Triply-methine pyridinium cyanine dyes, methods of making and using the same

Abstract

The application discloses a kind of trimethylenepyridinium cyanine dyes, its preparation method and application, by regulating the structure of nitrogen-containing heterocycle, can obtain a series of dyes molecules after binding DNA, ultraviolet absorption and fluorescence emission in red light region, spectrum is adjustable, synthesis is convenient;Specifically bind DNA, fluorescence quantum yield is high, brightness is big;Cell permeability is good, can dye cell nucleus, dyeing speed is fast, background interference is small, without repeated washing. It can be applied to fluorescence imaging, marking and tracing, blood cell analysis, clinical medical diagnosis, immune analysis detection field based on cell nucleus dyeing, thereby for nucleic acid research and clinical detection provides a more sensitive, safe and economic and efficient new dye scheme.

Description

Technical Field

[0001] The present invention relates to the field of fine chemicals, in particular to a type of trimethine pyridinium cyanine dye, a preparation method and application thereof. Background Art

[0002] DNA-labeling fluorescent dyes are widely used in biomedical fields such as gel electrophoresis, quantitative PCR, flow cytometry, imaging and tracking of cell nuclei, and blood cell analysis. Commercially available DNA dyes primarily include phenanthridines (EB, PI), acridines (AO), imidazoles (Hoechst, DAPI), and cyanines (Cy, TOTO, SYTO). For nuclear labeling, the DNA-specific blue fluorescent dyes DAPI and Hoechst 33342 remain the preferred choice for many applications due to their ease of use and low cost. However, these require ultraviolet lasers, and due to their high-energy excitation, ultraviolet light can cause DNA damage and cell death. In recent years, fluorescent dyes emitting at longer wavelengths (preferably in the red / near-infrared (NIR) region) have become increasingly popular. Compared to short-wavelength fluorescent dyes, these dyes exhibit less autofluorescence interference and allow for deeper tissue imaging depths. Furthermore, longer wavelength excitation light reduces dye photobleaching and photodamage to biological samples.

[0003] Currently, commercial red DNA dyes are monopolized by foreign countries, with limited products and high prices (SYTO TM DeepRed). Therefore, it is necessary to develop DNA-labeled fluorescent dyes that emit in the red / near-infrared region to achieve domestic substitution. The common strategy for constructing red DNA-labeled fluorescent dyes is to connect fluorescent dyes excited by visible light wavelengths to Hoechst dyes through linkers. For example, the far-infrared emitting silorhodamine dye is covalently linked to Hoechst 33342 to achieve specific labeling of nuclear DNA (Nat. Commun., 2015, 6, 8497.), but this method will reduce the binding strength of the dye to DNA (K D=8.4±0.5μM). Cyanine dyes can be excited by visible light and near-infrared light. By extending the methine chain on the basis of cyanine dyes, red fluorescent dyes that specifically bind to nucleic acids can be obtained, but they have low discrimination between DNA and RNA and cannot specifically stain cell nuclei. The pyridine cyanine dyes reported in 2021 improved the discrimination between DNA and RNA (Nat. Commun., 2021, 12, 2650.), but the fluorescence brightness after binding to DNA is still insufficient. Although researchers have made a series of progress in the development of DNA-labeled fluorescent dyes emitting in the red / near-infrared region, as the absorption / emission wavelength of the fluorophore red-shifts, it is a huge challenge to maintain the high brightness of the fluorophore (narrow band gap molecules usually have longer conjugated hydrophobic backbones, and their larger molecular charge transfer makes it easy for them to interact with external molecules, resulting in an increased probability of non-radiative transitions). Therefore, the development of an ideal dye with high sensitivity, high specificity, high brightness and excellent biocompatibility still faces huge challenges. In-depth research in this field requires not only the careful design and optimization of the dye's molecular structure to improve its binding affinity with DNA and fluorescence efficiency, but also the resolution of dye stability and targeting issues in complex biological environments. For different application scenarios such as living cell and in vivo imaging, new DNA dyes must also possess characteristics such as low toxicity, low background interference, and good penetration depth. Summary of the Invention

[0004] In view of the problems existing in the existing technology, the red specific DNA dye should have the following characteristics: (1) high discrimination between DNA and RNA; (2) high fluorescence quantum yield and high brightness after binding to DNA;

[0005] (3) long-wavelength absorption and fluorescence emission; (4) high fluorescence brightness for DNA staining in vivo; the present invention provides a class of trimethine pyridinium cyanine dyes and their preparation methods and applications. These dyes have an absorption wavelength between 550 and 700 nm, have a high degree of differentiation between DNA and RNA, exhibit good cell permeability in both live and fixed cell staining, and can achieve specific labeling of nuclear DNA.

[0006] The trimethine pyridinium cyanine dyes described in the present invention have the following structure of general formula I:

[0007]

[0008] In formula I, A is a nitrogen-containing heterocycle selected from At least one of;

[0009] R1 is selected from C 1-6 Alkyl, -(CH2) p COOR3, -(CH2)p OR3, -(CH2) p At least one of NR3 or a benzyl group substituted with R4, and p is selected from any integer from 1 to 6;

[0010] R2 is selected from H, C 1-6 alkyl, or at least one of phenyl groups;

[0011] R3 is H, C 1-6 At least one of an alkyl group or a phenyl group;

[0012] R4 is at least one of H, halogen, alkoxy, amide or nitro;

[0013] X - Selected from halogen anions, ClO4 - PF6 - CF3 - 、BF4 - , or OTs - At least one of .

