Nucleic acid probes with large stokes shift, methods of making and using same

Abstract

The application discloses a nucleic acid probe with a large stokes shift, and preparation and application thereof. The fluorescent probe is selected from a conjugated bridge of styrene, an electron acceptor of benzindole, benzothiazole, N-methylpyridine and the like, and a dimethylamino and a derivative thereof as an electron donor, so as to form a Donor-Acceptor structure, and the fluorescent probe has the advantages of a large stokes shift and good nucleic acid response. The molecular structure of the series of nucleic acid probes is shown in a structure of a general formula I.

Description

Technical Field

[0001] This invention relates to the field of fine chemicals, specifically to a class of nucleic acid probes with large Stokes shift, their preparation methods and applications, and particularly to a class of nucleic acid probe molecules for light-driven mitochondrial-nuclear cascade imaging, their preparation methods and applications in mitochondrial and nuclear fluorescence imaging. Background Technology

[0002] Nucleic acids are essential genetic material in living organisms, divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They store, transmit, and express genetic information through replication, transcription, and translation. Nucleic acids exist stably in normal cells, controlling cell proliferation and expression. They are mainly located in mitochondria and the cell nucleus, controlling normal physiological activities within the cell. Nucleic acids located in mitochondria play a crucial role in controlling mitochondrial replication, aging, and protein expression. The cell nucleus and mitochondria are spatially separated from the cytoplasm, with clear boundaries. However, nucleic acids in mitochondria and the cell nucleus are difficult to observe using an optical microscope. Normal cells have relatively stable amounts and types of nucleic acids, but when cellular physiological activities are abnormal or cancerous changes occur, intracellular nucleic acids undergo abnormal alterations. Furthermore, damage to mitochondrial and nuclear nucleic acids is closely related to cell death mechanisms such as apoptosis, ferroptosis, and autophagy. Anticancer drugs, such as paclitaxel, doxorubicin, cisplatin, and carboplatin, primarily induce cell death through interactions with intracellular nucleic acids. Therefore, the detection of nucleic acids in mitochondria and the cell nucleus plays a vital role in understanding mitochondrial functional impairment, genetic variation, abnormal gene expression, and abnormal protein synthesis.

[0003] For many years, numerous nucleic acid-based molecular probes have been developed for specific targeting of mitochondria or the cell nucleus. However, few probes simultaneously detect nucleic acids in both mitochondria and the nucleus. Most probes used for real-time imaging of mitochondria and the nucleus rely on changes in mitochondrial membrane potential (MMP), tending to accumulate in mitochondria with high MMP, but releasing and migrating to the nucleus or nucleolus when MMP decreases. While these subcellular organelle migration probes have made progress in monitoring subcellular organelle states, their application remains limited due to a lack of effective design strategies. Furthermore, currently developed small-molecule nucleic acid probes have certain limitations: firstly, a significant number of dyes cannot pass through the cell membrane of living cells, requiring cell fixation to increase membrane permeability for fluorescent labeling of the nucleic acid probes, making real-time imaging of nucleic acids within living cells difficult; secondly, commercially available nucleic acid probes, such as SYTO Green and Pico-green, exhibit small Stokes shifts, resulting in poor signal-to-noise ratios and severe background interference; and thirdly, most dyes currently exhibit significant dark toxicity and carcinogenicity, limiting their application in living cells. Summary of the Invention

[0004] To address the problems of small Stokes shifts, severe background interference, and poor nucleic acid response in existing nucleic acid probes, this invention aims to provide a class of nucleic acid probes with large Stokes shifts. This is achieved by introducing various nucleic acid affinity units and alkyl chains on both sides of a styrene backbone, resulting in the synthesis of a series of nucleic acid probe molecules with excellent nucleic acid responsiveness. This series exhibits excellent biocompatibility and can be used to track changes in mitochondrial and nuclear nucleic acids during apoptosis and ferroptosis, while possessing large Stokes shifts.

[0005] To achieve the above objectives, the technical solution of the present invention is: a type of nucleic acid probe with a large Stokes shift, the general structural formula of which is shown in Figure I:

[0006]

[0007] Wherein, R1 is selected from H or cyano; preferably, R1 is selected from H;

[0008] R2 is selected from dimethylamino, N,N-di(2-chloroethyl)amino, N,N-di(3-thiopentyl)amino or morpholine ring; preferably, R2 is selected from dimethylamino or N,N-di(3-thiopentyl)amino;

[0009] R3 is selected from either structural formula II or structural formula III;

[0010] In structural formula II, R4 is selected from C1-C8 alkyl, C1-C8 carboxyl, C1-C8 hydroxy, phenyl, 4-biphenyl or 4-aminophenyl; preferably, R4 is selected from phenyl, 4-biphenyl or 4-aminophenyl.

