CN118085600A

CN118085600A - Trimethyl pyridinium cyanine dye, preparation method and application thereof

Info

Publication number: CN118085600A
Application number: CN202410141492.XA
Authority: CN
Inventors: 樊江莉; 李子鹏; 张长玉; 彭孝军
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2024-02-01
Filing date: 2024-02-01
Publication date: 2024-05-28

Abstract

The invention discloses a trimethapyr cyanine dye, a preparation method and application thereof, wherein a series of dye molecules which are combined with DNA and emit in a red light region through ultraviolet absorption and fluorescence can be obtained by regulating and controlling the structure of a nitrogen-containing heterocycle, the spectrum is adjustable, and the synthesis is convenient; the specific binding DNA has high fluorescence quantum yield and high brightness; the cell permeability is good, the cell nucleus can be dyed, the dyeing speed is high, the background interference is small, and repeated washing is not needed. Can be applied to the fields of fluorescence imaging, marking and tracing, blood cell analysis, clinical medical diagnosis and immunoassay detection based on nuclear staining, thereby providing a more sensitive, safe, economical and efficient novel dye scheme for nucleic acid research and clinical detection.

Description

Trimethyl pyridinium cyanine dye, preparation method and application thereof

Technical Field

The invention relates to the field of fine chemical engineering, in particular to a trimethapyr cyanine dye, a preparation method and application thereof.

Background

The DNA marked fluorescent dye is widely applied in the biomedical fields such as gel electrophoresis, quantitative PCR, flow cytometry, imaging and tracing of cell nuclei, blood cell analysis and the like. Commercial DNA dyes are mainly phenanthridines (EB, PI), acridines (AO), imidazoles (Hoechst, DAPI), cyanines (Cy, TOTO, SYTO), and the like. For nuclear marker dyes, the DNA specific blue fluorescent dyes DAPI and Hoechst 33342 remain the first choice for many applications due to the advantages of simplicity and low cost of use. However, ultraviolet lasers are required, which can cause DNA damage and cell death due to high energy excitation. In recent years, there has been a great demand for fluorescent dyes that emit at long wavelengths, preferably in the red/Near Infrared (NIR) region, with less autofluorescence interference and deeper tissue imaging depths than short wavelength fluorescent dyes. And longer wavelength excitation light can reduce photobleaching of dyes and photodamage of biological samples.

Currently, red DNA dyes that have been commercialized are monopolized abroad, are less expensive (SYTO ^TM Deep Red). Therefore, there is a need to develop DNA-tagged fluorescent dyes that emit in the red/near infrared region to achieve domestic substitution. While a common strategy for constructing red DNA-labeled fluorochromes is to attach a fluorescent dye excited by visible wavelengths to Hoechst dye via a linking group. For example, covalent attachment of far-infrared-emitting silarhodamine dye to Hoechst 33342 achieves specific labeling of nuclear DNA (nat. Commun.,2015,6,8497.), but this approach reduces the binding strength of dye to DNA (K _D =8.4±0.5 μm). The cyanine dyes can be excited by visible light and near infrared light, and red fluorescent dyes which specifically bind nucleic acid can be obtained by extending a methine chain based on the cyanine dyes, but the cyanine dyes have low differentiation degree on DNA and RNA, and cannot specifically dye cell nuclei. The pyridinium dye reported in 2021 improved the degree of differentiation between DNA and RNA (nat. Commun.,2021,12,2650.) but the fluorescence intensity after DNA binding was still insufficient. Although researchers have made a series of advances in developing DNA-labeled fluorescent dyes that emit in the red/near infrared region, maintaining fluorophores with high brightness as their absorption/emission wavelengths red shift is a significant challenge (narrow bandgap molecules typically have longer conjugated hydrophobic backbones and their larger molecular charge transfer makes them susceptible to interactions with external molecules, resulting in an increased probability of non-radiative transitions). Thus, developing an ideal dye with high sensitivity, high specificity, high brightness and excellent biocompatibility still faces a great challenge. The intensive research in the field not only needs to carry out fine design and optimization on the molecular structure of the dye so as to improve the binding force and fluorescence efficiency of the dye and DNA, but also needs to solve the stability and targeting problems of the dye in complex biological environments, and the novel DNA dye also needs to have the characteristics of low toxicity, low background interference, good penetration depth and the like aiming at different application scenes such as living cells and in-vivo imaging.