[0014] For the technical solution described above, further specific compound structures of the compounds represented by general formula I are given, but the present invention is not limited to these specific examples:

[0015]

[0016] Another aspect of the present application is to protect the preparation method of the above-mentioned trimethine pyridine cyanine dye, comprising the following steps:

[0017]

[0018] In a first reaction solvent and a base, in the presence of a first catalyst, the compound represented by Formula IV and the compound represented by Formula V undergo a condensation reaction at a molar ratio of 1:1-2 (more preferably a molar ratio of 1:1.2) to obtain the target compound represented by I;

[0019] For the technical solution described above, more preferably, the first reaction solvent is at least one of dichloromethane, methanol or pyridine; the amount of the first reaction solvent is 4-10 times the mass of the total reaction compounds; more preferably, the multiple is 5 times.

[0020] For the technical solution described above, more preferably, the first catalyst is selected from at least one of p-toluenesulfonic acid, sodium acetate or acetic anhydride; the molar ratio of the first catalyst to the compound represented by formula IV is 1:1-2; the most preferred molar ratio is 1:1.5;

[0021] For the technical solution described above, more preferably, the base is at least one of triethylamine, diethylamine, pyridine, dimethylaminopyridine or N,N-diisopropylethylamine; the molar ratio of the base to the compound represented by formula IV is 1:1-3, and the more preferred molar ratio is 1:2;

[0022] For the technical solution described above, more preferably, the reaction time is 0.5-3 h and the reaction temperature is 10-60°C; more preferably, the temperature is 20-40°C.

[0023] For the technical solution described above, further, the compound represented by formula IV is prepared by the following method:

[0024]

[0025] In a second reaction solvent, 4-methylpyridine and 2,4-dinitrohalobenzene react at a molar ratio of 1:1-2 (more preferably a molar ratio of 1:1.2) to obtain a compound represented by Formula II;

[0026] In a second reaction solvent, the compound represented by formula II and R2-substituted aniline are reacted at a molar ratio of 1:1-2 (more preferably a molar ratio of 1:1.2) to obtain an N-aryl pyridinium salt represented by formula III;

[0027] In the presence or absence of a third reaction solvent, the compound represented by formula III is reacted with N,N-diphenylformamidine at a molar ratio of 1:1-2 (more preferably a molar ratio of 1:1.2) to obtain the compound represented by formula IV;

[0028] In the presence or absence of a second reaction solvent, the nitrogen-containing heterocycle and the R1-substituted halogenated hydrocarbon are reacted in a molar ratio of 1:1-2 (more preferably a molar ratio of 1:1.5) to obtain the compound represented by formula V.

[0029] For the technical solution described above, the preferred second reaction solvent is at least one of methanol, ethanol, acetonitrile or toluene; the third reaction solvent is acetic acid and / or acetic anhydride;

[0030] For the technical solution described above, more preferably, the amount of the second reaction solvent added is 5-10 times the mass of the total reaction compound; the amount of the third reaction solvent added is 1-2 times the mass of the total reaction compound;

[0031] For the technical solution described above, the preferred reaction time of 4-methylpyridine and 2,4-dinitrohalobenzene is 6-24 h, and the reaction temperature is 70-110° C., and the further preferred temperature is 90° C.

[0032] For the technical solution described above, the preferred reaction time of the compound represented by formula II and the R2-substituted aniline is 3-24 h, and the reaction temperature is 10-100° C.; the temperature is more preferably 70-100° C.

[0033] For the technical solution described above, the reaction time of the compound represented by formula III and N,N-diphenylformamidine is 1-5 hours, and the reaction temperature is 60-160°C; the most preferred reaction temperature is 150°C.

[0034] For the technical solution described above, more preferably, the reaction time of the nitrogen-containing heterocycle and the R1-substituted halogenated hydrocarbon is 4-24 hours, and the reaction temperature is 80-130°C; more preferably, the temperature is 100°C;

[0035] Another aspect of the present application is to protect the use of the above-mentioned trimethine pyridinium cyanine dye.

[0036] For the technical solution described above, further, the application is to apply the trimethine pyridinium cyanine dye to the fields of fluorescence imaging, labeling and tracing based on cell nucleus staining, blood cell analysis, clinical medical diagnosis, immunoassay detection, etc.; specifically:

[0037] 1. The fluorescence imaging based on cell nuclear staining utilizes its strong absorption and emission characteristics in the red region after binding to DNA to achieve accurate visualization of cell nuclear DNA;

[0038] 2. The in vivo cell labeling and tracing utilizes its high affinity and specificity with DNA to achieve the location and labeling of specific cells or nucleic acid molecules;

[0039] 3. The blood cell analysis is to perform refined detection and differentiation of cell types with specific nuclear structures, such as white blood cell subtypes;

[0040] 4. The clinical medical diagnosis mentioned above utilizes its fluorescence enhancement property after binding to DNA to label and quantify the nuclear structures of different cells, thereby improving and innovating existing clinical in vitro detection methods and increasing the accuracy and sensitivity of disease diagnosis;

[0041] 5. The immunoassay detection described above utilizes its high affinity and specificity for DNA to improve and innovate existing fluorescent immunoassay and fluorescent in situ hybridization detection technologies, thereby increasing the accuracy and sensitivity of disease diagnosis.

