Dye containing cyanine structure and application thereof

Abstract

The invention provides a dye containing a cyanine structure and application thereof. Wherein the dye containing the cyanine structure comprises a fluorophore mother nucleus and a functional group, the functional group comprises a membrane positioning group, and the functional group is connected to the fluorophore mother nucleus; the fluorophore mother nucleus is a cyanine structure mother nucleus. The problem that long-term living cell fluorescence imaging is difficult to realize by cell membrane dyes in the prior art can be solved, and the method is suitable for the field of cell membrane imaging.

Description

Technical Field

[0001] The present invention relates to the field of cell membrane imaging, and in particular to a dye containing a cyanine structure and applications thereof. Background Art

[0002] The cell membrane is a double-layered membrane structure that envelops the protoplast. It maintains a stable intracellular environment, controls the flow of substances into and out of the cell, and participates in intercellular signal transduction, performing important physiological functions. The cell membrane is composed of lipids and proteins, with cholesterol and proteins distributed within a phospholipid bilayer scaffold. Cells of different species or at different stages of the cell cycle often exhibit significant differences or variations in cell membrane properties, such as morphology, composition, membrane tension, and order. Cell membrane interactions also occur between specific cell types. Using cell membrane dyes, dynamic changes and interactions in the cell membrane can be detected in situ and in real time, making live cell imaging a crucial tool for studying the physiological functions and activities of the cell membrane.

[0003] Super-resolution and long-term imaging can investigate the fine structure of cell membranes or track cell membrane activity over long timescales. These applications, such as imaging dynamic cell migration and super-resolution imaging of migrasomes or exosomes, place higher demands on the phototoxicity and photostability of dyes. After absorbing excitation light and transitioning to the S1 state, fluorescent molecules can not only release photons back to the S0 state through radiative transitions but also transition to the T1 state through intersystem crossing. Compared to the S1 state, the T1 state is more reactive and has a longer lifetime, allowing it to undergo photophysical and photochemical reactions with surrounding oxygen. This process generates a range of reactive oxygen species, including singlet oxygen and superoxide radicals. These reactive oxygen species can damage the fluorescent molecules and surrounding cellular structures, leading to decreased fluorescence brightness and cell death. These phenomena are known as photobleaching and phototoxicity. Therefore, reducing the generation of reactive oxygen species during imaging can meet the requirements for reduced phototoxicity and improved photostability. The amount of reactive oxygen species produced is directly related to the total light dose. Super-resolution imaging uses stronger excitation light, and long-term imaging takes longer. Due to the significantly increased total light dose, both methods are more likely to produce large amounts of reactive oxygen species, greatly limiting the ability to observe living cells. Therefore, super-resolution and long-term imaging require a particular reduction in reactive oxygen species production. Currently, there are several methods to reduce the production of reactive oxygen species during imaging. Previous methods have involved deoxygenation or the addition of millimolar triplet quenchers, but these two methods are not suitable for live-cell imaging. Summary of the Invention

[0004] The main purpose of the present invention is to provide a dye containing a cyanine structure and its application, so as to solve the problem in the prior art that cell membrane dyes are difficult to achieve long-term living cell fluorescence imaging.

[0005] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a dye containing a cyanine structure is provided, wherein the dye containing a cyanine structure comprises a fluorophore core and a functional group, wherein the functional group comprises a membrane-localizing group, and the functional group is connected to the fluorophore core; the fluorophore core is a cyanine structure core.

[0006] Furthermore, the cyanine structure mother core has a structure shown in Formula I, Formula II or Formula III,

[0007]

[0008] Furthermore, the membrane-locating group is connected to the cyanine structure core via a linking group, and the linking group includes an amide bond or an ester bond; preferably, the linking group is connected to any one or more positions of position 1, position 2, or position 3 of the structure represented by Formula I, Formula II, or Formula III; more preferably, the cyanine structure core connected to the linking group has the following structure:

[0009]

[0010]

[0011] Wherein R1 is NH or O.

[0012] Furthermore, the membrane-locating group is an AZ group, and the AZ group has a structure shown in Formula IV, wherein R is an alkyl group consisting of 4 to 18 C atoms; preferably, the AZ group has a structure shown in Formula V,

[0013]

[0014] Furthermore, a triplet quenching group is connected to the cyanine structure core; the triplet quenching group is connected to the 3-position and / or 4-position of the structure shown in Formula I, Formula II or Formula III; preferably, the triplet quenching group is a cyclooctatetraene group, and the cyclooctatetraene group has a structure shown in Formula VI or Formula VII,

[0015]

[0016] Furthermore, the functional group also includes a negatively charged group, which is directly connected to the linking group or connected to the linking group through a carbon chain; preferably, the carbon chain is an alkylene group composed of 1 to 4 C atoms.

[0017] Furthermore, the negatively charged group is COO - or SO3 - ; Preferably, the dye containing a cyanine structure has the following structure:

[0018]

[0019] Among them, R2 is O or NH, R3 is COO - or SO3 - , X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + More preferably, the dye containing a cyanine structure is AZ-Cy3.5-COT, AZ-Cy3-COT or AZ-Cy5-COT,

[0020]

[0021] Among them, X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + .

[0022] Furthermore, the negatively charged group is SO3 - The negatively charged group is directly connected to the cyanine structure mother core or connected to the cyanine structure mother core through a carbon chain; preferably, the negatively charged group is connected to the 2-position or 4-position of the structure shown in Formula I, Formula II or Formula III.

[0023] Furthermore, the dye containing the cyanine structure is

[0024]

[0025]

[0026] wherein n=1 or 2, m is a positive integer of 1-5, and R2 is independently selected from O or NH. Further, the dye containing a cyanine structure is

[0027]

[0028] Among them, Y - is an anion;

[0029] Preferably, Y is trifluoroacetic acid, PF6, BH4, ClO4, Cl, Br or I.

[0030] In order to achieve the above object, according to a second aspect of the present invention, there is provided a use of the above dye containing a cyanine structure in cell membrane imaging.

[0031] Furthermore, the application includes: staining living cells with dyes containing cyanine structures to obtain stained cells; imaging the stained cells using microscopic imaging technology; preferably, the microscopic imaging technology includes fluorescence imaging; more preferably, the fluorescence imaging includes confocal imaging technology, super-resolution imaging technology, wide-field imaging technology or two-photon living imaging technology; preferably, the imaging includes long-term imaging, and more preferably, the time of long-term imaging is ≥4 hours.

[0032] By applying the technical solution of the present invention and optimizing the structure of cell membrane dyes, a new type of cell membrane dye containing a cyanine structure has been obtained. Compared with cell membrane dyes in the prior art, this cell membrane dye has improved photostability and reduced phototoxicity, enabling longer-term imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 The graph shows the comparative results of the photostability of the commercial MemGlow dye, PPM-Cy and AZ-Cy-Me cell membrane dyes on Hela cells according to Example 4 of the present invention.

[0035] Figure 2 The graph shows the in vitro ROS production rate detection results of PPM-Cy, AZ-Cy-Me and reference according to Example 5 of the present invention.

[0036] Figure 3 The confocal fluorescence imaging results of AZ-Cy-COT, PPM-Cy or NPM-Cy on Hela cells according to Example 6 of the present invention are shown.

[0037] Figure 4 The graph shows the results of Hela cell activity under different concentrations of AZ-Cy-COT according to Example 7 of the present invention.