Disclosure of Invention

In view of the problems of the prior art, 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 DNA binding;

(3) Absorption and fluorescence emission with long wavelength; (4) The DNA is dyed with higher fluorescence brightness in organisms; the invention provides a trimethapyr cyanine dye, a preparation method and application thereof. The dye has absorption wavelength of 550-700 nm, high differentiation degree of DNA and RNA, good cell permeability in living cell and fixed cell staining, and can realize specific labeling of cell nucleus DNA.

The trimethyl pyridinium cyanine dye has the structure shown in the following general formula I:

in the formula I, A is various nitrogen-containing heterocycles selected from At least one of (a) and (b);

R ₁ is selected from at least one of C _1-6 alkyl 、-(CH₂)_pCOOR₃、-(CH₂)_pOR₃、-(CH₂)_pNR₃ or benzyl containing R ₄ substitution, and p is selected from any integer from 1 to 6;

R ₂ is selected from H, C _1-6 alkyl, Or at least one of phenyl;

R ₃ is at least one of H, C _1-6 alkyl or phenyl;

R ₄ is at least one of H, halogen, alkoxy, amido or nitro;

X ^- is selected from at least one of halogen anions, clO ₄ ^-、PF₆ ^-、CF₃ ^-、BF₄ ^- or OTs ^-.

Further, the above-described embodiments further include specific compound structures of the compounds represented by the general formula I, but the present invention is not limited to these specific examples:

Another aspect of the present application is a process for the preparation of the trimethapyr cyanine dye described hereinabove comprising the steps of:

in a first reaction solvent and alkali, under the action of a first catalyst, carrying out condensation reaction on a compound shown in a formula IV and a compound shown in a formula V in a molar ratio of 1:1-2 (more preferably, a molar ratio of 1:1.2) to obtain a target compound shown in I;

For the technical scheme described above, more preferably, the first reaction solvent is at least one of dichloromethane, methanol or pyridine; the dosage of the first reaction solvent is 4-10 times of the total reaction compound; more preferably by a factor of 5.

For the technical solutions described above, more preferably, the first catalyst is selected from at least one of p-toluene sulfonic acid, sodium acetate or acetic anhydride; the molar ratio of the first catalyst to the compound shown in the formula IV is 1:1-2; the most preferred molar ratio is 1:1.5;

For the technical scheme 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 of formula IV is 1:1-3, more preferably a molar ratio of 1:2;

For the technical scheme, more preferably, the reaction time is 0.5-3h, and the reaction temperature is 10-60 ℃; more preferably the temperature is 20-40 ℃.

For the technical scheme, the compound shown in the formula IV is further prepared by the following method:

In the second reaction solvent, 4-methylpyridine and 2, 4-dinitrohalobenzene are reacted in an amount of 1:1-2 molar ratio (more preferably, the molar ratio is 1:1.2) to obtain a compound shown in a formula II;

In a second reaction solvent, the compound of formula II and R ₂ substituted aniline are prepared in a reaction mixture of 1:1-2 molar ratio (more preferably 1:1.2 molar ratio) to obtain N-aryl pyridine salt shown in formula III;

The compound of formula III is reacted with N, N-diphenylformamidine with or without a third reaction solvent in an amount of 1:1-2 molar ratio (more preferably, the molar ratio is 1:1.2) to obtain a compound shown in a formula IV;

with or without a second reaction solvent, the nitrogen-containing heterocycle and the R ₁ -substituted halocarbon are substituted with 1:1-2 molar ratio (more preferably 1:1.5) to give the compound of formula V.

For the technical scheme 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;

for the technical scheme described above, more preferably, the addition amount of the second reaction solvent is 5 to 10 times the mass of the total reaction compound; the addition amount of the third reaction solvent is 1-2 times of the mass of the total reaction compound;

For the technical scheme described above, the preferred reaction time of 4-methylpyridine and 2, 4-dinitrohalobenzene is from 6 to 24 hours, the reaction temperature is from 70 to 110 ℃, and the more preferred temperature is 90 ℃;

For the technical scheme, the preferable reaction time of the compound shown in the formula II and the R ₂ substituted aniline is 3-24h, and the reaction temperature is 10-100 ℃; the temperature is more preferably 70-100 ℃.

For the technical scheme, the reaction time of the compound shown in the formula III and N, N-diphenyl formamidine is 1-5h, and the reaction temperature is 60-160 ℃; the most preferred reaction temperature is 150 ℃.

For the technical scheme, more preferably, the reaction time of the reaction of the nitrogen-containing heterocycle and the R ₁ substituted halohydrocarbon is 4-24h, and the reaction temperature is 80-130 ℃; more preferably the temperature is 100 ℃;

another aspect of the application is the use of the trimethapyr cyanine dye described hereinabove.