[0042] Regarding the technical solution described above, further, the blood cell analysis includes: (1) identifying whether the cell has a nucleus; (2) distinguishing various subtypes of white blood cells, including eosinophils, basophils, neutrophils, lymphocytes and monocytes, and clearly identifying the different morphological characteristics of these cell nuclei; (3) identifying other cell types with specific nuclear structures.

[0043] The dye technology can not only accurately label and distinguish different white blood cell subtypes in peripheral blood cells, but also has broad application potential. It can be adapted to the refined detection and diagnosis of various cell types with specific nuclear structures, such as distinguishing cells with different functions in the immune system, such as epithelial cells, endothelial cells, nerve cells, stem cells and tumor cells, thereby broadening the scope of research and clinical applications in various biomedical fields.

[0044] After binding to DNA, the trimethine pyridinium cyanine dye exhibits significant absorption characteristics in the ultraviolet-visible light absorption spectrum within the wavelength range of 550-700 nm, a red shift of the maximum absorption wavelength by 10-30 nm, and an increase in the molar extinction coefficient by 10-120%. The fluorescence quantum yield is higher than 50%, the fluorescence brightness is greater than 40,000, and even reaches 71,820. The DNA / RNA discrimination is greater than 2, more preferably greater than 5, and even more preferably greater than 7.

[0045] Even reaching 11.1;

[0046] The working concentration of the trimethine pyridinium cyanine dye for staining cell nuclei is 0.1-1 μM, preferably 0.1-0.5 μM, so as to reduce the amount of dye used while ensuring sufficient labeling of the cell nucleus, thereby reducing potential cytotoxicity and economic costs.

[0047] When the trimethine pyridinium cyanine dye is used for laser confocal nuclear staining imaging, it is recommended to use a laser intensity of 0.6 to 2.0 μW for effective excitation, wherein the preferred laser intensity range is 0.6 to 1.0 μW; under further optimized conditions, the most suitable laser intensity is set to 0.8 μW.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] First, by manipulating the structure of the nitrogen-containing heterocyclic ring, the trimethine pyridinium cyanine dye disclosed in the present invention can produce a series of dye molecules with UV absorption wavelengths between 550 and 700 nm after binding to DNA, a red-shifted maximum absorption wavelength (10-30 nm), and an increased molar extinction coefficient (10%-120%). These dye molecules are spectrally tunable and easy to synthesize. They can be excited by a red semiconductor laser, increasing the tissue penetration depth of the dye and reducing photodamage to biological samples. The red-shifted absorption wavelength and increased molar extinction coefficient further reduce background fluorescence signals.

[0050] Second, the trimethine pyridine cyanine dye disclosed in the present invention specifically binds to DNA and has a high fluorescence quantum yield. Among them, the fluorescence quantum yield of dye 2 and dye 3 after binding to DNA reached 54% and 56% respectively; the brightness reached 71820 and 40752, which is significantly improved compared with the comparative example and the existing technical solution. It is the red DNA labeling dye with the highest fluorescence brightness known. It is of great significance to improve detection sensitivity and reduce background interference. In particular, dye 3 has a discrimination degree of 11.1 for binding to DNA / RNA, which is much higher than 1.98 of the comparative example. It reveals that the regulation of the dye structure makes it more specific and can effectively distinguish DNA and RNA, which is of great value for nucleic acid research and clinical detection.

[0051] Third, the trimethine pyridinium cyanine dye disclosed in the present invention has good cell permeability, can stain cell nuclei, has a fast staining speed, low background interference, does not require repeated washing, and has a low working concentration. Among them, the control sample has poor cell permeability and cannot mark cell nuclei in live cell imaging, while dye 2 can clearly mark cell nuclei, especially different subtypes of peripheral blood leukocyte nuclei, and can also be clearly marked; the staining effect is comparable to that of the blue DNA marker dye Hochest 33342 and the red SYTO TM It is comparable to Deep Red, but has a lower working concentration than Hoechst 33342, and can be used for real-time and high-fidelity imaging of cell nuclei at an extremely low dose (100nM) (the examples show that the effective concentration of dye 2 is 1 / 3 of that of Hoechst 33342 (0.1μM vs 0.3μM), that is, the concentration requirement of dye 2 is reduced by about 3 times for the same effect); TM Compared with Deep Red, the laser working intensity is lower (wherein, the embodiment proves that the effective intensity of dye 2 is SYTO TM The laser power requirement for Dye 2 is approximately 85 times lower than that of Deep Red (0.2% vs 17%), significantly reducing cytotoxicity and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Figure 2 is a comparative example, and a nucleic acid (DNA and RNA) response test diagram of dyes 2-6;

[0053] Figure 2 This is a live cell uptake experiment of dye 2;

[0054] Figure 3 This is a diagram of the co-localization experiment of dye 2 and the commercial nuclear dye Hoechst 33342 in living cells;

[0055] Figure 4 This is a diagram showing the colocalization experiment of dye 2 and the commercial nuclear dye Hoechst 33342 in fixed cells;

[0056] Figure 5 Images of living cells stained with different concentrations of dye 2 and the commercial nuclear dye Hoechst 33342;

[0057] Figure 6 Figure 2 shows the experiment of dye 2 and commercial red DNA labeling dye SYTOTM Deep Red staining cells.