[0011] Y1 - Selected from halide anions, ClO4 - PF6 - CH3COO - or OTs - One of them; preferably, Y1 - Selected from halogen anions, more preferably, Y1 - Selected from chloride anions;

[0012] In structural formula III, X is selected from C(CH3)2, O, S or Se; preferably, X is selected from S;

[0013] R5 is selected from H, C1-C8 alkyl, C1-C8 hydroxy, C1-C8 carboxyl, C1-C8 NR5R6, benzyl or substituted benzyl, wherein the substituent of the substituted benzyl is selected from C1-C8 alkyl, C1-C8 alkoxy, CN, COOH, NH2, NO2, OH, SH, halogen or C1-C8 haloalkyl; preferably, R5 is selected from methyl;

[0014] R6 is selected from H, C1-C8 alkyl or phenyl; preferably, R6 is selected from methyl or phenyl.

[0015] Y2 - Selected from halide anions, ClO4 - PF6 - CH3COO - or OTs - One of them, preferably, Y2 - Selected from iodine anions.

[0016] Furthermore, the large Stokes shift nucleic acid probe is selected from one of the following structural formulas N1-N13:

[0017]

[0018] Furthermore, when R3 is selected from structural formula II, R1 is selected from H or cyano, R2 is selected from dimethylamino or N,N-di(3-thiopentyl)amino, and in structural formula II, R4 is selected from 4-biphenyl or 4-aminophenyl;

[0019] When R3 is selected from structural formula III, R1 is selected from H, R2 is selected from N,N-di(3-thiopentyl)amino, and in structural formula III, R6 is selected from C1-C8 alkyl or phenyl.

[0020] Furthermore, the mitochondrial-nuclear cascade nucleic acid probes are selected from one of the following structural formulas N3-N8:

[0021]

[0022] The method for preparing the nucleic acid probe molecule includes the following steps: in the presence of an organic solvent and a catalyst, a compound of formula IV containing R2 substitution is reacted with a compound of formula V containing R5 or R6 substitution, or a compound of formula VI containing R4 substitution, at a molar ratio of 1:1.0-1.2, for a reaction time of 4-10 h and a reaction temperature of 80-100 °C, to obtain the corresponding compound of formula I; the organic solvent is selected from ethanol, methanol, acetonitrile, or mixtures thereof, and the catalyst is selected from piperidine, sodium carbonate, or sodium bicarbonate;

[0023]

[0024] In formula V, R7 is selected from H or cyano.

[0025] Further, the compound of formula V is prepared by the following method: a haloalkane is reacted with a compound of formula VII in an organic solvent at a molar ratio of 1:2-5 to obtain the corresponding compound of formula V; the reaction time is 12-24 h, the reaction temperature is 80-100 °C, and the organic solvent is selected from at least one of methanol, ethanol, acetonitrile, and ethyl acetate; the organic solvent is selected from methanol, ethanol, acetonitrile, ethyl acetate, or a mixture of the above solvents.

[0026]

[0027] Furthermore, the compound of formula VI is prepared by the following method:

[0028] (1) 4-methylpyridine and 1-chloro-2,4-dinitrobenzene were reacted in a reaction solvent at a molar ratio of 1:1-2 to prepare the compound shown in formula VIII;

[0029] (2) The compound of formula VIII obtained in step (1) is reacted with an aromatic compound with para-amino substitution in a reaction solvent at a molar ratio of 1:1-5 to obtain the compound of formula VI.

[0030]

[0031] Furthermore, in step (1), the reaction conditions are: the reaction temperature is 70-90℃, the reaction time is 6-24h, and the reaction solvent is methanol or ethanol;

[0032] In step (2), the reaction conditions are: reaction temperature of 70-90℃, reaction time of 6-24h, and reaction solvent of methanol or ethanol.

[0033] The application of the nucleic acid probe molecule, which is used for mitochondrial-nuclear cascade nucleic acid imaging under light induction.

[0034] Furthermore, the nucleic acid probe molecules migrate from the mitochondria to the cell nucleus under the drive of a wavelength of 468-515 nm.