For the technical scheme, the application is further that the trimethapyr cyanine dye is applied to the fields of fluorescence imaging, labeling and tracing, hemocyte analysis, clinical medical diagnosis, immunoassay detection and the like based on nuclear staining; specifically:

1. The fluorescence imaging based on the cell nucleus staining realizes the accurate visualization of the cell nucleus DNA by utilizing the strong absorption and emission characteristics of the combined DNA in a red light region;

2. The in vivo cell marking and tracing is to realize the positioning and marking of specific cells or nucleic acid molecules by utilizing the high affinity and specificity of the in vivo cell marking and tracing with DNA;

3. the blood cell analysis is to carry out refined detection and differentiation on cell types with specific nuclear structures, such as leukocyte subtypes;

4. The clinical medical diagnosis is to use the fluorescence enhancement characteristic of the combined DNA to label and quantify the nuclear structures of different cells, improve and innovate the existing clinical in-vitro detection means, and improve the accuracy and sensitivity of disease diagnosis;

5. the immunoassay detection utilizes the high affinity and specificity of the fluorescent antibody and DNA to improve and innovate the existing fluorescent immunity and fluorescent in-situ hybridization detection technology, thereby improving the accuracy and sensitivity of disease diagnosis.

For the technical solution described above, further, the blood cell analysis includes: (1) identifying whether the cell has a nucleus; (2) Differentiating between various subtypes of leukocytes, including eosinophils, basophils, neutrophils, lymphocytes and monocytes, and enabling distinct differentiation of the different morphological characteristics of these nuclei; (3) identifying other cell types having a specific nuclear structure.

The dye technology not only can realize accurate marking and distinguishing of different leukocyte subtypes in peripheral blood cells, but also has wide application potential, can be suitable for carrying out refined detection and diagnosis on various cell types with specific nuclear structures, for example, distinguishing cells with different functions in an immune system, such as epithelial cells, endothelial cells, nerve cells, stem cells, tumor cells and the like, thereby widening the research and clinical application range in various biomedical fields.

After DNA is combined with the trimethapyr cyanine dye, the ultraviolet-visible light absorption spectrum of the trimethapyr cyanine dye shows remarkable absorption characteristics in a wavelength range of 550-700nm, the maximum absorption wavelength is red-shifted by 10-30nm, and the molar extinction coefficient is increased by 10-120%; fluorescence quantum yield higher than 50%; the fluorescence brightness is more than 40000 and even up to 71820; the degree of DNA/RNA discrimination is greater than 2, more preferably greater than 5, still more preferably greater than 7,

Even up to 11.1;

The working concentration of the trimethapyr cyanine dye for dyeing the cell nucleus is 0.1-1 mu M, preferably 0.1-0.5 mu M, so that the dye consumption is reduced while the cell nucleus is ensured to be fully marked, and the potential cytotoxicity and economic cost are further reduced.

The trimethapyr cyanine dye is recommended to be effectively excited by using the laser intensity of 0.6-2.0 mu W in the laser confocal cell nucleus staining imaging, wherein the preferred laser intensity range is 0.6-1.0 mu W; under further optimization conditions, the optimum laser intensity was set to 0.8. Mu.W.

Compared with the prior art, the invention has the following beneficial effects:

According to the first, the trimethylpyridinium cyanine dye disclosed by the invention, through regulating and controlling the structure of the nitrogen-containing heterocycle, a series of dye molecules with the ultraviolet absorption wavelength between 550 and 700nm, the maximum absorption wavelength red shift (10-30 nm) and the molar extinction coefficient increase (10% -120%) after being combined with DNA can be obtained, the spectrum is adjustable, and the synthesis is convenient. The red semiconductor laser can be used for excitation, so that the tissue penetration depth of the dye is increased and the photodamage of the biological sample is reduced; the red shifted absorption wavelength and the increased molar extinction coefficient may further reduce the background fluorescence signal.

Secondly, the methine pyridine cyanine dye disclosed by the invention specifically binds to DNA, and has high fluorescence quantum yield, wherein the fluorescence quantum yield after the dye 2 and the dye 3 bind to DNA reaches 54% and 56% respectively; the brightness reaches 71820 and 40752, and compared with the comparative example and the prior technical proposal, the fluorescent dye has obviously improved brightness, and is the currently known red DNA marker dye with the maximum fluorescent brightness. Has great significance for improving the detection sensitivity and reducing the background interference. In particular, dye 3, which has a degree of discrimination of 11.1 with DNA/RNA binding, is far higher than that of comparative example 1.98, revealing that the regulation of dye structure makes it more specific, and can effectively discriminate DNA from RNA, which is of great value for nucleic acid research and clinical detection.