[0058] Figure 7 This is a live cell staining experiment diagram for the comparative example;

[0059] Figure 8 This is a diagram of the peripheral blood cell staining experiment using dye 2. DETAILED DESCRIPTION

[0060] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-8 , not all possible implementations. Nevertheless, any technician familiar with the field, without making any creative efforts, should consider all other implementations that can be obtained based on the embodiments disclosed in the present invention to fall within the scope of protection of the present invention.

[0061] Example 1

[0062] The synthetic route of the compound represented by general formula I of the present invention is:

[0063]

[0064] Starting from 4-methylpyridine, it reacts with 2,4-dinitrohalobenzene to prepare the compound of formula II;

[0065] The compound of formula II reacts with aniline substituted with R2 to prepare a compound of formula III; then undergoes a condensation reaction with N,N-diphenylformamidine to prepare a compound of formula IV; the nitrogen-containing heterocycle reacts with a halogenated hydrocarbon substituted with R1 to prepare a compound of formula V; finally, the compound represented by formula IV undergoes a condensation reaction with the compound represented by formula V to prepare a compound of formula I.

[0066] The compound represented by general formula I of the present invention can be synthesized by the method described below.

[0067] The compound shown in Ⅱ involved in this application is prepared by the following method:

[0068] S1: Synthesis of the compound represented by formula II

[0069] 50 mmol of 2,4-dinitrochlorobenzene and 55 mmol of 4-methylpyridine were dissolved in 40 mL of ethanol and refluxed for 6 h. The reaction was stopped. 100 mL of tert-butyl methyl ether was added and stirred for another 2 h. The mixture was filtered and air-dried to obtain 9.6 g of a black solid (yield: 65%).

[0070] Synthesis of the compound represented by formula III

[0071]

[0072] S2: Synthesis of III-1

[0073] 10.15 mmol of the compound of formula II obtained in step S1 was dissolved in 20 mL of ethanol, and 20.29 mmol of solid aniline was added. The reaction was heated under reflux for 3-8 h, then the reaction was stopped. The mixture was cooled to room temperature, the solvent was dried, and purified by column chromatography to obtain 0.79 g of compound III-1 (yield: 36%).

[0074] S3: Synthesis of III-2

[0075] 10.15 mmol of the compound of formula II obtained in step S1 was dissolved in 20 mL of ethanol, and 12.18 mmol of solid N,N-dimethylaniline was added. The reaction was heated under reflux for 3-8 h, then the reaction was stopped. The mixture was cooled to room temperature, the solvent was dried, and the mixture was purified by column chromatography to obtain 1.6 g of compound III-2 (yield: 63%).

[0076] Synthesis of the compound represented by formula IV

[0077]

[0078] S4: Synthesis of VI-1

[0079] 0.729 mmol of III-1 and 1.09 mmol of N,N-diphenylformamidine were placed in a 50 mL round-bottom flask and heated to 160°C for 2 h before stopping the reaction. After cooling to room temperature, 25 mL of ethanol was added and stirring continued for 2 h. After filtration and drying, 0.19 g of a black solid was obtained (yield: 84%).

[0080] S5: Synthesis of VI-2

[0081] 0.603 mmol of III-2 and 0.904 mmol of N,N-diphenylformamidine were placed in a 50 mL round-bottom flask and heated to 160°C for 2 h before stopping the reaction. After cooling to room temperature, 25 mL of ethanol was added and stirring continued for 2 h. The mixture was filtered and air-dried to obtain 0.17 g of a black solid (80% yield).

[0082] Synthesis of the compound represented by formula V

[0083]

[0084] S6: Synthesis of V-1

[0085] Weigh 10.52 mmol of 5-iodo-2,3,3-trimethyl-3H-indole and dissolve it in 10 mL of acetonitrile solution. Add 21.04 mmol of iodoethane and reflux for 24 hours before stopping the reaction. Cool to room temperature, filter, and wash with a mixed solvent of ethyl acetate and tert-butyl methyl ether to obtain 4 g of yellow solid (yield:

[0086] 86%).

[0087] S7: Synthesis of V-2

[0088] Weigh 13.4 mmol of 2-methylbenzothiazole and 16.08 mmol of bromoethanol into a 100 mL round-bottom flask, heat to 110°C, stir for 6 hours, and then stop the reaction. Cool to room temperature, filter, and wash with a mixed solvent of ethyl acetate and tert-butyl methyl ether to obtain 1.03 g of a white solid (yield:

[0089] 25%).

[0090] S8: Synthesis of V-3

[0091] Weigh 1.55 mmol of 5-methoxy-2-methylbenzoselenazole and 2.32 mmol of iodoethane into a 25 mL round-bottom flask. Heat to 120°C and stir for 6 hours before stopping the reaction. Cool to room temperature, filter, and wash with a mixture of ethyl acetate and tert-butyl methyl ether to obtain 0.316 g of a white solid (yield: 53%).

[0092] S9: Synthesis of V-4

[0093] Weigh 20.95 mmol of 2-methylquinoline and 62.85 mmol of iodoethane into a 50 mL round-bottom flask. Heat to 100°C and stir for 10 hours before stopping the reaction. Cool to room temperature, filter, and wash with a mixture of ethyl acetate and tert-butyl methyl ether to obtain 2.5 g of a pale yellow solid (yield: 39%).

