctatetraene (COT), diphenylhexatriene, or nickel ions, all of which are photoprotective by energy transfer between the dye and the quencher. Photochemical triplet quenchers require a reagent that has both oxidizing and reducing capabilities. For example: trolox (TX), ascorbic acid, ferrocene, p-nitrobenzyl alcohol, nitrophenylalanine, methyl viologen. The triplet state of the dye is quenched by a mechanism of photo-induced electron transfer (PET) to form a radical anion or cation intermediate, which is then restored to the ground state of the dye through successive redox reactions.

3. Self-healing dye: the prior art is to covalently connect a triplet quencher Cyclooctatetraene (COT) in the molecule of a cyanine dye (mainly Cy5 and Cy 3), wherein the triplet state of the dye and the triplet quencher are quenched in the molecule, so that the energy transfer of the triplet state and the triplet state is reduced, and the self-repairing capability of the dye molecule is realized, and the dye is called a self-repairing dye. Under deoxygenated conditions, the triplet lifetime of the Cy5-COT conjugate was around 0.15 μs, whereas the triplet lifetime of Cy5 was about 110 μs, and the photostability of Cy5-COT under aerobic conditions was improved by around 5-fold over Cy 5. Two COT molecules are symmetrically connected at two ends of Cy3 and Cy5, and the obtained molecules respectively reduce phototoxicity by 3 times and 5 times.

However, the above oxygen scavenging system is not suitable for living cell imaging, and in the method of adding a triplet quencher in the imaging system, the quenching efficiency depends on intermolecular collisions, and thus a higher concentration of triplet quencher is required in the system. Such high concentrations can be cytotoxic and poorly biocompatible. Self-healing dyes also suffer from certain drawbacks such as: (1) lack of universality: COT is covalently linked to cyanine dyes of different parent nuclei, which have a greatly different degree of triplet quenching of the dye molecules, with little apparent improvement. (2) limitations of living cell dyes: the cyanine dye naturally has a positive charge, can accumulate on the inner mitochondrial membrane rich in negative charge, and is not suitable for imaging other organelles, proteins and DNA.

Thus, there remains a need to provide new solutions for reducing the phototoxicity of fluorescent dyes.

Disclosure of Invention

The main purpose of the invention is to provide a rhodamine fluorescent dye and application thereof, so as to provide a new scheme for reducing phototoxicity of the fluorescent dye in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a rhodamine-based fluorescent dye including a rhodamine-based dye molecule and a triplet quencher cyclooctatetraene covalently coupled to the rhodamine-based dye molecule.

Further, rhodamine dye molecules are covalently coupled with cyclooctatetraene through a base ring; preferably, the covalent coupling is with cyclooctatetraene via ortho positions on the bottom ring.

Further, the rhodamine dye molecule is selected from any one of the following: oxyrhodamine, carborhodamine or silarhodamine; preferably, the oxyrhodamine class includes any one or more of: TMR, rho110, map555, JF503, JF525, JF536, JF549, JF522, JF571, 500R, 510R, or 515R; preferably, the carborhodamine class includes any one or more of: 6-CPY, 5-CPY, CRhp, 580CP, JF585, JF612, JF608 or Map618; preferably, the silicon rhodamine class includes any one or more of: siR, JF626, JF629, JF630, JF635, JF646, JF669 or SiR.

Further, the singlet oxygen yield of the rhodamine fluorescent dye is 0.0014 to 0.018.

According to a second aspect of the present application there is provided a fluorescence detection kit comprising any of the rhodamine fluorescent dyes described above.

According to a third aspect of the present application, there is provided a biomarker molecule comprising a biomolecule and a fluorescent dye for labelling the biomolecule, wherein the fluorescent dye of the biomolecule is any of the rhodamine fluorescent dyes described above.

According to a fourth aspect of the present application there is provided a method of bioluminescence imaging using any of the aforementioned rhodamine fluorescent dyes to label cells.

Further, the cells are living cells; preferably, the rhodamine-type fluorescent dye-labeled cells are labeled intracellular organelles, wherein the organelles include any one or more of the following: mitochondria, nuclei, cytoskeleton, golgi, endoplasmic reticulum, lysosomes, endosomes, ribosomes, or migratory bodies; preferably, the rhodamine fluorescent dye marks the cell to be a molecular marker in the cell, and the molecular marker is a protein molecule, a sugar molecule or a nucleic acid molecule; preferably, the bioluminescence imaging method further comprises placing the rhodamine fluorescent dye-labeled cells under a microscope for imaging observation, the microscope comprising any one or more of the following: confocal microscopy, random optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.

According to a fifth aspect of the present application there is provided the use of any one of the rhodamine fluorescent dyes, or kits, or bioluminescence imaging methods, as hereinbefore described, in live cell imaging studies.

Further, imaging is under any one or more of the following microscopes: confocal microscopy, random optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.

By applying the technical scheme of the invention, on one hand, because rhodamine can be applied to all living cells, the application range of the dye is widened; on the other hand, the space conformation of rhodamine enables the distance between the chromophore and the triplet quencher to be further shortened, so that phototoxicity can be reduced more effectively, and therefore, the rhodamine fluorescent dye which is formed by covalently coupling the triplet quencher cyclooctatetraene to rhodamine dye molecules can realize low phototoxicity imaging of various organelles and proteins at the living cell level.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIGS. 1a to 1c show the chemical structural formulae of (a) TMR-TSQs, respectively; (b) Under the irradiation of an LED lamp with the temperature of 520+/-10 nm and 50mW/cm < 2 >, the absorption decay curve of DPBF (hydrogen peroxide solution) with time has the slope reflecting the yield of singlet oxygen corresponding to each compound; (c) an absolute singlet oxygen yield table for TMR-TSQs.

FIG. 2 shows a comparison of phototoxicity of TMR-TSQs to Hela cells.