Thirdly, the trimethapyr cyanine dye disclosed by the invention has the advantages of good cell permeability, high dyeing speed, small background interference, no need of repeated washing and low working concentration, and can dye cell nuclei. Wherein, the comparative example has poor cell permeability, the cell nucleus can not be marked in living cell imaging, and the dye 2 can clearly mark the cell nucleus, especially different subtypes of peripheral blood leukocyte cell nucleus, and can also clearly mark; the staining effect was comparable to blue DNA marker dye Hochest 33342 and Red SYTO ^TM Deep Red, but lower than the working concentration of Hochest 33342, and could be used for real-time and high-fidelity imaging of the nuclei at very low doses (100 nM) (where the examples demonstrate that the effective concentration of dye 2 is 1/3 (0.1 μΜ vs 0.3 μΜ) of Hoechst 33342, i.e. the concentration requirement of dye 2 is reduced by about 3-fold with equivalent effect); the laser operating intensity was lower compared to the Red SYTO ^TM Deep Red (where the examples demonstrate that the effective intensity of dye 2 is 1.2% (0.2% vs 17%) of the SYTO ^TM Deep Red, i.e. the laser power requirement of dye 2 is reduced by about 85 times with equal effect). Greatly reduces cytotoxicity and use cost.

Drawings

FIG. 1 is a graph of test experiments of nucleic acid (DNA and RNA) responses of comparative examples, dyes 2-6;

FIG. 2 is a graph of live cell uptake experiments for dye 2;

FIG. 3 is a graph showing the co-localization of dye 2 with the commercial nuclear dye Hoechst 33342 in living cells;

FIG. 4 is a graph showing co-localization experiments of dye 2 in fixed cells with the commercial nuclear dye Hoechst 33342;

FIG. 5 is an image of different concentrations of dye 2 and commercial nuclear dye Hoechst 33342 staining living cells;

FIG. 6 is a diagram showing the experiment of dye 2 and commercial red DNA marker dye SYTOTMDeep Red for staining cells

FIG. 7 is a diagram of a comparative example of a live cell staining experiment;

FIG. 8 is a diagram of peripheral blood cell staining experiments with dye 2.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the following description will be taken in conjunction with fig. 1 to 8 of the embodiments of the present invention, but not all possible implementations. Nevertheless, any other implementations that can be obtained based on the disclosed embodiments of the invention without undue burden to those skilled in the art are considered to be within the scope of the invention.

Example 1

The synthetic route of the compound shown by the general formula I is as follows:

Starting from 4-methylpyridine, reacting with 2, 4-dinitrohalobenzene to prepare a compound of formula II;

Reacting a compound of formula II with R ₂ substituted aniline to prepare a compound of formula III; then carrying out condensation reaction with N, N-diphenyl formamidine to prepare a compound shown in a formula IV; reacting the nitrogen-containing heterocycle with a halogenated hydrocarbon substituted by R ₁ to prepare a compound of formula V; and finally, carrying out condensation reaction on the compound shown in the formula IV and the compound shown in the formula V to prepare the compound shown in the formula I.

The compounds of the present invention represented by the general formula I can be synthesized by the methods described below.

The compound shown as II is prepared by the following method:

S1: synthesis of Compound of formula II

50Mmol of 2, 4-dinitrochlorobenzene and 55mmol of 4-methylpyridine are dissolved in 40mL of ethanol, and the reaction is stopped after reflux for 6 h. After adding 100mL of t-butyl methyl ether and stirring for 2 hours, 9.6g (yield: 65%) of a black solid was obtained after air-drying by suction filtration.

Synthesis of Compound of formula III

S2: synthesis of III-1

10.15Mmol of the compound of formula II obtained in step S1 was dissolved in 20mL of ethanol, 20.29mmol of aniline solid was added, the reaction was stopped after heating reflux reaction for 3-8 hours, cooled to room temperature, the solvent was dried by spinning, and the compound represented by III-1 was purified by column chromatography to obtain 0.79g (yield: 36%).

S3: synthesis of III-2

10.15Mmol of the compound of formula II obtained in the step S1 is dissolved in 20mL of ethanol, 12.18mmol of N, N-dimethylaniline solid is added, the reaction is stopped after heating reflux reaction for 3-8h, the reaction solution is cooled to room temperature, the solvent is dried by spinning, and 1.6g (yield: 63%) of the compound shown in III-2 is obtained by column chromatography purification.