[0094] S10: Synthesis of V-5

[0095] Weigh 34.92 mmol of 4-methylquinoline and 48.89 mmol of iodoethane into a 100 mL round-bottom flask, heat to 100°C, and stir for 4 hours before stopping the reaction. Cool to room temperature, filter, and wash with a mixture of ethyl acetate and tert-butyl methyl ether to obtain 10.35 g of a pale yellow solid (yield: 99%).

[0096] Example 1

[0097] Synthesis of Dye 1:

[0098]

[0099] 1.46 mmol of compound VI-1 was dissolved in 6 mL of dichloromethane. 1.89 mmol of compound V-1 was added, followed by 0.6 mL of N,N-diisopropylethylamine and 0.3 mL of acetic anhydride. The reaction was stirred at room temperature for 4 hours before terminating. The product was then spin-dried and purified by column chromatography to yield 0.53 g of a purple-black solid (yield: 58%). 1 H NMR (400MHz, DMSO-d6) δ8.66(d,J=6.9Hz,2H),8.14(t,J=13.3Hz,1H),7.96(d,J= 6.7Hz,2H),7.84(s,1H),7.79(d,J=7.7Hz,2H),7.70(t,J=7.5Hz,2H),7.66(d,J= 7.0Hz,1H),7.60(d,J=8.2Hz,1H),6.95(d,J=8.3Hz,1H),6.45(d,J=14.1Hz,1H), 5.94(d,J=12.5Hz,1H),3.89(q,J=7.1Hz,2H),1.64(s,6H),1.18(t,J=7.1Hz,3H). 13 C NMR (101 MHz, DMSO) δ

[0100] 165.36,154.25,143.09,142.88,142.76,141.92,137.02,131.18,130.64,130.53,1 24.39,121.03,117.22,111.43,98.74,85.61,47.60,37.61,28.35,11.90.HR-MS:m / z calcd for C 26 H 26 N2I + [M] + :493.1136,found:493.1148.

[0101] Example 2

[0102] Synthesis of Dye 2:

[0103]

[0104] 0.226 mmol of compound VI-1 was dissolved in 4 mL of dichloromethane, followed by the addition of 0.226 mmol of compound V-2. 0.4 mL of triethylamine and 0.4 mL of acetic anhydride were then added. The mixture was heated to 30°C and stirred for 2 h before stopping the reaction. The residue was then spin-dried and purified by column chromatography to yield 0.030 g of a purple-black solid (30% yield). 1 H NMR(400MHz, DMSO-d6)δ8.49(d,J=7.1Hz,2H),8.02–7.91(m,1H),7.82(d,J=6.8H z,1H),7.75(d,J=7.8Hz,2H),7.71(s,2H),7.67(t,J=7.5Hz,2H),7.60(t,J=7.3Hz ,1H),7.52(d,J=8.2Hz,1H),7.45(t,J=8.4Hz,1H),7.25(t,J=7.0Hz,1H),6.28(d, J=6.4Hz,1H),6.25(d,J=4.7Hz,1H),4.48(t,J=5.1Hz,2H),4.40(t,J=5.0Hz,2H). 13 C NMR (101MHz, DMSO) δ159.76,153.21,143.43,142.67,141.57,140.71,130.60,130.05,127.9 8,125.04,124.12,123.91,122.95,119.43,113.24,112.26,96.47,40.77,32.57.HR-MS:m / z calcd for C 22 H 19 N2OS + [M] + :359.1213,found:359.1229.

[0105] Example 3

[0106] Synthesis of Dye 3:

[0107]

[0108] 0.369 mmol of compound VI-2 was weighed and dissolved in 4 mL of dichloromethane. 0.369 mmol of compound V-2 was added, followed by 0.4 mL of triethylamine and 0.4 mL of acetic anhydride. The temperature was raised to 30°C and stirred for 7 hours before stopping the reaction. The residue was then spin-dried and purified by column chromatography to obtain 0.072 g of a purple-black solid (yield: 40%). 1H NMR (400MHz, DMSO-d6) δ8.45(d,J=7.3Hz,2H),7.85(dd,J=14.0,12.0Hz,1H),7.77(d ,J=8.3Hz,1H),7.70(d,J=6.8Hz,2H),7.53(d,J=9.1Hz,2H),7.45(t,J=6.1Hz,1H),7. 40(d,J=7.0Hz,1H),7.21(t,J=6.7Hz,1H),6.88(d,J=9.2Hz,2H),6.27(d,J=14.0Hz,1 H), 6.17 (d, J = 12.0Hz, 1H), 4.48 (t, J = 5.1Hz, 2H), 4.40 (t, J = 5.0Hz, 2H), 3.00 (s, 6H). 13 C NMR(101MHz,DMSO)δ158.27,152.39,151.18,142.11,141.65,140.64,131.61,127.83,124.81,1 24.26,123.72,122.81,119.82,113.60,112.80,111.84,95.50,40.51,40.47,32.42.HR-MS:m / z calcd for C 24 H 24 N3OS + [M] + :402.1635,found:402.1655.