[0038] Figure 5 The confocal imaging results of the NPM-Cy dye according to Example 8 of the present invention and the commercial Cell Mask Deep Red on Hela cells are shown. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0040] Currently, mainstream commercially available cell membrane fluorescent dyes typically consist of a fluorophore core and a membrane-targeting group. The fluorophore core is often a cyanine, fluorescein, or rhodamine dye with excellent optical properties and high biocompatibility. The membrane-targeting group is a group that interacts strongly with the cell membrane or forms a covalent bond, responsible for transferring the dye from the aqueous dye solution to the cell membrane. Membrane-targeting groups can be small molecules or proteins, and they non-covalently bind to phospholipid molecules in the cell membrane through hydrophobic and electrostatic interactions, thereby targeting the cell membrane. However, in the prior art, there is little targeted optimization or improvement of the properties of cell membrane fluorophores. During long-term imaging or super-resolution imaging, these dyes are internalized rapidly. Under intense and prolonged excitation light, they convert to triplet states and generate a large amount of reactive oxygen species, further causing phototoxicity and photobleaching, ultimately interfering with normal cellular physiological activities and leading to a decrease in fluorescence brightness. In live-cell imaging of cell membranes, this manifests as decreased fluorescence brightness, reduced cellular oxidative and metabolic activity, and loss of cell membrane integrity. It can be seen that existing commercial dyes often have defects such as strong phototoxicity and weak photostability, which make it difficult to meet the requirements of long-term live cell fluorescence imaging.

[0041] In a first typical embodiment of the present application, a dye containing a cyanine structure is provided, wherein the dye containing a cyanine structure includes a fluorophore core and a functional group, wherein the functional group includes a membrane-localizing group, and the functional group is connected to the fluorophore core; the fluorophore core is a cyanine structure core.

[0042] The dyes containing a cyanine structure include a fluorophore core and a functional group, wherein the functional group includes a membrane-localizing group. The fluorophore core is a group with excellent optical properties, including but not limited to cyanine, fluorescein, or rhodamine compounds, which can emit fluorescence at a specific excitation wavelength, thereby enabling fluorescence detection. The membrane-localizing group is a group that has a strong interaction with the cell membrane or can form a covalent bond. It is responsible for transferring the dye from the aqueous phase of the dye solution to the cell membrane and preventing the dye from penetrating the cell membrane and entering the cell interior as much as possible.

[0043] In a preferred embodiment, the cyanine structure mother core has a structure shown in Formula I, Formula II or Formula III,

[0044]

[0045]

[0046] In a preferred embodiment, the membrane-locating group is connected to the cyanine structure core via a linking group, and the linking group includes an amide bond or an ester bond; preferably, the linking group is connected to any one or more positions of position 1, position 2, or position 3 of the structure represented by Formula I, Formula II, or Formula III; more preferably, the cyanine structure core connected to the linking group has the following structure:

[0047] Wherein R1 is NH or O.

[0048] The dye containing cyanine structure prepared using the above-mentioned cyanine structure mother core can achieve fluorescence imaging in three bands of 550nm, 590nm or 650nm. The three colors are conducive to compatibility with other fluorescent probes in multi-color imaging processes such as immunofluorescence or ordinary fluorescence.

[0049] In a preferred embodiment, the membrane-locating group is an AZ group, and the AZ group has a structure shown in Formula IV, wherein R is an alkyl group consisting of 4 to 18 C atoms, including but not limited to an alkyl group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 C atoms, including but not limited to a normal chain alkyl group or an isochain alkyl group; preferably, the AZ group has a structure shown in Formula V,

[0050]

[0051] Preferably, the membrane-localizing group is connected to the amide bond of the cyanine structure core through the position of "~~~". The membrane-localizing group localizes the dye containing the cyanine structure near the cell membrane.

[0052] In the prior art, membrane-localizing groups (AZ groups) consisting of a dodecyl chain, a nitrogen cation, and a sulfonic acid anion have been used to modify Nile red, squaric acid, cyanine, and BODIPY to construct corresponding cell membrane dyes, all of which achieve good cell membrane localization. However, as described above, the inventors of this application discovered that all of the cell membrane dyes modified with AZ groups in the prior art are positively charged dyes, and without further modification of the fluorophore core. Consequently, they suffer from drawbacks such as dye internalization, photobleaching, and phototoxicity that are detrimental to long-term imaging.

[0053] In a preferred embodiment, a triplet quenching group is further connected to the cyanine structure core; the triplet quenching group is connected to the 3-position and / or 4-position of the structure shown in Formula I, Formula II or Formula III; preferably, the triplet quenching group is a cyclooctatetraene group, and the cyclooctatetraene group has a structure shown in Formula VI or Formula VII,

[0054]

[0055] In the above-mentioned dyes containing a cyanine structure, the triplet quencher cyclooctatetraene is connected to the cyanine structure mother core. Cyclooctatetraene can convert the T1 state of the dye into the S0 state through energy transfer, avoiding the generation of reactive oxygen species, thereby further reducing the phototoxicity of the dye, improving the photostability, and extending the time that the dye containing a cyanine structure can image living cells.

[0056] In a preferred embodiment, the functional group further includes a negatively charged group, which is directly connected to the linking group or connected to the linking group through a carbon chain; preferably, the carbon chain is an alkylene group composed of 1 to 4 C atoms.

[0057] Existing fluorophore cores with excellent optical properties, such as cyanine dyes and rhodamine dyes, all carry a positive charge. Therefore, when modified with membrane-targeting groups to create cell membrane dyes, the overall charge of the dye is often +1 or even +2. In other words, current cell membrane dyes are often positive overall. However, due to groups such as phosphate, the cell membrane itself is electronegative. This interaction between positive and negative charges enables contact between the fluorophore core and the cell membrane and fluorescence imaging. However, existing cell membrane dyes are difficult to use over long periods of time, and their cytotoxicity can affect the normal physiological activities of living cells.

[0058] Based on in-depth research on cell membrane dyes, the inventors unexpectedly discovered that when the dye carries an overall negative charge, the rate at which the dye enters the cell can be greatly reduced. Therefore, in this application, the inventors attempted to attach negatively charged groups to the fluorophore core of a dye containing a cyanine structure. By using negatively charged groups such as carboxyl anions, the overall charge of the dye is converted from positive to negative. This, through electrostatic interactions between the dye and the cell membrane, hinders the dye from penetrating into or even passing through the cell membrane, further reducing the dye's internalization rate and increasing the dye's retention time on the cell membrane, thereby achieving longer-term imaging.

[0059] In a preferred embodiment, the negatively charged group is COO - or SO3 - ; Preferably, the dye containing a cyanine structure has the following structure:

[0060]

[0061] Among them, R2 is O or NH, R3 is COO - or SO3 - , X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + ;

[0062] COO - and / or SO3 - The group is connected to the connecting group connected to the cyanine structure mother core through a carbon chain (including but not limited to methylene, ethylene, propylene or butylene), which can change the electrical properties of the cyanine structure mother core into negative charge, reduce the impact on the conjugated structure of the mother core, and do not affect the luminescent properties and capabilities of the mother core.

[0063] More preferably, the dye containing a cyanine structure is AZ-Cy3.5-COT, AZ-Cy3-COT or AZ-Cy5-COT as follows,

[0064] Among them, X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + .

[0065] By coupling the membrane-localizing group AZ and the triplet quencher cyclooctatetraene to a cyanine dye, the carboxylate group imparts a negative charge to the dye, and the introduction of a cationic group balances the overall charge in the dye solution. This results in a series of negatively charged cyanine-containing dyes, AZ-Cy-COT, with high retention times, low phototoxicity, and high photostability at different wavelengths. These dyes, including AZ-Cy3.5-COT, AZ-Cy3-COT, and AZ-Cy5-COT, are suitable for long-term and super-resolution imaging.

[0066] In a preferred embodiment, the negatively charged group is SO3 - The negatively charged group is directly connected to the cyanine structure mother core or connected to the cyanine structure mother core through a carbon chain; preferably, the negatively charged group is connected to the 2-position or 4-position of the structure shown in Formula I, Formula II or Formula III.

[0067] The above SO3 - Negative charges can be attached directly to the cyanine core (the six-membered ring) or through carbon chains, including but not limited to methylene, ethylene, propylene, or butylene groups, to change the charge of the cyanine core from positive to negative or neutral without affecting its luminescence. Although sulfonic acid groups have been used in some prior art, they are attached to membrane-localizing groups to increase the dye's hydrophilicity, rather than to adjust the overall charge.

[0068] In a preferred embodiment, the dye containing a cyanine structure is

[0069]

[0070]

[0071] wherein n=1 or 2, m is a positive integer of 1-5, and R2 is independently selected from O or NH.

[0072] In a preferred embodiment, the dye containing a cyanine structure is

[0073]

[0074] Among them, Y - is an anion; preferably, Y is trifluoroacetic acid, PF6, BH4, ClO4, Cl, Br or I.

[0075] In a second typical embodiment of the present application, a use of the above-mentioned dye containing a cyanine structure in cell membrane imaging is provided.

[0076] In a preferred embodiment, the application includes: staining living cells with a dye containing a cyanine structure to obtain stained cells; imaging the stained cells using microscopic imaging technology; preferably, the microscopic imaging technology includes fluorescence imaging; more preferably, the fluorescence imaging includes confocal imaging technology, super-resolution imaging technology, wide-field imaging technology or two-photon living imaging technology; preferably, the imaging includes long-term imaging; more preferably, the time of long-term imaging is ≥4 hours.

[0077] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0078] Example 1

[0079] AZ-Cy-COT series membrane dyes were prepared by organic synthesis.

[0080] Taking the synthesis route of AZ-Cy3.5-COT as an example, the reaction formula is as follows:

[0081]

[0082] Compound S1: Triethylamine (6.97 g, 68.8 mmol, 4 eq) and 1-bromododecane (8.58 g, 34.4 mmol, 2 eq) were added to a solution of tert-butyl 2-(methylamino)ethylcarbamate (3.0 g, 17.2 mmol) in 20 mL of dichloromethane and stirred at room temperature for 12 h. The reaction system was washed with saturated brine, and the aqueous phase was extracted with 2 × 20 mL of dichloromethane. The organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and rotary evaporated to obtain a pale yellow oil. The pale yellow oil was dissolved in 20 mL of dichloromethane, and 4 mL of TFA was added. The mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography using 0-6% MeOH / DCM to obtain 1.81 g of S1 as a colorless oil in a 43% yield.

[0083] 1 H NMR(400MHz,Chloroform-d)δ3.66(s,3H),2.65(t,J=6Hz,2H),2.30(t,J=6Hz,2H),2.17( t,J=7.6Hz,2H),2.04(s,3H),1.28(t,J=6.4Hz,2H),1.09(s,18H),0.71(t,J=6.8Hz,2H). 13 C NMR (400MHz, Chloroform-d) δ58.68,57.88,41.81,38.62,31.73,29.49,29.46,29.17,27.31,27.06,22.48,14.90.

[0084]

[0085] Compound S2: Dissolve 5-tert-butyl N-Boc-L-glutamate (100 mg, 0.330 mmol), S1 (160 mg, 0.659 mmol, 2 eq), triethylamine (133 mg, 1.318 mmol, 4 eq), and 4-dimethylaminopyridine (4 mg, 0.033 mmol, 0.1 eq) in 10 mL of dichloromethane. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI; 151 mg, 0.396 mmol, 1.2 eq) and stir at room temperature for 12 h. The solvent was removed under reduced pressure, and the product was purified by column chromatography using a PE:EA ratio of 5:1 to afford 51 mg of S2 as a light yellow oil (29% yield).

[0086] ESI mass spectrometry results, S2(M+H) + Calculated for C 29 H 58 N3O5 + ,527.4,observed 527.6.

[0087]

[0088] Compound S3: S2 (43 mg, 0.0814 mg), 1,3-propane sultone (50 mg, 0.407 mmol, 5 eq), and potassium carbonate (56 mg, 0.407 mmol, 5 eq) were dissolved in 5 mL of acetonitrile and heated under reflux at 90°C for 12 h. The solvent was removed under reduced pressure, and the resulting solid was added with 2 mL of dichloromethane and 0.5 mL of trifluoroacetic acid, and stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the product was separated by HPLC (eluent, linear gradient from 20% to 70% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 214 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 11 mg of the desired product S3 in a 27% yield.

[0089] ESI mass spectrometry results, S3 (M+H) + Calculated for C 23 H 48 N3O6S+,493.3, observed 493.5.

[0090]

[0091] Compound S4: A solution of NaNO2 (111 mg, 1.6 mmol, 1 eq) in 3 mL of concentrated hydrochloric acid was added dropwise to a solution of 6-amino-2-naphthoic acid (300 mg, 1.6 mmol) in 2 mL of water at 0°C in an ice bath. The mixture was stirred at 0°C for 1 h. A solution of SnCl2 (1518 mg, 9.0 mol, 5 eq) in 2 mL of concentrated hydrochloric acid was added dropwise at 0°C. The mixture was reacted for 5 h at 0°C. The precipitate was collected by filtration and washed sequentially with 2 × 20 mL of water, 20 mL of ethanol, and 20 mL of ether. This afforded 140 mg of an off-white solid, S4, in a 43% yield.

[0092] 1 H NMR (400MHz, DMSO-d6) δ10.51(s,3H),8.87(s,1H),8.49(s,1H),8.01(d,J=8.8Hz,1 H),7.93(d,J=8.8Hz,1H),7.77(d,J=8.8Hz,1H),7.31(s,1H),7.28(d,J=8.8Hz,1H). 13 CNMR(400MHz,DMSO-d6)δ167.45,145.45,135.87,130.50,130.45,127.61,126.55,126.18,125.76,117.46,106.92.

[0093]

[0094] Compound S5: Dissolve p-hydrazinobenzoic acid (200 mg, 1.32 mmol) and 3-methyl-2-butanone (342 mg, 3.96 mmol, 3 eq) in acetic acid and heat with stirring at 90°C for 4 h. The solvent was removed under reduced pressure, and the product was purified by column chromatography using 50% EA / PE to afford 162 mg of S5 as a pale yellow solid (60% yield).

[0095] 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=1.6Hz,1H),8.18(d,J=8.8Hz,1H),8.10(d,J=8.8H z,1H),8.05(dd,J=8.8,1.6Hz,1H),7.77(d,J=8.8Hz,1H),2.31(s,3H),1.45(s,6H). 13 C NMR (400MHz, DMSO-d6) δ191.02,167.44,152.71,138.84,132.10,130.73,130.37,130.04,126.36,125.81,122.94,120.04,54.99,22.00,15.05.