Synthesis of Compound of formula IV

S4: synthesis of VI-1

0.729Mmol of III-1 and 1.09mmol of N, N-diphenylformamidine were placed in a 50mL round-bottomed flask, and the reaction was stopped after heating to 160℃for 2h. After cooling to room temperature and adding 25mL of ethanol and stirring for 2 hours, 0.19g (yield: 84%) of a black solid was obtained after air-drying by suction filtration.

S5: synthesis of VI-2

0.603Mmol of III-2 and 0.904mmol of N, N-diphenylformamidine were placed in a 50mL round-bottomed flask and reacted for 2h at 160℃to terminate the reaction. After cooling to room temperature and adding 25mL of ethanol and stirring for 2 hours, 0.17g (yield: 80%) of a black solid was obtained after air-drying by suction filtration.

Synthesis of Compound shown in V

S6: synthesis of V-1

10.52Mmol of 5-iodo-2, 3-trimethyl-3H-indole was weighed and dissolved in 10mL of acetonitrile solution, 21.04mmol of ethyl iodide was added, and the reaction was stopped after 24 hours of reflux reaction. Cooled to room temperature, suction filtered, and washed with a mixed solvent of ethyl acetate and t-butyl methyl ether to give 4g of a yellow solid (yield:

86％)。

S7: synthesis of V-2

13.4Mmol of 2-methylbenzothiazole and 16.08mmol of bromoethanol are weighed into a 100mL round bottom flask and the reaction is stopped after stirring for 6h at 110 ℃. Cooled to room temperature, suction filtered, and washed with a mixed solvent of ethyl acetate and t-butyl methyl ether to give 1.03g of a white solid (yield:

25％)。

S8: synthesis of V-3

1.55Mmol of 5-methoxy-2-methylbenzselenazole and 2.32mmol of ethyl iodide were weighed into a 25mL round bottom flask, heated to 120℃and stirred for 6h, and the reaction was stopped. Cooled to room temperature, suction filtered, and washed with a mixed solvent of ethyl acetate and t-butyl methyl ether to obtain 0.316g (yield: 53%) of a white solid.

S9: synthesis of V-4

20.95Mmol of 2-methylquinoline and 62.85mmol of iodoethane are weighed into a 50mL round bottom flask and the reaction is stopped after stirring for 10h at 100 ℃. Cooled to room temperature, suction filtered, and washed with a mixed solvent of ethyl acetate and tert-butyl methyl ether to obtain 2.5g (yield: 39%) of pale yellow solid.

S10: synthesis of V-5

34.92Mmol of 4-methylquinoline and 48.89mmol of iodoethane were weighed into a 100mL round bottom flask and the reaction was stopped after stirring for 4h at 100 ℃. Cooled to room temperature, suction filtered, and washed with a mixed solvent of ethyl acetate and tert-butyl methyl ether to obtain 10.35g (yield: 99%) of pale yellow solid.

Example 1

Synthesis of dye 1:

1.46mmol of compound VI-1 was weighed out and dissolved in 6mL of methylene chloride solution, 1.89mmol of compound V-1 was added, and 0.6mL of N, N-diisopropylethylamine and 0.3mL of acetic anhydride were continuously added, followed by stirring at room temperature for 4 hours, and the reaction was stopped. Column chromatography purification after spin drying to obtain purple black solid 0.53g (yield) ：58％).¹H 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).¹³C NMR(101MHz,DMSO)δ

165.36,154.25,143.09,142.88,142.76,141.92,137.02,131.18,130.64,130.53,124.39,121.03,117.22,111.43,98.74,85.61,47.60,37.61,28.35,11.90.HR-MS:m/z calcd for C₂₆H₂₆N₂I⁺[M]⁺:493.1136,found:493.1148.

Example 2

Synthesis of dye 2:

0.226mmol of compound VI-1 was weighed out and dissolved in 4mL of methylene chloride solution, 0.226mmol of compound V-2 was added, 0.4mL of triethylamine and 0.4mL of acetic anhydride were further added, and the reaction was stopped after heating to 30℃and stirring for 2 hours. Column chromatography purification after spin drying to obtain purple black solid 0.030g (yield) ：30％).¹H NMR(400MHz,DMSO-d₆)δ8.49(d,J＝7.1Hz,2H),8.02–7.91(m,1H),7.82(d,J＝6.8Hz,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).¹³C NMR(101MHz,DMSO)δ159.76,153.21,143.43,142.67,141.57,140.71,130.60,130.05,127.98,125.04,124.12,123.91,122.95,119.43,113.24,112.26,96.47,40.77,32.57.HR-MS:m/z calcd for C₂₂H₁₉N₂OS⁺[M]⁺:359.1213,found:359.1229.