[0109] Example 4

[0110] Synthesis of dye 4:

[0111]

[0112] 0.310 mmol of compound VI-1 was dissolved in 4 mL of pyridine, followed by the addition of 0.373 mmol of compound V-3. 0.4 mL of N,N-diisopropylethylamine and 0.4 mL of acetic anhydride were then added. The mixture was stirred at room temperature for 2 h, then the reaction was terminated and poured into 50 mL of tert-butyl methyl ether. The filter cake was filtered and purified by column chromatography to yield 0.152 g of a purple-black solid (87% yield). 1H NMR (400MHz, DMSO-d6) δ8.50(d,J=7.0Hz,2H),7.88(t,J=13.6Hz,1H),7.81–7.72(m,5H),7.67(t,J=7.6Hz,2H),7.61(t,J=7.3Hz,1H),7.01(d,J=2. 1Hz,1H),6.83(d,J=8.6Hz,1H),6.40(d,J=11.9Hz,1H),6.30(d,J=13.8Hz ,1H),4.21(q,J=7.2Hz,2H),3.84(s,3H),1.26(t,J=7.1Hz,3H).HR-MS:m / z calcd for C 24 H 23 nnJC + [M] + :435.0971,found:435.0987.

[0113] Example 5

[0114] Synthesis of Dye 5:

[0115]

[0116] 0.284 mmol of compound VI-2 was dissolved in 4 mL of pyridine, followed by the addition of 0.341 mmol of compound V-4. 0.4 mL of N,N-diisopropylethylamine and 0.4 mL of acetic anhydride were then added. The mixture was stirred at room temperature for 2 h, then the reaction was terminated and poured into 50 mL of tert-butyl methyl ether. The filter cake was filtered and purified by column chromatography to yield 0.055 g of a purple-black solid (yield: 45%). 1 H NMR (400MHz, DMSO-d6) δ8.43(t,J=13.0Hz,1H),8.35(d,J=7.1Hz,2H),8.03(d,J=9.7Hz,1H),7.75–7.55(m,6H),7.51(d,J=9.1Hz,2H),7.27(t, J=7.1Hz,1H),6.87(d,J=9.2Hz,2H),6.29(d,J=13.7Hz,1H),6.00(d,J=12.2Hz,1H),4.24(t,J=14.1Hz,2H),2.99(s,6H),1.33(t,J=7.0Hz,3H). 13C NMR (101MHz, DMSO) δ152.38,151.04,149.49,142.96,140.03,139.57,133.17,132.09,131.71,129.06,1 24.27,124.18,123.75,120.79,119.24,115.29,114.13,112.83,102.20,42.20,40.48,12.20.HR-MS:m / z calcd for C 27 H 28 N3 + [M] + :394.2278,found:394.2294.

[0117] Example 6

[0118] Synthesis of Dye 6:

[0119]

[0120] 0.241 mmol of compound VI-2 was dissolved in 4 mL of pyridine, followed by the addition of 0.289 mmol of compound V-5. 0.4 mL of N,N-diisopropylethylamine and 0.4 mL of acetic anhydride were then added. The mixture was stirred at room temperature for 2 h before tertiary butyl methyl ether was added. The resulting mixture was then poured into 50 mL of tert-butyl methyl ether. The filter cake was filtered and purified by column chromatography to yield 0.100 g of a purple-black solid (yield: 79%). 1 H NMR(400MHz, DMSO-d6)δ8.43(t,J=13.2Hz,1H),8.34(d,J=7.0Hz,2H),8.22(d, J=8.4Hz,1H),7.74–7.66(m,5H),7.52(d,J=9.0Hz,2H),7.41(dt,J=8.2,3.6Hz ,1H),7.36(d,J=7.6Hz,1H),6.87(d,J=9.2Hz,2H),6.66(d,J=12.5Hz,1H),6.3 0(d,J=13.8Hz,1H),4.26(q,J=7.0Hz,2H),2.99(s,6H),1.34(t,J=7.0Hz,3H). 13C NMR (101MHz, DMSO) δ151.93,151.04,145.82,141.54,139.93,138.65,138.26,132.20,131.75,125.15,125. 09,124.15,124.08,119.44,116.84,115.09,112.85,107.57,107.04,47.73,40.50,14.70.HR-MS:m / zcalcd for C 27 H 28 N3 + [M] + :394.2278,found:394.2293.

[0121] Comparative Example

[0122] Synthesis of comparative example:

[0123]

[0124] Referring to the synthesis method for dye 2, 0.244 mmol of the formamidine intermediate and 0.293 mmol of N-ethyl-4-methylquinolate were weighed and dissolved in 4 mL of dichloromethane. 0.4 mL of triethylamine and 0.4 mL of acetic anhydride were added. The mixture was heated to 30°C and stirred for 2 hours before stopping the reaction. The product was then dried and purified by column chromatography to obtain 0.100 g of a purple-red solid (yield: 84%). 1 H NMR (400MHz, DMSO-d6) δ8.48(d,J=7.4Hz,1H),8.44(d,J=7.2Hz,1H),8.16(t,J=12.8Hz,1H),8.09(d ,J=8.8Hz,1H),7.96(t,J=7.2Hz,1H),7.88(t,J=7.2Hz,2H),7.71(t,J=7.5Hz,1H),7.61(d,J=8.2Hz ,1H),7.49(t,J=7.2Hz,1H),7.31(t,J=7.6Hz,1H),7.14(d,J=13.3Hz,1H),6.54(d,J=12.3Hz,1H),4 .61(q,J=7.1Hz,2H),4.30(q,J=7.1Hz,2H),1.45(t,J=7.1Hz,3H),1.33(t,J=7.0Hz,3H).HR-MS:m / z calcd for C 23 H 23 N2S + [M] + :359.1577,found:359.1593.