FIGS. 3a to 3f show the chemical structural formulae of (a) Rho110-COT, rho123, siR-COOMe, siR-COT, respectively; (b) Photo toxicity profile of Rho110-COT and Rho123 on HeLa cells; (c) Photo toxicity contrast plots of SiR-COOMe and SiR-COT on Hela cells; (d) Singlet oxygen yield plot of Rho110-COT and Rho123 at 520+ -10 nm,50mW/cm2 LED lamp irradiation; (e) Singlet oxygen yield graphs of SiR-COOMe and SiR-COT under the irradiation of an LED lamp of 620+/-10 nm and 5mW/cm < 2 >, wherein MB represents methylene blue and is taken as a quantitative reference; (f) Absolute singlet oxygen yields of the respective compounds were obtained with reference to known singlet oxygen yields of Rho123 and MB, respectively.

Figures 4a to 4c show that the fluorescent dyes of the present application show low phototoxicity as well when they label protein molecules by coupling to protein tags, respectively. Wherein FIG. 4a shows the stable transgenic cell line U2OS of Map555-COT-Halo and the reported compound MaP-Halo-tagged stably expressing H2B-Halo; FIG. 4b shows that under the same light conditions, there is a small amount of punctiform aggregation of XRCC1 in the Map555-COT-Halo and MaP-Halo labeled nuclear region DNA damage, while there is a significant punctiform aggregation distribution of XRCC1 in the MaP-555-Halo labeled nuclear region; FIG. 4c is a quantitative analysis of FIG. 4b showing that the aggregation of XRCC1 (increase in aggregation number by about 5-fold) was significantly less in the Map555-COT-Halo labeled experimental group than in the MaP555-Halo (increase in aggregation number by about 20-fold) labeled control group.

FIG. 5 shows that the fluorescent dye MaP-555-COT-DNA of the present application also exhibits low phototoxicity when labeling nucleic acid molecules.

In FIGS. 4 and 5, maP-COT-Halo means Map555-COT-Halo, and MaP-COT-DNA means MaP555-COT-DNA.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

As mentioned in the background art, the existing schemes for reducing phototoxicity of fluorescent dyes have the problems of limited application range or poor biocompatibility, and the like, and in order to improve the situation, the present application provides a new improvement idea, which is specifically described in detail below.

In light of the above-described deficiencies in the prior art, the present application applies the method of COT coupling to rhodamine-based dyes to achieve low phototoxicity imaging of various organelles and proteins at the living cell level. Among them, rhodamine dyes were chosen as the precursors of the study for the following two main reasons:

first, rhodamine dyes are popular tools for live cell imaging. Most of the fluorescent probes reported to date to be suitable for use in living cell microscopes are rhodamine-based derivatives. The key point is that rhodamine and derivatives exist in dynamic balance of non-fluorescent spirolactone form and fluorescent zwitterionic form. At first sight, the presence of this dynamic balance appears to be disadvantageous, as it reduces the brightness of the fluorophore. However, hydrophobic, non-fluorescent spirolactone structures have higher cell permeability than zwitterionic structures, and binding of probes to their targets will typically shift the equilibrium towards fluorescent zwitterions, rendering such probes fluorescent, effectively reducing the imaging background signal.

Second, the steric conformation of rhodamine enables it to further pull the chromophore closer to the triplet quencher. According to the previous study structure, the rate of triplet-triplet energy transfer increases with decreasing distance of triplet quencher from fluorescent dye. Due to the planar lamellar structure of the cyanine dye, the COT is far from the chromophore of the cyanine dye. The base ring of rhodamine can rotate freely, the space distance between the base ring substituent and the conjugated chromophore is shortened by the torsion angle, and the space positions of the triplet quencher group and the chromophore are changed by the substitution of different positions of the base ring of rhodamine.

Thus, in this application, first, taking N, N tetramethyl rhodamine (TMR) as an example, COT is coupled in the form of an ester bond at the 3,4,5 positions of the bottom ring of TMR, respectively, to give the compounds o-TMR-COT, m-TMR-COT and p-TMR-COT. In order to compare the improvement effect of different triplet quenchers on phototoxicity of rhodamine dyes, parallel comparisons were also made for o-TMR-NB (FIG. 1 a) synthesized in this application. As a result, it was found that the compounds o-TMR-COT, m-TMR-COT, p-TMR-COT all exhibited lower singlet oxygen yields than the commercially available mitochondrial dye tetramethyl rhodamine methyl ester (TMRM), tetramethyl rhodamine ethyl ester (TMRE). And the COT is connected to the No. 3 carbon atom of rhodamine, so that the singlet oxygen yield is reduced by about 6 times compared with TMRM.

Further, it was found by cell experiments that TMRM decreased to 50% in HeLa survival at about 2 minutes and decreased to 50% in HeLa survival at about 10 minutes of o-TMR-COT. Consistent with the in vitro singlet oxygen yield results, the COT is connected to the rhodamine bottom ring 3 carbon, and the phototoxicity can be reduced by 5-6 times.

To further broaden the spectrum of low phototoxicity fluorescent dyes, the application of the COT coupling strategy to other rhodamine dyes in this application was also found to reduce phototoxicity.

Based on the above results, the applicant has proposed a series of solutions for the present application. In one exemplary embodiment, a rhodamine-based fluorescent dye is provided that includes a rhodamine-based dye molecule and a triplet quencher cyclooctatetraene covalently coupled to the rhodamine-based dye molecule.

Because rhodamine can be applied to all living cells, the application range of the dye is widened. In addition, the spatial conformation of rhodamine enables further closer distance of chromophore and triplet quencher, thus enabling more effective reduction of phototoxicity, and thus, the rhodamine-based fluorescent dye covalently coupling the triplet quencher cyclooctatetraene to rhodamine-based dye molecule of the present application enables low phototoxicity imaging of various organelles and proteins at living cell level.