Example 3

Synthesis of dye 3:

0.369mmol of compound VI-2 is weighed and dissolved in 4mL of dichloromethane, 0.369mmol of compound V-2 is added, 0.4mL of triethylamine and 0.4mL of acetic anhydride are continuously added, the temperature is raised to 30 ℃ and stirring is carried out for 7 hours, and then the reaction is stopped. Column chromatography purification after spin drying to obtain purple black solid 0.072g (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,1H),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).¹³C NMR(101MHz,DMSO)δ158.27,152.39,151.18,142.11,141.65,140.64,131.61,127.83,124.81,124.26,123.72,122.81,119.82,113.60,112.80,111.84,95.50,40.51,40.47,32.42.HR-MS:m/z calcd for C₂₄H₂₄N₃OS⁺[M]⁺:402.1635,found:402.1655.

Example 4

Synthesis of dye 4:

0.310mmol of compound VI-1 was weighed and dissolved in 4mL of pyridine, 0.373mmol of compound V-3 was added, 0.4mL of N, N-diisopropylethylamine and 0.4mL of acetic anhydride were continuously added, the reaction was stopped after stirring at room temperature for 2 hours, and the reaction solution was poured into 50mL of t-butyl methyl ether. Filtering, purifying with filter cake column chromatography to obtain purple black solid 0.152g (yield) ：87％).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 C₂₄H₂₃N₂OSe⁺[M]⁺:435.0971,found:435.0987.

Example 5

Synthesis of dye 5:

0.284mmol of compound VI-2 was weighed and dissolved in 4mL of pyridine, 0.341mmol of compound V-4 was added, 0.4mL of N, N-diisopropylethylamine and 0.4mL of acetic anhydride were continuously added, the reaction was stopped after stirring at room temperature for 2 hours, and the reaction solution was poured into 50mL of t-butyl methyl ether. Filtering, purifying with filter cake column chromatography to obtain purple black solid 0.055g (yield) ：45％).¹H 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)δ152.38,151.04,149.49,142.96,140.03,139.57,133.17,132.09,131.71,129.06,124.27,124.18,123.75,120.79,119.24,115.29,114.13,112.83,102.20,42.20,40.48,12.20.HR-MS:m/z calcd for C₂₇H₂₈N₃ ⁺[M]⁺:394.2278,found:394.2294.

Example 6

Synthesis of dye 6:

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0.241mmol of compound VI-2 was weighed and dissolved in 4mL of pyridine, 0.289mmol of compound V-5 was added, 0.4mL of N, N-diisopropylethylamine and 0.4mL of acetic anhydride were continuously added, the reaction was stopped after stirring at room temperature for 2 hours, and the reaction solution was poured into 50mL of t-butyl methyl ether. Filtering, purifying with filter cake column chromatography to obtain purple black solid 0.100g (yield) ：79％).¹H 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),6.30(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/z calcd for C₂₇H₂₈N₃ ⁺[M]⁺:394.2278,found:394.2293.

Comparative example

Synthesis of comparative example:

Referring to the synthesis method of dye 2, 0.244mmol of formamidine intermediate and 0.293mmol of N-ethyl-4-methylquinoline salt are respectively weighed and dissolved in 4mL of dichloromethane solution, 0.4mL of triethylamine and 0.4mL of acetic anhydride are continuously added, and the reaction is stopped after the temperature is raised to 30 ℃ and stirring is carried out for 2 hours. Column chromatography purification after spin drying to obtain 0.100g (yield) of purple solid ：84％).¹H 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 C₂₃H₂₃N₂S⁺[M]⁺:359.1577,found:359.1593.

Test example for Performance detection

Test example 1

The nucleic acid (DNA and RNA) response experimental methods of comparative examples, dyes 2, 3, 4, 5 and 6 were: comparative examples, dyes 2, 3, 4, 5 and 6 were added to PBS buffer (ph=7.4) to give a final dye concentration of 4 μm, uv absorbance spectrum and fluorescence spectrum data were collected, then calf thymus DNA or yeast RNA was added to the system to give a concentration of 100 μg/mL, uv absorbance spectrum and fluorescence spectrum data were collected, the test results are shown in fig. 1, and the data were collated, and the results are shown in the following table. The maximum absorption wavelength of the dyes 2, 3, 4, 5 and 6 after being combined with DNA is 600-700nm, and the fluorescence emission wavelength is 620-720 nm; the degree of discrimination of binding DNA/RNA was 7.09/11.10/10.11/2.49/5.46, respectively, which was higher than that of the comparative example (1.98). And the fluorescence quantum yields of dye 2 and dye 3 after binding to DNA were 0.540 and 0.566, respectively, which were significantly higher than that of the comparative example (0.137). And the dye brightness reaches 71820 and 40752, and compared with the comparative example and the prior technical proposal, the dye has obvious improvement after fully researching the literature, and is the red DNA marker dye with the maximum fluorescence brightness known at present. This has great significance in improving detection sensitivity and reducing background interference.