[0125] Performance test example

[0126] Test Example 1

[0127] The nucleic acid (DNA and RNA) response experiment of the comparative example, dyes 2, 3, 4, 5 and 6 was performed as follows: the comparative example, dyes 2, 3, 4, 5 and 6 were added to PBS buffer (pH = 7.4) to a final concentration of 4 μM, and UV absorption and fluorescence spectrum data were collected. Calf thymus DNA or yeast RNA was then added to the system to a concentration of 100 μg / mL, and UV absorption and fluorescence spectrum data were collected. The test results are shown in Figure 1 The data were sorted and the results are shown in the table below. The maximum absorption wavelengths of dyes 2, 3, 4, 5 and 6 after binding to DNA are between 600-700nm, and the fluorescence emission wavelengths are between 620-720nm; the discrimination between DNA / RNA is 7.09 / 11.10 / 10.11 / 2.49 / 5.46, respectively, which are all higher than the discrimination between DNA / RNA in the control example (1.98). In addition, the fluorescence quantum yields of dyes 2 and 3 after binding to DNA are 0.540 and 0.566, respectively, which are significantly higher than the fluorescence quantum yield of the control example after binding to DNA (0.137). In addition, the dye brightness reaches 71820 and 40752. After a thorough investigation of the literature, it was found that compared with the control example and the existing technical solutions, it has been significantly improved. It is the red DNA labeling dye with the highest fluorescence brightness known so far. This is of great significance for improving detection sensitivity and reducing background interference.

[0128] Table 1 Comparative Example, Nucleic Acid Response Data of Dyes 2-6

[0129]

[0130]

[0131] Test Example 2

[0132] Live cell uptake experiments of dye 2

[0133] The experimental method is as follows: 0.5 μM dye 2 was added to a culture dish containing incubated MCF-7 cells (cell culture density 105 cells / mL, 70-80% coverage of the dish bottom), and representative areas were imaged using a laser confocal microscope. The excitation wavelength of dye 2 was 600 nm, and the receiving wavelength was 610-700 nm. Images were taken every 5 minutes, and the results were displayed on Figure 2 It can be seen that dye 2 completely entered the cell nucleus after 20 minutes, the cell permeability was high, and the staining speed was fast.

[0134] Test Example 3

[0135] Live cell staining experiments with dye 2

[0136] The experimental method is as follows: 1 μM Hoechst 33342 (a widely used commercial nuclear dye) and 0.5 μM dye 2 were added to the incubated MCF-7 cells and MCF-10A cells (cell culture density 105 cells / ml, dish bottom coverage 70-80%), incubated and stained for 20 minutes at 37°C, 5% CO2, and representative areas were selected for imaging using a laser confocal microscope. The results are shown in Figure 3 It can be seen that Dye 2 and Hoechst 33342 have a good colocalization effect whether staining MCF-7 or MCF-10A cells, with correlation coefficients reaching 0.92 and 0.94, respectively. This shows that Dye 2 can specifically target the cell nucleus and has a staining effect comparable to that of commercial nuclear dyes, and can be used for specific labeling of nuclear DNA.

[0137] Test Example 4

[0138] Fixed cell staining experiments with dye 2

[0139] The experimental method is: the cultured cells (cell culture density 10 5 cells / ml, with 70-80% coverage of the dish bottom) were first treated with cooled ethanol (-20°C) for 20 minutes, and then washed twice with phosphate buffer solution to complete the cell fixation. Then, 1μM Hoechst 33342 and 0.5μM dye 2 were added to the incubated MCF-7 and MCF-10A cells and incubated for 20 minutes at 37°C, 5% CO2. Representative areas were selected and imaged using a laser confocal microscope. The results are shown in Figure 4 It can be seen that the dye can specifically stain the nuclei of fixed cells, with a staining effect comparable to that of commercial nuclear dyes.

[0140] Test Example 5

[0141] Cell staining experiment with different concentrations of dye 2

[0142] The experimental method is: cells were incubated with different concentrations (0.1, 0.3, 0.5, 1 μM) of Hoechst33342 and dye 2, and then imaged using a laser confocal microscope. The results are shown in Figure 5As shown in the figure, when the concentration of dye 2 is reduced to 0.1 μM, it still effectively labels the cell nucleus. However, for Hochest 33342, when the dye concentration is reduced to 0.3 μM, the cell nucleus is no longer clearly labeled. This shows that dye 2 can effectively label the cell nucleus even at low doses, which not only reduces costs but also improves the biocompatibility of the dye.

[0143] Test Example 6

[0144] Dye 2 and commercial red DNA labeling dye SYTO TM Deep Red staining cell experiment

[0145] The experimental method is: TM The Deep Red dye instruction manual does not provide a specific concentration of the dye. Therefore, cells were incubated with the concentration recommended in the instruction manual and 0.5 μM of dye 2 as a comparison. The cells were then imaged using a laser confocal microscope (laser rated power 0.4 mW). The results are shown in Figure 6 As shown in the figure, under the condition of fixed PMT detector gain, using the same laser intensity (0.2% of the rated power, 0.8μW), dye 2 can label the cell nucleus well, but for SYTO TM Deep Red, no fluorescence signal can be seen. When the laser intensity is further increased to 17% (68μW), a bright fluorescence signal of the cell nucleus can be observed. This shows that the same red light cell nuclear DNA labeling dye can be used with extremely low laser power (SYTO TM Deep Red dye (1.2%) labels the cell nucleus, greatly reducing photodamage to cells.