[0096]

[0097] Compound S6: Dissolve S4 (76 mg, 0.30 mmol) and 2-iodoethanol (155 mg, 0.90 mmol, 3 eq) in 5 mL of acetonitrile and heat at 90°C in a sealed tube for 48 h. The solvent was removed under reduced pressure, and the resulting solid was washed twice with diethyl ether to afford 56 mg of a green solid, S6, in a 44% yield.

[0098] 1 H NMR (400MHz, DMSO-d6) δ8.87(s,1H),8.50(d,J=7.2Hz,2H),8.22(t,J=8.4Hz,2H),4.74(br,2H),3.94(br,2H),2.94(s,3H),1.79(s,6H). 13 C NMR(400MHz,DMSO-d6)δ198.93,166.93,140.35,136.76,132.33,132.10,129.0 4,127.41,123.91,121.81,115.00,114.41,57.99,55.71,50.56,21.52,14.34.

[0099]

[0100] Compound S7: N,N-dibenzamide (20 mg, 0.10 mmol) and 0.5 mL of pyridine were added to a solution of S6 (91 mg, 0.21 mmol, 2.1 eq) in 3 mL of acetic anhydride and stirred at 75°C for 12 h. The mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and the mixture was washed twice with ether to obtain a purple-red powder. The powder was dissolved in 3 mL of methanol, and sodium methoxide (43 mg, 0.80 mmol, 8 eq) was added. The mixture was stirred at room temperature for 2 h. 1 M dilute hydrochloric acid was added to neutralize the mixture, and the solvent was removed by rotary evaporation. The resulting solid was separated by HPLC (eluent, linear gradient from 20% to 70% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 590 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to obtain 35 mg of purple-red solid S7 in 33% yield.

[0101] 1 H NMR (400MHz, DMSO-d6) δ8.73(s,2H),8.67(t,J=13.2Hz,1H),8.37(d,J=8.8Hz,2H),8.30(d,J=8.8Hz,2H),8. 12(d,J=6.0Hz,2H),7.84(d,J=6.0Hz,2H),6.63(d,J=9.2Hz,2H),4.38(br,4H),3.89(br,4H),2.03(s,12H). 13 C NMR(400MHz,DMSO-d6)δ176.47,167.14,149.08,142.31,132.95,132.51,131.89,13 0.61,129.26,127.01,126.72,122.53,113.26,103.27,58.37,50.62,48.56,27.06.

[0102]

[0103] Compound S8: 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 260 mg, 0.683 mmol, 10 eq) and triethylamine (70 mg, 0.683 mmol, 10 eq) were added to a solution of COTCOOH (80 mg, 0.546 mmol, 8 eq) in 5 mL of DMF. The mixture was stirred at room temperature for 10 min, followed by the addition of a solution of S7 (50 mg, 0.0683 mmol) in 1 mL of pyridine. The mixture was stirred at room temperature for 48 h. The resulting mixture was separated by HPLC (eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 20 mg of a purple-red solid, yielding 33%.

[0104] ESI mass spectrometry results, S8 (M+H) + Calculated for C 55 H 49 N2O8 + ,865.4,observed 865.8.

[0105]

[0106] AZ-Cy3.5-COT: HATU (5 mg, 0.0122 mmol, 2.2 eq) and TEA (22 mg, 0.0333 mmol, 6 eq) were added to a solution of S8 (5 mg, 0.00556 mmol) in 1 mL of DMF and stirred at room temperature for 10 min. Subsequently, S3 (11 mg, 0.0222 mmol) and 0.5 mL of pyridine were added and stirred at room temperature for 12 h. After completion of the reaction, the product was separated by HPLC (eluent, linear gradient from 40% to 90% solvent B over 30 min; flow rate, 10 mL / min; detection wavelength, 590 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford AZ-Cy3.5-COT as a purple-red solid (180 nmol, 3% yield).

[0107] ESI mass spectrometry results, AZ-Cy3.5-COT (M+2H) 2+ Calculated for C 100 H 138 N8O 18 S2 2+ ,902.0,observed902.4.

[0108] The synthetic routes for AZ-Cy3-COT and AZ-Cy5-COT are essentially the same as for AZ-Cy3.5-COT. The only difference is that the substrate S4 is replaced with p-carboxyphenylhydrazine in the synthesis of AZ-Cy3-COT and p-carboxyphenylhydrazine in the synthesis of AZ-Cy5-COT. In the synthesis of S7, the substrate N,N-dibenzamide is replaced with pentadienaldehyde diphenylamine hydrochloride. The reaction conditions remain unchanged.

[0109] ESI mass spectrometry results, AZ-Cy3-COT (M+2H) 2+ Calculated for C 92 H 134 N8O 18 S2 + ,852.0,observed852.2.

[0110] ESI mass spectrometry results, AZ-Cy5-COT (M+2H) 2+ Calculated for C 94 H 136 N8O 18 S2 + ,865.0,observed865.4.

[0111] Example 2

[0112] The NPM-Cy series membrane dyes are prepared by organic synthesis. The preparation route is shown in the following reaction formula:

[0113]

[0114]

[0115] Compound S9: Dissolve 2,3,3-trimethyl-3H-indole (200 mg, 1.26 mmol) and 3-bromopropionic acid (384 mg, 2.51 mmol, 2 equiv.) in 5 mL of acetonitrile and reflux at 90°C for 12 h. The reaction mixture was spin-dried and the residue washed twice with ether to yield 213 mg of a brown solid, S9, in a 47% yield. ESI mass spectrometry results: S9 (M) + Calculated for C 14 H 18 NO2 + ,232.1,observed 232.3.

[0116] Compound S10: S9 (1000 mg, 3.20 mmol), triethylamine (974 mg, 9.61 mmol, 3 equiv.), and HATU (2434 mg, 6.41 mmol, 2 equiv.) were dissolved in 20 mL of DMF and stirred at room temperature for 15 min. S1 (1554 mg, 6.41 mmol, 2 equiv.) was added and stirred at room temperature for 12 h. The reaction system was quenched by dropwise addition of saturated brine, extracted with ethyl acetate, and dried. The resulting organic phase was purified by column chromatography using a 20:1 ratio of DCM to MeOH to afford 634 mg of S10 as a light red oily liquid in a 40% yield. ESI mass spectrometry results showed that S10 (M) + Calculated for C 29 H 50 N3O + ,456.4,observed 456.7.

[0117] Compound S11: 2,3,3-Trimethylindole (100 mg, 0.632 mmol) and 3-bromopropionic acid (154 mg, 1.26 mmol, 2 equiv.) were dissolved in 5 mL of acetonitrile and refluxed at 90°C for 12 h. The reaction mixture was spin-dried and the residue washed twice with ether to afford 93 mg of a purple solid, S11, in a 52% yield. ESI mass spectrometry results for S11 (M+H) + , Calculated for C 14 H 20 NO3S + ,282.1,observed 282.4.