Rhodamine dye molecules such as oxyrhodamine (e.g., TMR, rho110, map555, JF503, JF525, JF536, JF549, JF522, JF571, 500R, 510R, 515R, etc.), carborhodamine (e.g., 6-CPY, 5-CPY, CRhp, 580CP, JF585, JF612, JF608, map618, etc.), silicon rhodamine (e.g., siR, JF626, JF629, JF630, JF635, JF646, JF669, siRhp, etc.), and having a base ring (i.e., a benzene ring with a carboxylic acid group or carboxylate group attached) that is free to rotate, the twist angle being capable of pulling the base ring substituent closer to the spatial distance of the conjugated chromophore. According to the previous study results: the rate of triplet-triplet energy transfer increases with decreasing distance of the triplet quencher from the fluorescent dye, and thus the shortening of the distance contributes to the improvement of the oxidative quenching effect, and more contributes to the reduction of phototoxicity. And substitution of the base ring of rhodamine in different positions changes the spatial position of the triplet quencher group to the chromophore. Thus, in one preferred embodiment of the present application, the coupling is covalent with cyclooctatetraene via the bottom ring; more preferably, the cyclooctatetraene is covalently coupled through an ortho position on the bottom ring. Experiments prove that the cyclooctatetraene is covalently coupled to the ortho position on the bottom ring, and has more excellent low phototoxicity performance than para position and meta position.

(Oxorrhodamine: rho110 as an example)

(silicon rhodamine: siR as an example)

(carborhodamine: map618 is taken as an example)

In a preferred embodiment, the rhodamine dye molecule is selected from any one of the following: oxyrhodamine (TMR, rho110, map555, JF503, JF525, JF536, JF549, JF522, JF571, 500R, 510R, 515R, etc.), carborhodamine (6-CPY, 5-CPY, CRhp, 580CP, JF585, JF612, JF608, map618, etc.) or silarhodamine (SiR, JF626, JF629, JF630, JF635, JF646, JF669, siRhp, etc.)

The singlet oxygen yield of the rhodamine fluorescent dye provided by the application is lower than that of the existing self-repairing dye, and is in the range of 0.0014-0.018. It is possible to detect the properties of the rhodamine fluorescent dye of the present application by this index.

In a second exemplary embodiment of the present application, a biomarker molecule is provided that is a biomolecule with any of the rhodamine-based fluorescent dyes described above. Such biomarker molecules are capable of fluorescent imaging under a microscope of relatively long duration and/or high light intensity. The specific biomolecules may be macromolecules such as proteins or nucleic acids. The distribution of such biomolecules on cells may be in cellular membrane, cytoplasm, nucleus or organelle of mitochondria.

In a third exemplary embodiment of the present application, a fluorescence detection kit is provided, the kit comprising any of the rhodamine-based fluorescent dyes described above. The dye of the kit is adopted for fluorescence labeling imaging, and can realize fluorescence imaging of various cell sub-microstructures on the living cell level.

In a fourth exemplary embodiment of the present application, a method of bioluminescence imaging is provided, the method employing any of the rhodamine-based fluorochromes described above to label cells. The rhodamine fluorescent dye is adopted to mark cells for fluorescence imaging, so that the fluorescence imaging of various cell sub-microstructures on the living cell level can be realized.

Thus, preferably, the cells are living cells; more preferably, rhodamine-based fluorescent dyes of the present application label intracellular organelles, wherein the organelles include, but are not limited to, any one or more of the following: mitochondria, nuclei, cytoskeleton, golgi, endoplasmic reticulum, lysosomes, inclusion bodies, ribosomes, or migration bodies, and the like. In other preferred embodiments, the rhodamine fluorescent dye labels a molecular marker on the cells, and the specific molecular marker is a protein molecule, a sugar molecule, or a nucleic acid molecule.

It should be noted that, when the rhodamine fluorescent dye with low phototoxicity is used for labeling organelles, the specific labeling principle is based on the following steps: the microenvironment of different organelles, such as different pH values, the charged properties and the hydrophilicity and hydrophobicity are different. For example, the pH of lysosomes, early inclusion bodies, late inclusion bodies is between 4.0-5.5, while the organelle environment except for the above is around ph=7.4; the inner and outer mitochondrial membranes have a potential difference of negative inside and positive outside; endoplasmic reticulum, golgi apparatus, lipid droplets have a relatively hydrophobic environment. The specific marking mode is as follows: by further coupling groups specific to each organelle on rhodamine fluorescent dye, the dye can specifically label one or more organelles. The following is an exemplary illustration of mitochondria, lysosomes, and endoplasmic reticulum: rhodamine molecules with positive charges can target the inner mitochondrial membrane to realize mitochondrial localization; the rhodamine dye is coupled with the glibenclamide drug molecule, so that the endoplasmic reticulum positioning can be realized by combining with a sulfonylurea receptor on the endoplasmic reticulum; rhodamine molecules are added with a few weak alkaline amines, so that the molecules can be gathered in a low-pH small chamber to realize the positioning of the acidic cell organelles, and the specific positioning of different acidic cell organelles can be realized by adjusting the alkalinity of different amines.

In the case of labeling molecular markers, the rhodamine fluorescent dye is coupled with corresponding labeling molecules to realize the labeling. For example, when a protein molecule is labeled, the protein may be labeled by coupling to a tag carried on the protein. When labeling nucleic acid molecules, labeling is achieved by coupling small molecules that specifically intercalate into the strands of the DNA molecule.

Because rhodamine fluorescent dyes have low phototoxicity, the rhodamine fluorescent dyes are not only suitable for conventional fluorescent microscopes, such as common optical microscopes or laser confocal microscopes, but also have more advantages in application to super-resolution imaging microscopes, such as random optical reconstruction microscopes or stimulated emission loss fluorescent microscopes.

In a fifth exemplary embodiment of the present application, there is provided the use of any one of the rhodamine fluorescent dyes, or the kit, or any one of the bioluminescence imaging methods, as described above, in live cell imaging studies. Preferably, the imaging is under any one or more of the following microscopes: confocal microscopy, random optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.