TABLE 1 nucleic acid response data sheet for comparative examples, dyes 2-6

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Test example 2

Living cell uptake assay for dye 2

The experimental method comprises the following steps: 0.5. Mu.M dye 2 was added to a dish of MCF-7 cells (cell culture density 105cells/mL, dish bottom cover 70-80%) which had been incubated, and representative areas were imaged using a confocal laser microscope, with excitation wavelength of dye 2 being 600nm, reception band 610-700nm, and the results being shown in FIG. 2, taken every 5 minutes. It can be seen that dye 2 completely enters the nucleus after 20min, and has high cell permeability and high dyeing speed.

Test example 3

Living cell staining experiment of dye 2

The experimental method comprises the following steps: 1. Mu.M Hoechst 33342 (a widely used commercial nuclear dye) and 0.5. Mu.M dye 2 were added to the well-incubated MCF-7 cells and MCF-10A cells (cell culture density 105cells/ml, dish bottom covered 70-80%), incubated at 37℃for 20min with 5% CO ₂, and representative areas were imaged using a confocal laser microscope, and the results are shown in FIG. 3. It can be seen that the dye 2 has good co-localization effect with Hoechst 33342 no matter the dye is used for staining MCF-7 or MCF-10A cells, and the correlation coefficient reaches 0.92 and 0.94 respectively, which indicates that the dye 2 can specifically target cell nuclei, has the same staining effect as commercial cell nucleus dye and can be used for specific labeling of cell nucleus DNA.

Test example 4

Fixed cell staining experiment of dye 2

The experimental method comprises the following steps: the cultured cells (cell culture density 10 ⁵ cells/ml, dish bottom cover 70-80%) were treated with cooled ethanol (-20 ℃) for 20min, and then washed twice with phosphate buffer solution, to complete cell fixation. Then 1. Mu.M Hoechst 33342 and 0.5. Mu.M dye 2 were added to the already incubated MCF-7 cells and MCF-10A cells, and the incubation was performed at 37℃for 20min under 5% CO ₂, and the selected representative areas were imaged using a confocal laser microscope, the results of which are shown in FIG. 4. It can be seen that the dye specifically stains the nuclei of the fixed cells, comparable to the commercial nuclear dye staining effect.

Test example 5

Dye 2 staining cell experiments at different concentrations

The experimental method comprises the following steps: cells were incubated with Hoechst33342 and dye 2 at different concentrations (0.1, 0.3, 0.5, 1 μm), respectively, and then imaged using a confocal laser microscope, the results shown in fig. 5. As shown, when the concentration of dye 2 was reduced to 0.1. Mu.M, the nuclei could still be well labeled. However, for Hochest 33342, the nuclei were not clearly labeled already when the dye concentration was reduced to 0.3. Mu.M. This suggests that dye 2 can label the nucleus well even at low doses, which can reduce cost and increase biocompatibility of the dye.

Test example 6

Dye 2 and commercial Red DNA labeling dye SYTO ^TM Deep Red staining cell experiment

The experimental method comprises the following steps: since no specific concentration of dye is given on the instructions for use of the SYTO ^TM Deep Red dye, cells were incubated at the concentrations recommended on the instructions while cells were incubated with 0.5 μm dye 2 as a control and then imaged using a confocal laser microscope (laser rated 0.4 mW), the results are shown in fig. 6. As shown, dye 2 marks the nucleus well with the same laser intensity (0.2% rated power, 0.8 μw) with a fixed PMT detector gain, but no fluorescence signal was seen for SYTO ^TM Deep Red. When the laser intensity was further increased to 17% (68. Mu.W), a bright fluorescent signal of the nucleus could be observed. This shows that the same Red light nuclear DNA marker dye, due to the ultra high brightness of dye 2, can mark the nuclei with very low laser power (1.2% of SYTO ^TM Deep Red dye), greatly reducing photodamage to cells.