[0118] Compound S12: S11 (50 mg, 0.178 mmol) and N-(3-phenylamino-2-propenylidene)aniline hydrochloride (46 mg, 0.178 mmol, 1 equiv.) were dissolved in 1.5 mL of acetic acid and 1.5 mL of acetic anhydride and heated at 75°C in a sealed tube for 6 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 70% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 380 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 32 mg of a brown solid S12 in a 40% yield. ESI mass spectrometry results showed that S12 (M+H) + Calculated for C 25 H 29 N2O4S + ,453.2,observed 453.8.

[0119] Compound S13: 2,3,3-Trimethyl-3H-indole-5-carboxylic acid (1000 mg, 4.92 mmol) and iodomethane (1429 mg, 9.85 mmol, 2 equiv.) were dissolved in 10 mL of acetonitrile and refluxed at 90°C for 12 h. The reaction mixture was spin-dried and the residue washed twice with ether to yield 642 mg of a brown solid S13 in a 40% yield. ESI mass spectrometry results: S13 (M) + Calculated for C 13 H 17 NO + ,203.1,observed 203.3.

[0120] Compound S14: S13 (300 mg, 0.87 mmol), triethylamine (263 mg, 2.61 mmol, 3 equiv.), and HATU (397 mg, 1.04 mmol, 1.2 equiv.) were dissolved in 5 mL of DMF and stirred at room temperature for 15 min. S1 (421 mg, 1.74 mmol, 2 equiv.) was added and stirred at room temperature for 12 h. The reaction system was quenched by dropwise addition of saturated brine, extracted with ethyl acetate, and dried. The resulting organic phase was purified by column chromatography using a 20:1 ratio of DCM to MeOH to afford 113 mg of S14 as a pale yellow oil, with a 27% yield. ESI mass spectrometry results showed that S14 (M) + Calculated for C 28 H 48 N3O + ,442.4,observed 442.7.

[0121] Compound S15: Dissolve 2,3,3-trimethyl-3H-indole-5-sulfonic acid (200 mg, 0.96 mmol) and iodomethane (271 mg, 1.91 mmol, 2 equiv.) in 5 mL of acetonitrile and reflux at 90°C for 12 h. The reaction mixture was spin-dried and the residue washed twice with ether to afford 193 mg of a pink solid, S11, in a 79% yield. ESI mass spectrometry results for S15 (M+H) + Calculated for C 12 H 16 NO3S + ,254.1,observed 254.4.

[0122] Compound S16: S15 (50 mg, 0.20 mmol) and N-(3-phenylamino-2-propenylidene)aniline hydrochloride (61 mg, 0.24 mmol, 1.2 equiv.) were dissolved in 1.5 mL of acetic acid and 1.5 mL of acetic anhydride and heated at 75°C in a sealed tube for 6 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 70% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 380 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 44 mg of a brown solid S16 in a 47% yield. ESI mass spectrometry results showed that S16 (M+H) + Calculated for C 22 H 25 N2O4S + ,425.1,observed 425.5.

[0123] Compound S17: S10 (10 mg, 0.020 mmol) and S12 (9 mg, 0.02 mmol, 1 equiv.) were dissolved in 0.5 mL of pyridine and 1.5 mL of acetic anhydride and heated at 90°C in a sealed tube for 4 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 80% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 4 mg of a blue solid, S17, in a 26% yield. ESI mass spectrometry results showed that S17 (M+H) + Calculated for C 46 H 67 N4O4S + ,772.5,observed 772.9.

[0124] Compound NPM1-Cy5: S17 (4 mg, 0.0052 mmol), potassium carbonate (7 mg, 0.052 mmol, 10 equiv.), and 1,3-propane sultone (6 mg, 0.052 mmol, 10 equiv.) were dissolved in 1 mL of acetonitrile and heated at 65°C in a sealed tube for 12 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN, linear gradient over 30 min). The flow rate was 10 mL / min, and the detection wavelength was 650 nm. The yield of NPM1-Cy5 was 1816 nmol, a blue solid, with a yield of 35%. ESI mass spectrometry results showed that NPM1-Cy5 (M+H)+ Calculated for C 49 H 75 N4O7S2 + ,895.5,observed 896.0.

[0125] Compound S18: S10 (10 mg, 0.020 mmol) and S16 (8 mg, 0.02 mmol, 1 equiv.) were dissolved in 0.5 mL of pyridine and 1.5 mL of acetic anhydride and heated at 90°C in a sealed tube for 4 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 80% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 5 mg of a blue solid, S18, in a 34% yield. ESI mass spectrometry results showed that S18 (M+H) + Calculated for C 44 H 65 N4O4S + ,745.5,observed 745.8.

[0126] Compound NPM2-Cy5: S18 (5 mg, 0.0067 mmol), potassium carbonate (9 mg, 0.067 mmol, 10 equiv.), and 1,3-propane sultone (8 mg, 0.067 mmol, 10 equiv.) were dissolved in 1 mL of acetonitrile and heated at 65°C in a sealed tube for 12 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 80% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to yield 73 nmol of NPM2-Cy5 as a blue solid, with a reaction yield of 1%. ESI mass spectrometry results showed that NPM2-Cy5 (M+H) + , Calculated for C 47 H 71 N4O7S2 + ,867.5,observed 868.0.

[0127] Compound S19: S14 (10 mg, 0.020 mmol) and S12 (9 mg, 0.02 mmol, 1 equiv.) were dissolved in 0.5 mL of pyridine and 1.5 mL of acetic anhydride and heated at 90°C in a sealed tube for 4 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 80% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 3 mg of a blue solid, S19, in a 20% yield. ESI mass spectrometry results showed that S19 (M+H) + Calculated for C 45 H 67 N4O4S + ,759.5,observed 759.8.

[0128] Compound NPM3-Cy5: S19 (3 mg, 0.0040 mmol), potassium carbonate (9 mg, 0.040 mmol, 10 equiv.), and 1,3-propane sultone (8 mg, 0.040 mmol, 10 equiv.) were dissolved in 1 mL of acetonitrile and heated at 65°C in a sealed tube for 12 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN, linear gradient over 30 min). The flow rate was 10 mL / min, and the detection wavelength was 650 nm. The yield of NPM3-Cy5 was 1066 nmol, a blue solid, with a yield of 27%. ESI mass spectrometry results showed that NPM3-Cy5 (M+H) + Calculated for C 48 H 73 N4O7S2 + ,881.5,observed 880.7.

[0129] Compound S20: S14 (10 mg, 0.020 mmol) and S16 (8 mg, 0.02 mmol, 1 equiv.) were dissolved in 0.5 mL of pyridine and 1.5 mL of acetic anhydride and heated at 90°C in a sealed tube for 4 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent, linear gradient from 20% to 80% solvent B over 30 min; flow rate 10 mL / min; detection wavelength 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to afford 4 mg of a blue solid, S20, in a 20% yield. ESI mass spectrometry results showed that S20 (M+H) + Calculated for C43 H 63 N4O4S + ,731.5,observed 731.7.

[0130] Compound NPM4-Cy5: S20 (4 mg, 0.0052 mmol), potassium carbonate (7 mg, 0.052 mmol, 10 equiv.), and 1,3-propane sultone (6 mg, 0.052 mmol, 10 equiv.) were dissolved in 1 mL of acetonitrile and heated at 65°C in a sealed tube for 12 h. The reaction mixture was spin-dried to dryness, and the resulting mixture was separated by HPLC (eluent: linear gradient from 20% to 80% solvent B over 30 min; flow rate: 10 mL / min; detection wavelength: 650 nm; eluent A: ddH2O containing 0.1% (v / v) TFA; eluent B: ACN) to yield 112 nmol of NPM4-Cy5 as a blue solid in a 2% yield. ESI mass spectrometry results: NPM4-Cy5 (M+H) + , Calculated for C 46 H 69 N4O7S2 + ,853.5,observed 853.9.