The beneficial effects of the present application will be further described below in connection with specific examples.

Example 1

Alcohol derivatives of Cyclooctatetraene (COT) are synthesized first by organic synthesis means for subsequent coupling with rhodamine dyes. The following abbreviations for the compounds or procedures used in the synthetic procedure and their corresponding full names are respectively as follows:

CDI N, N' -carbonyldiimidazole

DMF N, N-dimethylformamide

THF tetrahydrofuran

COT cyclooctatetraene

DCM dichloromethane

MeOH methanol

PE Petroleum ether

EA ethyl acetate

MgSO ₄ Magnesium sulfate

Na ₂ SO ₄ Sodium sulfate

TsOH para-toluene sulfonic acid

n-BuLi n-butyllithium

HATU 2- (7-azabenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate

TEA triethylamine

DDQ 2, 3-dichloro-5, 6-dicyano-p-benzoquinone

EDCI. HCl 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride

HOBT 1-hydroxybenzotriazoles

HPLC high pressure liquid chromatography

CuBr ₂ Copper bromide

RT room temperature

Mass Mass

¹ H NMR hydrogen Spectroscopy

Chloroform-d deuterated Chloroform.

1.1 Synthesis of alcohol derivatives of Cyclooctatetraene (COT):

35.38mg of 221mmol of liquid bromine are dissolved in 150ml of DCM and, at-78℃a solution of commercially available compound 1 (23 g,221 mmol) in DCM is slowly added and after stirring for 1 hour, 300ml of 30mmol of potassium tert-butoxide are added dropwise. The reaction mixture was stirred at-60℃for 4 hoursAnd then heating to-10 ℃ and dripping into ice water. With a small amount of MgSO ₄ The emulsion phase was cleaved, the organic phase removed and the aqueous phase extracted with diethyl ether (3 x 200 ml). The extracted aqueous phase was subjected to MgSO ₄ Drying, filtration and concentration gave a yellow oily substance (40 g, 99% yield, i.e. compound 2) which was used in the next step without further purification.

¹ H NMR(400MHz,Chloroform-d):δ6.21(s,1H),5.77-5.92(m,5H),5.62(s,1H).

40g of Compound 2 was dissolved in 50mL of ultra-dry tetrahydrofuran and added drop-wise to previously ground magnesium strips (15.7 g, 65mmol) under ice bath and argon protection. After stirring the reaction mixture at room temperature for 3 hours until a dark blue-green solution appears. The resulting solution was cooled to-78 ℃ and then excess solid dry ice was added to produce the carboxylate derivative of COT. The reaction was then quenched with 250ml of aqueous solution and acidified to ph=2 with 1M hydrochloric acid solution. Extraction with dichloromethane and saturated brine, combined organic phases, dried over MgSO4, filtered with suction, concentrated, and column-chromatographed on silica gel (petroleum ether: ethyl acetate=4:1) to give compound 3 (17.2 g, 53% yield) as a yellow oil.

¹ H NMR(400MHz,Chloroform-d)7.16(s,1H),6.06–5.96(m,1H),5.93(d,J＝10.9Hz,3H),5.90–5.84(m,1H),5.81(s,1H).

400mg,2.70mmol of Compound 3 are dissolved in 5ml MeOH solution and added dropwise to the sulfonyl chloride at room temperature. After the completion of the dropwise addition, the reaction mixture was warmed to 60℃and stirred for 5 hours, followed by spin-drying to give 440mg of the crude product compound 4 for the next reaction.

440mg,2.7mmol of Compound 4 are dissolved in 10ml of THF and added to 150mg,4.05mmol of lithium aluminum hydride under ice-bath, nitrogen protection. After stirring for 30 minutes, the mixture was warmed to room temperature and stirred for 2 hours. 150ul of water, 15% NaOH solution, and 450ul of water were added in this order and stirring was continued for 5 hours. Filtration, washing of the filter cake with 20Ml of ethyl acetate, concentration, and silica gel column chromatography (petroleum ether: ethyl acetate=20:1) gave a colorless oily substance 5 (260 mg, yield 72%).

¹ H NMR(400MHz,Chloroform-d)δ5.81～5.94(s,7H),4.04(d,J＝5.6Hz,2H).

Preparation of COT sulfonamides

To a glass flask dried by a hot air gun was added a magnetic stirrer and an activated magnesium ribbon of 0.4g (6 eq), and a solution of bromocyclooctatetraene (compound 2) in 500mg (1 eq.) (prepared by the prior art method, nat. Methods 2012,9,68.) in 2mL of anhydrous THF was added under nitrogen, and after stirring at room temperature for 10-15min, the system color was darkened to dark green, stirring was continued for 4h to complete the Grignard reagent, followed by cooling to-78 ℃. To another nitrogen-protected dry round bottom flask was added SO ₂ Cl ₂ Is cooled to-78 ℃ in Dichloromethane (DCM) (1-2M). The above-mentioned Grignard reagent in THF solution is sucked up by syringe, and the above-mentioned SO is slowly added dropwise at-78 deg.C ₂ Cl ₂ The solution was stirred at-78℃for 15min after the completion of the dropwise addition. The system is restored to room temperature, and the yellow gummy crude COT sulfonyl chloride is obtained after vacuum drying at room temperature. The product was used directly for subsequent conversion without further purification. 5mL of DCM was added to prepare 5mL of a solution of the above crude sulfonyl chloride and 5mL of ammonia in THF (1.5M), each cooled to-78deg.C, and mixed and stirred at this temperature for 1h. The solvent and excess ammonia were removed by rotary evaporation and column chromatography on silica gel (petroleum ether: ethyl acetate=5:1-3:1) gave 130mg of COT sulfonamide as a brown oil in 25% yield (as COT-Br).