Test example 7

Viable cell staining experiments of comparative examples

The experimental method comprises the following steps: 1. Mu.M of the comparative example was added to the MCF-7 cells which had been incubated (cell culture density 105cells/ml, dish bottom covered with 70-80%), stained by incubation at 37℃for 20min under 5% CO ₂, and selected representative areas were imaged using a confocal laser microscope, the results of which are shown in FIG. 7. It can be seen that the comparative examples, although binding nucleic acids was possible in vitro experiments, only fluorescence signals were observed in cytoplasm under complex microenvironment of cells, and could not be used for specific labeling of nuclear DNA.

Test example 8

Peripheral blood cell staining experiment of dye 2

Peripheral blood cells diluted with PBS buffer were incubated with 0.5 μm dye 2 and then imaged using confocal laser microscopy, the results are shown in fig. 8. As shown, dye 2 labels only leukocytes having a nuclear structure, whereas no fluorescent signal was observed for platelets, erythrocytes and hemoglobin without a nuclear structure. And the different types of eosinophils, basophils, neutrophils, lymphocytes and monocytes in the white blood cells can be marked clearly, so that the distinction of the peripheral blood white blood cells is realized. This shows that the dye 2 can be used in the fields of blood cell analysis, clinical medical diagnosis, immunoassay detection and the like.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A class of trimethapyr cyanine dyes is characterized by having a structure of a general formula I:

in the formula I, A is selected from at least one of the following substituents:

R ₁ is selected from at least one of C1-6 alkyl 、-(CH₂)_pCOOR₃、-(CH₂)_pOR₃、-(CH₂)_pNR₃ or benzyl containing R ₄ substitution; and p is selected from any integer from 1 to 6;

R ₂ is selected from H, C-6 alkyl, Or at least one of phenyl;

r ₃ is selected from at least one of H, C-6 alkyl or phenyl;

R ₄ is selected from at least one of H, halogen, alkoxy, amido or nitro;

x-is at least one selected from halogen anions, clO ₄ ^-、PF₆ ^-、CF₃ ^-、BF₄ ^- or OTs ^-.

2. The preparation method of the trimethapyr cyanine dye as claimed in claim 1, which is characterized by comprising the following steps:

and in a first reaction solvent and alkali, under the action of a first catalyst, carrying out condensation reaction on the compound shown in the formula IV and the compound shown in the formula V in a molar ratio of 1:1-2 to obtain the target compound shown in the formula I.

3. The method according to claim 2, wherein the first catalyst is selected from at least one of p-toluene sulfonic acid, sodium acetate, or acetic anhydride; the molar ratio of the first catalyst to the compound shown in the formula IV is 1:1-2.

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

5. The preparation method according to claim 2, wherein the preparation method of the compound represented by formula iv comprises the steps of:

In the second reaction solvent, 4-methylpyridine and 2, 4-dinitrohalobenzene are reacted in an amount of 1:1-2 mol ratio to obtain a compound shown in a formula II;

In a second reaction solvent, the compound of formula II and R ₂ substituted aniline are prepared in a reaction mixture of 1:1-2 mol ratio to obtain N-aryl pyridine salt shown in a formula III;

The compound of formula III is reacted with N, N-diphenylformamidine with or without a third reaction solvent in an amount of 1:1-2 mol ratio reaction to obtain a compound shown in a formula IV;

With or without a second reaction solvent, the nitrogen-containing heterocycle and the R ₁ -substituted halocarbon are substituted with 1: and (3) reacting in a molar ratio of 1-2 to obtain the compound shown in the formula V.

6. The preparation method of the methine pyridinium 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; the third reaction solvent is acetic acid and/or acetic anhydride.

7. Use of a methine pyridinium cyanine dye according to claim 1, characterized in that the use comprises: fluorescent imaging, labeling and tracing based on nuclear staining, hemocyte analysis, clinical medical diagnosis and immunoassay detection fields.

8. The use according to claim 7, wherein said trimethapyr dye exhibits a significant absorption characteristic in the wavelength range of 550-700nm in the uv-vis absorption spectrum, a red shift of 10-30nm in the maximum absorption wavelength, and an increase in the molar extinction coefficient of 10-120% after binding to DNA; fluorescence quantum yield higher than 50%; the fluorescence brightness is greater than 40000; the degree of DNA/RNA discrimination was greater than 2.

9. The use according to claim 7, wherein the working concentration of the trimethapyr dye is 0.1-1 μm.

10. The use according to claim 7, wherein said trimethapyr dye is activated effectively by laser intensity of 0.6-2.0 μw during laser confocal nuclear dye imaging.