[0146] Test Example 7

[0147] Comparative live cell staining experiment

[0148] The experimental method is as follows: 1 μM of the control sample was added to the incubated MCF-7 cells (cell culture density 105 cells / ml, 70-80% coverage of the dish bottom), incubated and stained for 20 minutes at 37°C, 5% CO2, and representative areas were selected for imaging using a laser confocal microscope. The results are shown in Figure 7 It can be seen that although the comparative example can bind to nucleic acids in vitro, under the complex microenvironment of the cell, the fluorescence signal can only be observed in the cytoplasm and cannot be used for specific labeling of nuclear DNA.

[0149] Test Example 8

[0150] Peripheral blood cell staining experiment with dye 2

[0151] Peripheral blood cells diluted in PBS were incubated with 0.5 μM dye 2 and then imaged using a laser confocal microscope. The results are shown in Figure 8 As shown in the figure, Dye 2 only labels white blood cells with nuclear structures, while no fluorescent signal is observed for platelets, red blood cells, and hemoglobin, which do not have nuclear structures. Furthermore, the different nuclear morphologies of different white blood cell subtypes—eosinophils, basophils, neutrophils, lymphocytes, and monocytes—can be clearly labeled, enabling differentiation of peripheral blood white blood cells. This demonstrates that Dye 2 can be used in fields such as blood cell analysis, clinical medical diagnosis, and immunoassay testing.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A trimethine pyridine cyanine dye, characterized in that: Having the structure of general formula I: In Formula I, A is selected from at least one of the following substituents: R1 is selected from C1-6 alkyl, -(CH2) p COOR3, -(CH2) p OR3, -(CH2) p At least one of NR3 or benzyl substituted with R4; and p is selected from any integer from 1 to 6; R2 is selected from H, C1-6 alkyl, or at least one of phenyl groups; R3 is selected from at least one of H, C1-6 alkyl or phenyl; R4 is selected from at least one of H, halogen, alkoxy, amide or nitro; X - Selected from halogen anions, ClO4 - PF6 - CF3 - 、BF4 - , or OTs - At least one of .

2. The method for preparing the trimethine pyridine cyanine dye according to claim 1, wherein The steps include: In a first reaction solvent and a base, under the action of a first catalyst, the compound represented by formula IV and the compound represented by formula V undergo a condensation reaction at a molar ratio of 1:1-2 to obtain the target compound represented by I.

3. The preparation method according to claim 2, characterized in that The first catalyst is selected from at least one of p-toluenesulfonic acid, sodium acetate or acetic anhydride; the molar ratio of the first catalyst to the compound represented by formula IV is 1:1-2.

4. The preparation method according to claim 2, characterized in that The base is at least one of triethylamine, diethylamine, pyridine, dimethylaminopyridine or N,N-diisopropylethylamine; the molar ratio of the base to the compound represented by formula IV is 1:1-3.

5. The preparation method according to claim 2, characterized in that The preparation method of the compound represented by formula IV comprises the following steps: In a second reaction solvent, 4-methylpyridine and 2,4-dinitrohalobenzene react at a molar ratio of 1:1-2 to obtain a compound represented by formula II; In a second reaction solvent, the compound represented by formula II and R2-substituted aniline react at a molar ratio of 1:1-2 to obtain an N-arylpyridinium salt represented by formula III; In the presence or absence of a third reaction solvent, the compound represented by formula III reacts with N,N-diphenylformamidine at a molar ratio of 1:1-2 to obtain the compound represented by formula IV; In the presence or absence of a second reaction solvent, the nitrogen-containing heterocycle and the R1-substituted halogenated hydrocarbon are reacted in a molar ratio of 1:1-2 to obtain a compound represented by formula V.

6. The preparation method of trimethine pyridine cyanine dye according to claim 2, wherein The first reaction solvent is at least one of dichloromethane, methanol or pyridine; the second reaction solvent is at least one of methanol, ethanol, acetonitrile or toluene; and the third reaction solvent is acetic acid and / or acetic anhydride.

7. The use of the trimethine pyridine cyanine dye according to claim 1, wherein The applications include: fluorescence imaging based on cell nucleus staining, labeling and tracing, blood cell analysis preparations, clinical medical diagnostic preparations, and immunoassay detection preparations.

8. The use according to claim 7, characterized in that After binding to DNA, the trimethine pyridinium cyanine dye has an ultraviolet-visible light absorption spectrum showing significant absorption characteristics within the wavelength range of 550-700 nm, a red shift of the maximum absorption wavelength by 10-30 nm, and an increase in the molar extinction coefficient by 10-120%. The fluorescence quantum yield is higher than 50%, the fluorescence brightness is greater than 40,000, and the DNA / RNA discrimination is greater than 2.

9. The use according to claim 7, characterized in that The working concentration of the trimethine pyridinium cyanine dye is 0.1-1 μM.

10. The use according to claim 7, characterized in that The trimethine pyridinium cyanine dye is effectively excited by using a laser intensity of 0.6 to 2.0 μW during laser confocal nuclear staining imaging.

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