¹ H NMR(400MHz,Methanol-d ₄ )δ6.78(d,J＝3.6Hz,1H),6.16(dd,J＝11.3,3.5Hz,1H),6.04(d,J＝11.4Hz,1H),5.90(m,4H).

1.2 Synthesis of Compound o-TMR-COT

80mg,0.21mmol of Compound 6 in 3.0ml of DMF are taken and 118mg,0.310mmol of HATU,42.5mg,0.42mmol of EA and 41.6mg,0.310mmol of Compound 5 are added in succession. After stirring at room temperature for 24 hours, concentration and column chromatography on silica gel eluting with DCM: meOH=20:1 as developing solvent gave 36mg of the product. It was redissolved in 2.5ml DCM, slowly added dropwise to 25ml MTBE for recrystallization, filtered off with suction and concentrated to give 20mg of a red solid (yield: 19%).

¹ H NMR(400MHz,Chloroform-d)δ8.27(d,J＝7.6Hz,1H),7.81(t,J＝7.5Hz,1H),7.74(t,J＝7.5Hz,1H),7.35(d,J＝7.7Hz,1H),7.09(d,J＝10.0Hz,2H),6.87(m,4H),5.74(m,7H),4.39(s,2H),3.31(s,12H).

1.3 Synthesis of Compound o-TMR-NB

100mg,0.258mmol of Compound 6 in 5.0ml of DMF, 147mg,0.356mmol of HATU,78.3mg,0.774mmol of TEA and 51.4mg,0.356mmol of commercially available Compound 7 were taken in this order and stirred at room temperature for 18 hours. Concentration, column chromatography on silica gel eluting with DCM: meOH=50:1 as a developing solvent gave 40mg of a red solid (yield: 41%)

¹ H NMR(400MHz,DMSO-d6)δ8.31(dd,J＝7.7,1.5Hz,1H),8.06–8.01(m,2H),7.92(td,J＝7.5,1.5Hz,1H),7.87(td,J＝7.6,1.5Hz,1H),7.49(dd,J＝7.4,1.4Hz,1H),7.25–7.19(m,2H),7.04(dd,J＝9.5,2.4Hz,2H),6.97(d,J＝9.4Hz,2H),6.80(d,J＝2.3Hz,2H),5.08(s,2H),3.26(s,12H).

1.4 Synthesis of Compound m-TMR-COT

685mg 5.00mmol of Compound 8, 375mg2.5mmol of Compound 9, 76mg,0.441mmol of TsOH were dissolved in 15ml of propionic acid solution, respectively, and stirred at 65℃for 16 hours. The reaction mixture was cooled to room temperature and added dropwise to 100ml of 3mol/L sodium acetate solution, extracted with DCM, and the combined organic phases were washed with saturated brine, na ₂ SO4 is dried, filtered and concentrated to give a residue. The residue was dissolved in 50ml MeOH, dcm=3: 2 solution then add 295mg,1.20mmol of DDQ, stir at room temperature for 2 hours, then concentrate in vacuo to give the residue which is chromatographed on silica gel (DCM: meoh=50:1) to give a red solid (compound 10, 100mg, 10% yield).

The coupling reaction of the compound 10 and the compound 5 is carried out in the same synthesis step as the compound o-TMR-COT to obtain the compound m-TMR-COT.

¹ H NMR(300MHz,Acetonitrile-d ₃ )δ8.27(d,J＝7.9Hz,1H),8.01(s,1H),7.79(t,J＝7.7Hz,1H),7.66(d,J＝7.2Hz,1H),7.25(d,J＝9.5Hz,2H),6.94(dd,J＝9.5,2.4Hz,2H),6.79(d,J＝2.4Hz,2H),6.06–5.68(m,7H),4.75(s,2H),3.24(s,12H).

1.5 Synthesis of the Compound p-TMR-COT

Similar to the compound m-TMR-COT, no further description is given.

¹ H NMR(300MHz,Chloroform-d)δ8.27(d,J＝7.9Hz,1H),7.48(d,J＝8.2Hz,1H),7.33–7.19(m,1H),6.92(dd,J＝9.4,2.2Hz,1H),6.84(d,J＝2.3Hz,1H),5.92(m,,7H),4.81(s,2H),3.32(s,12H).

1.6 Synthesis of the Compound Rho110-COT

100mg,0.272mmol of Compound 13, which is commercially available, are dissolved in 5.0ml of DMF, 67mg,0.354mmol,47.9mg,0.354mmol of EDCI. HCl,47.9mg,0.354mmol of HOBT,110mg,1.09mmol of TEA are added in succession, after stirring for 15 minutes at RT, 43.8mg,0.327mmol of Compound 5 are added, stirring is continued for 24 hours, and after vacuum concentration, purification by carbooctadeca reverse HPLC (0.5% HCl) gives a red solid (8.0 mg, yield: 7%)

1.7 Synthesis of the Compounds SiR-COOMe and SiR-COT

1.7.1 Synthesis of Compound 16

2.0g,10.0mmol of compound 14 are dissolved in 30ml of THF and added dropwise to 6.9ml,11.0mmol of n-BuLi at-78℃under nitrogen, stirring is carried out for 1 hour, then 640 mg,5.0mmol of compound 15 are added and stirring is continued for ten minutes. The reaction was slowly returned to room temperature and stirring was continued for 2 hours. 10ml of water was added to the reaction system, followed by extraction with 3 times 30ml of EA, and the mixed organic phase was dried, suction-filtered, and concentrated in vacuo, followed by silica gel column chromatography (PE: EA=20:1 to 10:1 as eluent) to give a colorless oily substance (460 mg, yield: 31%)

1.7.2 Synthesis of Compound 18

Compound 16 (50 mg, 0.67 mmol), compound 17 (75 mg,0.501 mmol), and CuBr ₂ (3.5 mg,0.017 mmol) was added to the autoclave and reacted at 120℃for 6 hours under the protection of argon. Slowly cooled to room temperature and purified by silica gel column chromatography (DCM: meoh=30:1) to give a blue solid (12 mg, yield: 17%)

1.7.3 Synthesis of Compound 19

10mg,0.023mmol of compound 18 was dissolved in 2.0ml of overdry DCM and oxalyl chloride (148 mg,1.17 mmol) DMF (1.0 uL) was added and stirred at room temperature for 8 hours. The residue obtained by concentration in vacuo was redissolved in 2.0ml of ultra-dry DCM and used without purification in the next step.