[0131] The synthetic routes for the Cy3 derivatives of the NPM-Cy5 series of dyes are identical to those for their Cy5 versions. By replacing the substrate pentadienaldehyde diphenylamine with N,N-dibenzamide in steps S12 or S16 of the synthesis, the reaction conditions remain unchanged, allowing the synthesis of four NPM-Cy3 dyes.

[0132] ESI mass spectrometry results, NPM1-Cy3 (M+H) + Calculated for C 47 H 73 N4O7S2 + ,869.5,observed869.9.

[0133] ESI mass spectrometry results, NPM2-Cy3 (M+H) + Calculated for C 45 H 69 N4O7S2 + ,841.5,observed842.0.

[0134] ESI mass spectrometry results, NPM3-Cy3 (M+H) + Calculated for C 46 H 71 N4O7S2 +,855.5,observed854.7.

[0135] ESI mass spectrometry results, NPM4-Cy3 (M+H) + Calculated for C 44 H 67 N4O7S2 + ,827.5,observed827.9.

[0136] Example 3

[0137] The PPM-Cy series membrane dyes were prepared by organic synthesis.

[0138] Synthesis of PPM-Cy3

[0139] 1) Preparation of compound S21:

[0140]

[0141] Dissolve p-hydrazinobenzoic acid (200 mg, 1.32 mmol) and 3-methyl-2-butanone (342 mg, 3.96 mmol, 3 eq) in acetic acid, stir at room temperature for 30 minutes, and heat at 90°C for 4 hours. Remove the solvent under reduced pressure, and purify by 50% EA / PE column chromatography to afford 162 mg of S21 as a pale yellow solid (60% yield).

[0142] 1 H NMR (400MHz, DMSO-d6) δ8.12(s,1H),8.00(dd,J=8.0,1.2Hz,1H),7.61(d,J=8.0Hz,1H),2.47(s,3H),1.37(s,6H).

[0143] 13 C NMR (400MHz, DMSO-d6) δ196.07,167.37,151.18,144.92,130.31,129.41,123.71,118.07,54.10,22.20,15.29.

[0144] 2) Preparation of compound S22:

[0145]

[0146] 2,3,3-Trimethyl-5-carboxy-3H-indole (100 mg, 0.49 mmol) and 2-iodoethanol (169 mg, 0.98 mmol, 2 eq) were dissolved in 5 mL of acetonitrile and heated at 90°C in a sealed tube for 12 hours. After the reaction was complete, the system was cooled to room temperature and the solvent was removed by rotary evaporation. The product was washed twice with ether to obtain 149 mg of a light red solid S22 in 81% yield.

[0147] 1 H NMR (400MHz, DMSO-d6) δ8.12(s,1H),8.00(dd,J=8.0,1.2Hz,1H),7.61(d,J=8.0Hz,1H),4.63(s,2H),3.88(s,2H),2.87(s,3H),1.59(s,6H).

[0148] 13 C NMR (400MHz, DMSO-d6) δ200.78,166.43,144.33,142.17,131.62,130.31,124,36,115.86,57.76,54.60,50.61,21.83,14.90.

[0149] 3) Preparation of compound S23:

[0150]

[0151] N,N'-dibenzamide (13 mg, 0.0666 mmol) and 0.5 mL of pyridine were added to a solution of S22 (50 mg, 0.133 mmol, 2 eq) in 3 mL of acetic anhydride and heated at 75°C with stirring for 12 h. The mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and the mixture was washed twice with ether to obtain a dark red powder. The powder was dissolved in 2 mL of methanol, and sodium methoxide (29 mg, 0.533 mmol, 8 eq) was added and stirred at room temperature for 2 h. 1 M dilute hydrochloric acid was added to neutralize the mixture, the solvent was removed by rotary evaporation, the mixture was dissolved in water, and the mixture was separated by HPLC (water-acetonitrile system) to obtain 12 mg of S23 as a dark red solid in a 32% yield.

[0152] 1 H NMR (400MHz, Methanol-d4) δ8.63(t,J=13.6Hz,1H),8.12(d,J=1.2Hz,2H),8.10(dd,J=8.4,1.2Hz,2H),7 .44(d,J=8.4Hz,2H),6.55(d,J=13.6Hz,2H),4.31(t,J=5.2Hz,4H),3.96(t,J=5.2Hz,4H),1.79(s,12H).

[0153] 13 C NMR (400MHz, Methanol-d4) δ178.34,168.98,153.43,147.71,142.32,132.20,129.12,124.67,112.85,105.80,60.04,50.67,48.30,28.24.

[0154] 4) Preparation of compound S24:

[0155]

[0156] HATU (145 mg, 0.38 mmol, 3 eq) and triethylamine (100 mg, 0.76 mmol, 6 eq) were added to a solution of S23 (80 mg, 0.13 mmol) in 5 mL of DMF and stirred at room temperature for 10 min. Subsequently, S1 (90 mg, 0.38 mmol, 3 eq) was added and stirred at room temperature for 12 h. After completion of the reaction, separation by HPLC (water-acetonitrile system) afforded 103 mg of S24 as a dark red solid in 75% yield.

[0157] 1 H NMR (400MHz, Methanol-d4) δ8.66(t,J=13.2Hz,2H),8.03(s,2H),7.96(d,J=8.4Hz,2 H),7.42(d,J=8.4Hz,2H),6.54(d,J=13.2Hz,2H),4.30(t,J=4.8Hz,4H),3.77(t,J=4. 8Hz,4H),3.77(t,J=5.6Hz,4H),3.45(br,2H),3.27(s,4H),3.13(br,2H),2.94(s,6H) ,1.79(s,12H),1.72(t,J=8Hz,4H),1.34(s,4H),1.25(s,36H),0.85(t,J=6.8Hz,6H).

[0158] 13 C NMR(400MHz,Methanol-d4)δ178.06,170.09,153.23,147.04,142,40,131.66,129.93,122.68,112.94,105.73,60.06,5 7.75,56.89,50.74,48.29,41.07,36.39,33.04,30.71,30.60,30.48,30.44,30.18,28.25,27.49,25.13,23.71,14.43.

[0159] 5) Preparation of compound S25:

[0160]

[0161] HATU (42 mg, 0.11 mmol, 4 eq) and DIPEA (29 mg, 0.22 mmol, 8 eq) were added to a solution of COTCOOH (16 mg, 0.11 mmol, 4 eq) in 5 mL of DMF and stirred at room temperature for 10 min. S24 (30 mg, 0.028 mmol) and 0.5 mL of pyridine were then added and stirred at room temperature for 48 h. After completion of the reaction, HPLC separation (water-acetonitrile system) afforded 21 mg of S25 as a dark red solid in 50% yield.