1.7.4 Synthesis of Compound SIR-COOMe

1.0ml of compound 19 (5.5 mg,0.012 mmol) of the crude product of the previous step was taken under ice bath and nitrogen protection, and 1.0ml of MeOH was added. After the addition of the reactants was completed, the reaction system was slowly returned to room temperature, stirred for 30 minutes and concentrated in vacuo to give a residual solid. The product was isolated by silica gel column chromatography (DCM: meoh=10:1) to give a blue solid (1.8 mg, yield: 34%).

Mass:[M] ⁺ ＝443.25。

¹ H NMR(400MHz,Methanol-d4)δ8.25(dd,J＝7.8,1.5Hz,1H),7.78(td,J＝7.5,1.4Hz,1H),7.71(td,J＝7.7,1.4Hz,1H),7.34(d,J＝2.8Hz,2H),7.30(dd,J＝7.5,1.4Hz,2H),6.97(d,J＝9.6Hz,2H),6.73(dd,J＝9.6,2.8Hz,2H),3.62(s,3H),3.33(s,12H)。

Synthesis of 1.7.5 Compound SIR-COT

1.0ml of compound 19 (5.5 mg,0.012 mmol) of the crude product of the previous step was taken under ice bath and nitrogen protection, and compound 7 (1.90 mg,0.014 mmol) was added. After the addition of the reactants was completed, the reaction system was slowly returned to room temperature, stirred for 3 hours, and then concentrated in vacuo to give a residual solid. The product was isolated by silica gel column chromatography (DCM: meoh=10:1) to give a blue solid (1.2 mg, yield: 18%).

Mass:[M] ⁺ ＝545.12。

¹ H NMR(400MHz,Acetonitrile-d3)δ7.97(dd,J＝7.8,1.4Hz,1H),7.53(td,J＝7.5,1.5Hz,1H),7.47(td,J＝7.6,1.4Hz,1H),7.07(d,J＝1.3Hz,1H),7.03(d,J＝2.8Hz,2H),6.70(d,J＝9.6Hz,2H),6.41(dd,J＝9.7,2.8Hz,2H),5.63-5.24(m,7H),4.17(s,2H),3.03(s,12H),0.36(s,6H).

1.8 Synthesis of Compound MaP555-COT-Halo

25mg of compound 20 was dissolved in 1mL of DMF, and 16. Mu.L of triethylamine (2 eq) and 16mg of potassium carbonate (2 eq) were added. The reaction was cooled to 0℃and 7.5. Mu.L of allyl bromide (1.5 eq) was added, the reaction system was warmed to room temperature and reacted for 2 hours, and the reaction was terminated. After washing the reaction with water and extraction with DCM, the solvent was dried and chromatographed on silica gel (DCM: meoh=10:1) to finally give 25mg of the target product in 90% yield.

¹ H NMR(400MHz,Chloroform-d)δ8.28(dd,J＝8.0,1.4Hz,1H),8.11(d,J＝7.8Hz,1H),7.84(d,J＝1.4Hz,1H),6.65(d,J＝8.9Hz,2H),6.53(d,J＝2.5Hz,2H),6.45(dd,J＝8.9,2.5Hz,2H),5.98(ddt,J＝16.6,10.4,5.9Hz,1H),5.36(dq,J＝17.2,1.5Hz,1H),5.27(dq,J＝10.4,1.3Hz,1H),4.78(dt,J＝6.0,1.3Hz,2H),3.03(s,12H).

25mg of compound 21 obtained in the previous step was dissolved in 5mL of DCM, 100. Mu.L of phosphorus oxychloride (20 eq) was added, refluxed for 4 hours at 40 ℃, after the solvent was dried by spin-drying in vacuo, 3mL of acetonitrile was added to dissolve, 43mg of COT sulfonamide (4.4 eq) was added, 65. Mu.L of DIPEA (7 eq) was reacted overnight at 50℃without further separation, 3mL (DCM: meOH=5:1) of solvent was again used to dissolve after spin-drying, 30mg of 1,3 dimethylbarbituric acid (4.5 eq) was added, 25mg of tetrakis (triphenylphosphine) palladium (0.5 eq) was added, and after 4 hours of reaction, separation was performed by HPLC, finally 3.8mg of the product was obtained in 12% yield.

Mass:[M+H] ⁺ ＝596.21。

1mg of the above-obtained molecule was dissolved in 1mL of DMF, 2mg of Halo-NH2.HCl (4.6 eq), 1.5mg of a Kate condensing agent (2 eq) and 6. Mu.L of DIPEA (20 eq) were added, and after overnight reaction, the reaction solution was separated by HPLC, whereby 34. Mu.g of the final product was obtained in 3% yield.

Mass:[M+H] ⁺ ＝801.38。

1.9 Synthesis of Compound MaP555-COT-TMP

1mg of the above-obtained molecule was dissolved in 1mL of DMF, 1mg of TMP3-NH2.HCl (1 eq), 1.5mg of a Kate condensing agent (2 eq) and 6. Mu.L of DIPEA (20 eq) were added, and after overnight reaction, the reaction solution was separated by HPLC to give 85. Mu.g of the final product in a yield of 5%

Mass:[M+H] ⁺ ＝1070.34。

It should be noted that, the compound MaP-COT-TMP in step 1.9 and the compound MaP-COT-Halo in step 1.8 are both labeled proteins by coupling with the protein tag, and the only difference is that the protein tag Halo is different from TMP, so that the fluorescence effect is similar when both are attached to the same protein (see example 5 and FIGS. 4a and 4 b).

2.0 Synthesis of Compound MaP555-COT-DNA

Compound 24: ¹ H NMR(400MHz,Methanol-d4)δ8.93(d,J＝1.5Hz,1H),8.46(dd,J＝7.9,1.7Hz,1H),7.57(d,J＝7.9Hz,1H),7.14(d,J＝9.5Hz,2H),7.06(dd,J＝9.5,2.4Hz,2H),6.99(d,J＝2.3Hz,2H),6.15(ddt,J＝16.2,10.9,5.7Hz,1H),5.49(dq,J＝17.1,1.7Hz,1H),5.36(dq,J＝10.5,1.4Hz,1H),4.95(d,J＝5.7Hz,2H),3.31(s,12H).

compound 25: ¹ H NMR(400MHz,Methanol-d4)δ8.53(d,J＝1.5Hz,1H),8.40(d,J＝7.8Hz,1H),7.52(s,1H),7.20–6.74(m,6H),3.24(s,12H),2.63(s,6H).

compound 23 is used as a raw material to synthesize compound 25, which is completely identical to the synthetic steps of compounds 20-22 in 1.8, and no detailed description is required.

20mg,0.038mmol of Compound 26 are weighed out in 1ml of DMF and K is added ₂ CO ₃ (30 mg,0.218 mmol) was then stirred at room temperature for 30 minutes. Compound 27 (12.3 mg,0.049 mmol) was added to the reaction and stirred overnight at 60 ℃.

The reaction solution was purified by HPLC (solution A: H) ₂ O+0.1% tfa, b: ACN), 18mg was finally obtained in 75% yield.

1mg of the above-obtained molecule was dissolved in 1mL of DMF, 1.6mg of 28 (2 eq), 1.5mg of a Kate condensing agent (2 eq) and 6. Mu.L of DIPEA (20 eq) were added, and after overnight reaction, the reaction solution was separated by HPLC to obtain the final product MaP-555-COT-DNA (the DNA in this structural formula essentially means small molecule Hoechst targeting DNA, i.e., compound 28. Small molecule Hoechst fluoresces by intercalating into a small groove of DNA) in 321. Mu.g, yield 17.8%.

¹ H NMR(400MHz,Methanol-d4)δ8.42(d,J＝1.7Hz,1H),8.36(d,J＝1.7Hz,1H),8.21(dd,J＝8.0,1.8Hz,1H),8.11(d,J＝8.6Hz,2H),8.02(dd,J＝8.5,1.7Hz,1H),7.86(d,J＝8.6Hz,1H),7.71(d,J＝9.0Hz,1H),7.47(d,J＝7.9Hz,1H),7.38(dd,J＝9.0,2.3Hz,1H),7.31(d,J＝2.4Hz,1H),7.18(d,J＝8.5Hz,2H),6.98(d,J＝9.4Hz,2H),6.89(dd,J＝9.3,2.4Hz,2H),6.82(d,J＝2.5Hz,2H),6.64(s,1H),6.02–5.93(m,1H),5.91–5.84(m,1H),5.75–5.59(m,4H),4.21(t,J＝5.7Hz,2H),4.03-3.87(s,2H),3.75-3.62(m,2H),3.58(t,J＝6.4Hz,2H),3.44-3.34(m,2H),3.21(s,12H),3.16-3.08(m,2H),3.02(s,3H),2.01–1.89(m,4H)。

Example 2 singlet oxygen yield test

For the aforementioned coupling of COT in the form of ester bonds at positions 3,4,5 of the TMR's bottom ring, the resulting compounds o-TMR-COT, m-TMR-COT, p-TMR-COT and, as a control, o-TMR-NB (FIG. 1 a) were measured in vitro for the singlet oxygen yield of the solutions. Singlet oxygen yield was quantitatively determined by 1, 3-Diphenylisopropylfuran (DPBF).

As shown in FIGS. 1b and 1c, the compounds o-TMR-COT, m-TMR-COT, p-TMR-COT all exhibited lower singlet oxygen yields than the commercially available mitochondrial dye tetramethyl rhodamine methyl ester (TMRM), tetramethyl rhodamine ethyl ester (TMRE). From literature studies, TMRE is known to have a singlet oxygen yield of 0.012. According to the reference method, the singlet yield of TMRM was 0.0087, while the absolute singlet oxygen yield of the compound o-TMR-COT was only 0.0014, which is 1/6 of TMRM. It can be concluded that the strategy of ligating COT to rhodamine number 3 carbon atoms reduces singlet oxygen yield by about 6-fold.

Example 3

In this example, hela (human cervical cancer cell) cells were taken as an example, and the phototoxicity of this series of dyes was tested.

All compounds were diluted with medium to a concentration of 250nM and cells were incubated at 37 ℃ for 15min at 5% carbon dioxide for subsequent experiments. Cells from different wells were imaged in time series with 561nm laser using a high content living cell imaging system, with 10 second exposure time, time points of gradient were acquired. After 1 hour from the end of imaging, propidium Iodide (PI) staining was added, the number of PI positive signals was counted as dead cell number=ase:Sub>A, the number of cells before exposure was counted as B, and cell viability= (B-ase:Sub>A)/B100% was obtained, which is an important index for evaluating dye phototoxicity.

The result of the phototoxicity data analysis is shown in FIG. 2, the survival rate of TMRM is reduced to 50% in about 2 minutes, and the survival rate of o-TMR-COT is reduced to 50% in about 10 minutes, which is quite consistent with the yield of singlet oxygen in vitro. The data of the two aspects together prove that COT is connected on the No. 3 carbon of the rhodamine bottom ring, so that phototoxicity can be reduced by 5-6 times.