[0162] 1 H NMR (400MHz, Methanol-d4) δ8.62(t,J=13.6Hz,1H),8.09(d,J=1.6Hz,2H),8.02(dd,J=8.4,1. 6Hz,2H),7.52(d,J=8.4Hz,2H),6.45(d,J=13.6Hz,2H),5.78–5.68(m,14H),4.66(br,4H),4.5 8(br,4H),3.82(t,J=5.6Hz,4H),3.51–3.48(m,2H),3.42–3.32(m,4H),3.18–3.12(m,2H),2.9 8(s,6H),1.81(s,12H),1.78–1.74(m,4H),1.38(br,4H),1.29(s,36H),0.89(t,J=6.8Hz,6H).

[0163] 13 C NMR(400MHz,Methanol-d4)δ178.01,169.91,166.54,153.60,146.42,142.29, 135.40,134.35,133.84,133.01,132.56,131.94,130.90,130.22,130.07,122 .80,112.86,105.93,61.83,57.76,56.88,50.81,47.88,44.76,41.10,36.42,33.05,30.72,30.60,30.48,30.45,30.19,28.26,27.50,25.16,23.72,14.44.

[0164] 6) Preparation of PPM-Cy3:

[0165]

[0166] 1,3-Propane sultone (15 mg, 0.12 mmol, 5 eq) and potassium carbonate (17 mg, 0.12 mmol, 5 eq) were added to a solution of S25 (3 mg, 0.024 mmol) in acetonitrile and stirred at 55°C for 24 h. The solid was removed by filtration and purified by HPLC to afford 17 mg of PPM-Cy3 as a dark red solid in a 47% yield.

[0167] 1 H NMR (400MHz, Methanol-d4) δ8.62(t,J=13.2Hz,2H),8.09(s,2H),7.96(d,J=8.0Hz,2H ),7.53(d,J=8.0Hz,2H),6.57(d,J=12.4Hz,1H),5.81–5.67(m,14H),4.66(br,4H),4.5 9(br,4H),3.88(br,4H),3.66–3.62(m,4H),3.59(br,4H),3.18(s,6H),2.92(t,J=5.6H z,4H),2.27(br,4H),1.81(br,16H),1.38(s,4H),1.28(s,36H),0.89(t,J=7.2Hz,6H).

[0168] 13 C NMR(400MHz,Methanol-d4)δ177.98,169.11,166.53,153.59,146.42,144.9 4,142.38,135.40,134.31,133.84,133.03,132.58,131.98,130.87,130.10, 122.74,112.95,105.98,63.81,61.94,60.25,50.85,44.77,34.77,33.06,30.74,30.64,30.59,30.46,30.26,28.34,27.45,23.73,23.23,19.67,14.47.

[0169] The synthetic routes of PPM-Cy3.5 and PPM-Cy5 are exactly the same as those of PPM-Cy3, except that in the synthesis step S23, the substrate S22 is replaced by S6 or the substrate N,N-dibenzamide is replaced by pentadienaldehyde diphenylamine. The reaction conditions remain unchanged, and the synthesis of PPM-Cy3.5 or PPM-Cy5 dyes can be achieved.

[0170] ESI mass spectrometry results, PPM-Cy3.5 (M+H) 2+Calculated for C 91 H 126 N6O 12 S2 + ,779.9,observed 780.2.

[0171] ESI mass spectrometry results, PPM-Cy5 (M+H) 2+ Calculated for C 85 H 124 N6O 12 S2 + ,742.4,observed742.3.

[0172] Example 4

[0173] HeLa cells cultured in DMEM were fixed with paraformaldehyde for 10 minutes, washed with HBSS to remove residual paraformaldehyde, and then stained with 200 nM PPM-Cy, AZ-Cy-Me, or MemGlow staining solution for 15 minutes. Under illumination with the same excitation intensity, the fluorescence emission intensity changes of the three dyes labeled with PPM-Cy (PPM-Cy3, PPM-Cy3.5, or PPM-Cy5), MemGlow, and AZ-Cy-Me (AZ-Cy3-Me, AZ-Cy3.5-Me, or AZ-Cy5-Me) on the fixed HeLa cells were measured. Under illumination with the same excitation intensity, the fluorescence emission intensity changes of the three dyes labeled with PPM-Cy, MemGlow, and AZ-Cy-Me on the fixed HeLa cells were measured. The time at which the probe had maximum fluorescence intensity was defined as t = 0, and the fluorescence intensity was normalized.

[0174] The results of the photostability comparison between PPM-Cy and the commercial dye MemGlow on the cell membrane are as follows: Figure 1 As shown, the photostability of PPM-Cy is 2-3 times that of the commercial dye MemGlow, a significant improvement. The photostability improvement of the AZ-Cy-COT series of dyes is consistent with that of PPM-Cy.

[0175] AZ-Cy-Me is a negative control synthesized in this application. AZ-Cy-Me and AZ-CY-COT are otherwise structurally identical, but lack the COT group, making its photostability and phototoxicity unsuitable for imaging. The structure of AZ-Cy-Me is shown below. AZ-Cy-Me has three structures, differing only in the presence of Cy3, Cy5, or Cy3.5 as the parent nucleus.

[0176]

[0177] Example 5

[0178] The ability of the fluorophore to generate ROS was evaluated using 1,3-diphenylisobenzofuran (DPBF). -2 ;590-600nm,0.050W·cm -2 ;620-630nm,0.0125W·cm -2 ) irradiated a mixed solution containing 1 μM PPM-Cy dye. At regular time points, changes in the UV-visible absorption spectrum of DPBF were observed at 415 nm, resulting in a linear plot of absorption intensity versus time. The quantum yield of singlet oxygen was determined by comparing the slope of the sample with that of a standard.

[0179] 1,3-Diphenylisobenzofuran (DPBF) can be degraded by singlet oxygen oxidation, resulting in a decrease in the absorbance of its solution at 415nm. PPM-Cy or AZ-Cy-Me diluted in methanol reacts with DPBF and then illuminates it with an LED light at fixed time points. At fixed time points, the changes in the absorption spectrum of DPBF at 415nm are observed. The experimental results are as follows: Figure 2 As shown in Figure 2 , the absorption intensity shows a linear relationship with time, with tetramethylrhodamine ethyl ester (TMRE) and methylene blue (MB) as references. It was found that the photostability of AZ-Cy-Me and MemGlow (cytoskeleton, MG02-02, MG03-02, MG 04-02) was essentially the same, while the photostability of PPM-Cy was 2-3 times higher. In vitro phototoxicity experiments showed that the ROS generation rate of PPM-Cy was only approximately half that of AZ-Cy-Me in the same wavelength range.

[0180] Cell phototoxicity experiments showed that, using the same excitation intensity, the PPM-Cy series captured 2-3 times more frames before cell death than AZ-Cy-Me and MemGlow in the same wavelength range. These experiments demonstrate that coupling cyclooctatetraene with negatively charged groups to cyanine dyes significantly reduces phototoxicity and improves photostability, making them ideal for super-resolution and long-term imaging.

[0181] Example 6

[0182] Remove the DMEM medium from the HeLa cells and wash three times with HBSS or PBS buffer. Add 200 nM AZ-Cy-COT, PPM-Cy, or NPM-Cy solution and stain at 37°C for 15 minutes or at room temperature for half an hour. Imaging was performed directly under a confocal microscope.

[0183] The cell membrane localization of AZ-Cy-COT, PPM-Cy, or NPM-Cy was determined. All of the membrane dyes achieved good cell membrane localization on Hela cells, with uniform staining, minimal internalization, and the ability to clearly label structures such as plasmodesmata. Wash-free imaging was also achieved. Figure 3 shown.