Example 4

To further broaden the spectrum of the low phototoxicity fluorescent dye of the present application, the present example applied the COT coupling strategy to other rhodamine dyes, resulting in a similarly structured green fluorescent probe compound Rho110-COT (see 1.6 in example 1), a commercial green mitochondrial membrane potential probe Rho123 was available as a control compound for the compound Rho110-COT (as shown in FIG. 3 a); and far-red fluorescent probe compounds SiR-COOMe and SiR-COT (1.7 in example 1) (wherein compound SiR-COOMe is a control compound of compound SiR-COT, as shown in fig. 3 a), both compound Rho110-COT and compound SiR-COT have a significant reduction in phototoxicity compared to the control compound, as measured by ROS level in vitro and intracellular phototoxicity, wherein phototoxicity of Rho110-COT and Rho123 is characterized by their indicated mitochondrial membrane potential changes (see fig. 3b,3c,3d,3e and 3 f).

Example 5

The strategy of COT coupling is combined with protein tag technology, so that low toxicity imaging of specific proteins in living cells can be realized. Taking histone H2B with high abundance in a cell nucleus area as an example, a stable transfer cell line U2OS for stably expressing H2B-Halo is respectively marked by Map555-COT-Halo and a reported compound MaP-Halo as an example for experiments. (see FIG. 4 a) since H2B is tightly entangled with DNA, DNA damage will be rapidly initiated when active oxygen species such as singlet oxygen are generated around H2B. The damage condition of DNA is characterized by human X-ray staggered complementary repair factor (XRCC 1) of DNA repair protein, and when DNA in the nucleus is not damaged or is less damaged, XRCC1 is uniformly distributed in the nucleus area or is aggregated in a very small amount of spots; when DNA damage was greater, a punctate aggregation distribution of XRCC1 was clearly observed in the nuclear region (see fig. 4 b).

By comparison, the XRCC1 aggregation (increase in aggregation number by about 5-fold) was significantly less in the Map555-COT-Halo labeled experimental group than in the MaP555-Halo (increase in aggregation number by about 20-fold) labeled control group. After changing the Halo-Tag protein Tag to the TMP-Tag, map555-COT-TMP also showed less damage to the protein under light conditions, as shown in FIG. 4c.

In addition, other marker molecules can be linked to the ligand of the protein tag, and the compound MaP555-COT-DNA (using the commercially available SPY555-DNA as a control molecule) can be synthesized by taking nucleic acid as an example, and the same conclusion can be obtained by using the evaluation system described above, and the MaP555-COT-DNA shows lower DNA toxicity under the same labeling intensity and the same illumination condition (see FIG. 5).

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

1. the invention can be applied to living cell imaging systems without the need to improve the photostability and phototoxicity of the dye by deoxygenation.

2. The invention greatly improves the biocompatibility of imaging. On the one hand, covalent intramolecular attachment of the triplet quencher increases the reaction rate, reducing cytotoxicity resulting from high concentrations of triplet quencher; on the other hand, efficient collisions of triplet quenchers with dyes are more efficient in reducing phototoxicity generated by dye molecules.

3. The invention has wide application range. The strategy can be applied to specific markers of various organelles and various proteins in cells to realize super-resolution imaging of ultra-long time.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rhodamine fluorescent dye, characterized in that the rhodamine fluorescent dye comprises a rhodamine dye molecule and a triplet quencher cyclooctatetraene covalently coupled with the rhodamine dye molecule.

2. The rhodamine fluorescent dye according to claim 1, characterized in that the rhodamine dye molecule is covalently coupled to the cyclooctatetraene via a ground ring;

preferably, the cyclooctatetraene is covalently coupled through an ortho position on the bottom ring.

3. Rhodamine fluorescent dye according to claim 2, characterized in that the rhodamine dye molecule is selected from any one of the following: oxyrhodamine, carborhodamine or silarhodamine;

preferably, the oxyrhodamine comprises any one or more of the following: TMR, rho110, map555, JF503, JF525, JF536, JF549, JF522, JF571, 500R, 510R, or 515R;

preferably, the carborhodamine includes any one or more of: 6-CPY, 5-CPY, CRhp, 580CP, JF585, JF612, JF608 or Map618;

preferably, the silicon rhodamine comprises any one or more of the following: siR, JF626, JF629, JF630, JF635, JF646, JF669 or SiR.

4. A rhodamine fluorescent dye according to any one of claims 1 to 3, characterized in that the singlet oxygen yield of the rhodamine fluorescent dye is between 0.0014 and 0.018.

5. A fluorescence detection kit comprising a rhodamine-based fluorescent dye according to any one of claims 1 to 4.

6. A biomarker molecule comprising a biomolecule and a fluorescent dye for labelling the biomolecule, characterized in that the fluorescent dye of the biomolecule is a rhodamine fluorescent dye as defined in any one of claims 1 to 4.

7. A method of bioluminescence imaging, characterized in that cells are labeled with a rhodamine-based fluorescent dye as defined in any one of claims 1 to 4.

8. The method of bioluminescence imaging of claim 7, wherein said cell is a living cell;

preferably, the rhodamine fluorescent dye-labeled cells are organelles that label the cells, wherein the organelles include any one or more of the following: mitochondria, nuclei, cytoskeleton, golgi, endoplasmic reticulum, lysosomes, endosomes, ribosomes, or migratory bodies;

preferably, the rhodamine fluorescent dye marks cells and is a molecular marker in the marked cells, and the molecular marker is a protein molecule, a sugar molecule or a nucleic acid molecule;

preferably, the method of bioluminescence imaging further comprises placing the rhodamine fluorescent dye labeled cells under a microscope comprising any one or more of the following: confocal microscopy, random optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.

9. Use of a rhodamine fluorescent dye according to any one of claims 1 to 4, or a kit according to claim 5, or a bioluminescence imaging method according to claim 7 or 8, in a living cell imaging study.

10. The use according to claim 9, wherein the imaging is under any one or more of the following microscopes: confocal microscopy, random optical reconstruction microscopy, or stimulated emission depletion fluorescence microscopy.