[0184] Example 7

[0185] The cytotoxicity assay of PPM-Cy dye was detected using Cell Counting Kit 8 (CCK8). 24 hours before the cytotoxicity assay was performed in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, a total of 1x104 Hela cells / well were seeded in a 96-well plate and incubated at 37°C in a 5% CO2 incubator. Then, 100 μL DMEM containing 20nM, 200nM and 1000nM AZ-Cy-COT was added to the Hela cells and cultured in an incubator for 1 hour. 1% Triton was used as a positive control for cytotoxicity or a DMEM-treated cell with the same amount of DMSO (0.1% v / v) as a control solution using the test dye. After the dye was incubated for 1 hour, 10 μl CCK-8 solution was added to each well of the plate. Then, the plate was cultured in an incubator for 4 hours. The absorbance at 450nm was measured. Six replicates of each dye concentration were evaluated in three independent experiments. We calculated the percentage of cell viability for each concentration compared to the control DMEM + 0.1% DMSO.

[0186] The CCK-8 assay showed that even though the dye had very low cytotoxicity, even 1 μM of PPM-Cy did not cause cytotoxicity. Figure 4 shown.

[0187] Example 8

[0188] Remove the DMEM medium of HeLa cells and wash them three times with HBSS or PBS buffer. Add 200nM NPM-Cy dye or Cell Mask Deep Red (Invitrogen TM C10046) solution, stained at 37°C for 15 min, and imaged directly under a confocal microscope.

[0189] Confocal imaging results are as follows Figure 5As shown in the figure, the commercial Cell Mask DeepRed showed dye internalization at 30 and 60 minutes, and the fluorescence signal inside the cell increased over time. However, NPM1-Cy5 showed almost no dye internalization at 30 and 60 minutes, indicating that changing the overall charge of the dye to neutrality effectively inhibited dye internalization, facilitating cell membrane imaging.

[0190] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0191] The cell membrane defines the cell and plays many important biological functions. The visualization of the cell membrane will make an important contribution to the study of many important physiological events. Existing and commonly used cell membrane dyes have limitations such as poor localization effect, high phototoxicity, low photostability, and fast internalization. In this application, the negatively charged group COO - , membrane-localizing group AZ and triplet quencher cyclooctatetraene were coupled to the classic cyanine dye to synthesize small molecule dyes containing cyanine structure in three bands of 550, 590 and 650, which can be used as cell membrane dyes and named AZ-Cy-COT. The three colors are conducive to compatibility with other fluorescent probes in multi-color imaging processes such as immunofluorescence or ordinary fluorescence. In addition, by adding sulfonic acid groups to adjust the overall charge number of the dye, the NPM-Cy series of membrane dyes were synthesized. This type of electrically neutral dye is less likely to penetrate the cell membrane and enter the cell interior. Compared with commercial cell membrane dyes, it greatly inhibits the internalization of the dye, which is beneficial to the cell membrane imaging effect.

[0192] Experiments have shown that the AZ-Cy-COT series and PPM-Cy cell membrane dyes are superior to commonly used cell membrane dyes on the market in terms of phototoxicity, photostability, and membrane localization. The AZ-Cy-COT, PPM-Cy, and NPM-Cy series of dyes are slightly better than Memglow membrane dyes in terms of membrane localization, and significantly better than dyes such as DiR and Cell Mask. AZ-Cy-COT and PPM-Cy also increase photostability to three times that of Memglow and reduce phototoxicity to half that of MemGlow, making them more suitable for super-resolution and long-term fluorescence imaging. At the same time, the characteristics of the AZ-Cy-COT, PPM-Cy, and NPM-Cy series of dyes also determine that they are also excellent tools for various primary cells and stem cells, and can be used for advanced biological membrane imaging.

[0193] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A dye containing a cyanine structure, characterized in that The dye containing a cyanine structure includes a fluorophore core and a functional group, wherein the functional group includes a membrane-localizing group, and the functional group is connected to the fluorophore core; The fluorophore mother core is a cyanine structure mother core.

2. The dye containing a cyanine structure according to claim 1, wherein The cyanine structure mother core has a structure shown in Formula I, Formula II or Formula III, 3. The dye containing a cyanine structure according to claim 2, wherein The membrane-locating group is connected to the cyanine structure core via a linking group, wherein the linking group comprises an amide bond or an ester bond; Preferably, the linking group is connected to any one or more positions of position 1, position 2 or position 3 of the structure represented by Formula I, Formula II or Formula III; More preferably, the cyanine structure core connected with the linking group has the following structure: Wherein R1 is NH or O.

4. The dye containing a cyanine structure according to claim 3, wherein The membrane-locating group is an AZ group having a structure shown in Formula IV, wherein R is an alkyl group consisting of 4 to 18 C atoms, Preferably, the AZ group has a structure shown in Formula V, 5. The dye containing a cyanine structure according to claim 2, wherein The cyanine structure mother core is also connected to a triplet quenching group; The triplet quenching group is connected to the 3-position and / or 4-position of the structure shown in Formula I, Formula II or Formula III; Preferably, the triplet quenching group is a cyclooctatetraene group, and the cyclooctatetraene group has a structure shown in Formula VI or Formula VII, 6. The dye containing a cyanine structure according to any one of claims 1 to 5, characterized in that The functional group further comprises a negatively charged group, which is directly connected to the linking group or connected to the linking group via a carbon chain; Preferably, the carbon chain is an alkylene group consisting of 1 to 4 C atoms.

7. The dye containing a cyanine structure according to claim 6, characterized in that The negatively charged group is COO - or SO3 - ; Preferably, the dye containing a cyanine structure has the following structure: Among them, R2 is O or NH, R3 is COO - or SO3 - , X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + ; More preferably, the dye containing a cyanine structure is AZ-Cy3.5-COT, AZ-Cy3-COT or AZ-Cy5-COT, Among them, X + is a cationic group; preferably, the cationic group includes Na + , K + NH4 + or NMe4 + .

8. The dye containing a cyanine structure according to claim 6, characterized in that The negatively charged group is SO3 - , the negatively charged group is directly connected to the cyanine structure mother core or is connected to the cyanine structure mother core through a carbon chain; Preferably, the negatively charged group is connected to the 2-position or 4-position of the structure represented by Formula I, Formula II or Formula III.

9. The dye containing a cyanine structure according to claim 8, characterized in that The dye containing a cyanine structure is wherein n=1 or 2, m is a positive integer of 1-5, and R2 is independently selected from O or NH.

10. The dye containing a cyanine structure according to any one of claims 1 to 5, characterized in that The dye containing a cyanine structure is Among them, Y - is an anion; Preferably, Y is trifluoroacetic acid, PF6, BH4, ClO4, Cl, Br or I.

11. Use of the dye containing a cyanine structure according to any one of claims 1 to 10 in cell membrane imaging.

12. The use according to claim 11, characterized in that The application includes: staining living cells with the dye containing the cyanine structure to obtain stained cells; imaging the stained cells with a microscopic imaging technique; Preferably, the microscopic imaging technique includes fluorescence imaging; more preferably, the fluorescence imaging technique includes confocal imaging technique, super-resolution imaging technique, wide-field imaging technique or two-photon in vivo imaging technique; Preferably, the imaging comprises long-term imaging; More preferably, the long-term imaging time is ≥ 4 hours.

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