[0035] In summary, the present invention has the following beneficial effects:

[0036] 1. The fluorescent probe disclosed in this application uses styrene as a conjugate bridge to connect electron acceptors such as benzoindole, benzothiazole, and N-methylpyridine with dimethylamino and its derivatives as electron donors, forming a Donor-Acceptor structure. It has the advantages of large Stokes shift and good nucleic acid response, reduces background interference, and has good biocompatibility and low dark toxicity.

[0037] 2. The fluorescent probe disclosed in this application has the function of light-driven probe organelle localization and migration. It can migrate from mitochondria to the cell nucleus under light-driven conditions, realizing imaging of both mitochondria and the cell nucleus as sub-organelles. Under dark conditions, the fluorescent probe binds to nucleic acids in mitochondria to generate fluorescent signals. Furthermore, it can be used to track changes in nucleic acids in mitochondria and the cell nucleus during apoptosis and ferroptosis. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 These are UV-fluorescence test images of the mitochondrial-nuclear cascade nucleic acid probe molecules N1-13 disclosed in Examples 1-13 of this invention;

[0040] Figure 2 This is a comparison of the nucleic acid strength of the mitochondrial-nuclear cascade nucleic acid probe molecules N1-13 disclosed in Examples 1-13 of this invention;

[0041] Figure 3 These are comparative staining images of HepG2 live cells with the mitochondrial-nuclear cascade nucleic acid probe molecules N1-13 disclosed in Examples 1-13 of this invention;

[0042] Figure 4 This is an image of the mitochondrial-nuclear cascade imaging of HepG2 live cells using the mitochondrial-nuclear cascade of the mitochondrial-nuclear cascade nucleic acid probe molecule N1-13 disclosed in Examples 1 and 2 of this invention.

[0043] Figure 5 This is an image of the mitochondrial-nuclear cascade imaging of HepG2 live cells using the mitochondrial-nuclear cascade of the mitochondrial-nuclear cascade nucleic acid probe molecule N1-13 disclosed in Examples 3-6 of this invention.

[0044] Figure 6 This is an image of the mitochondrial-nuclear cascade imaging of HepG2 live cells by the mitochondrial-nuclear cascade of the nucleic acid probe molecule N1-13 disclosed in Examples 7-10 of this invention.

[0045] Figure 7 This is an image of the mitochondrial-nuclear cascade nucleic acid probe molecule N1-13 disclosed in Examples 11-13 of this invention, showing the light migration of mitochondrial-nuclear cascade in HepG2 live cells.

[0046] Figure 8This is a UV-fluorescence assay of different concentrations of the mitochondrial-nucleus cascade nucleic acid probe molecule N4 disclosed in Example 4 of this invention;

[0047] Figure 9 This is the UV-fluorescence spectrum of the mitochondrial-nuclear cascade nucleic acid probe molecule N4 disclosed in Example 4 of this invention in different solvents;

[0048] Figure 10 The absorption spectra of the mitochondrial-nuclear cascade nucleic acid probe molecule N4 disclosed in Example 4 of this invention for different concentrations of nucleic acid are shown.

[0049] Figure 11 The fluorescence spectra of the mitochondrial-nuclear cascade nucleic acid probe molecule N4 disclosed in Example 4 of this invention against nucleic acids of different concentrations are shown.

[0050] Figure 12 This is a comparative diagram of the selectivity of the mitochondrial-nuclear cascade nucleic acid probe molecule N4 to different substances disclosed in Example 4 of this invention;

[0051] Figure 13 This is a schematic diagram of mitochondrial colocalization of the mitochondrial-nucleus cascade nucleic acid probe molecule N4 and a commercial mitochondrial dye in HepG2 live cells, as disclosed in Example 4 of this invention.

[0052] Figure 14 This is a schematic diagram of cellular nucleic acid digestion imaging of the mitochondrial-nucleus cascade nucleic acid probe molecule N4 and commercial cell nuclear dye disclosed in Example 4 of the present invention;

[0053] Figure 15 This is a schematic diagram of the mitochondrial-nuclear cascade imaging of the nucleic acid probe molecule N4 disclosed in Example 4 of the present invention under time-dependent conditions.

[0054] Figure 16 This is a feature diagram of the mitochondrial-nuclear cascade nucleic acid probe molecule N4 disclosed in Example 4 of the present invention, which distinguishes between apoptosis and ferroptosis. Detailed Implementation

[0055] The present invention will be further described below with reference to the embodiments, but it should be understood that the scope of protection of the present invention is not limited to the embodiments.

[0056] In this invention, unless otherwise explicitly stated, percentages and contents are all by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available.

[0057] The synthesis process involved in this application is as follows:

[0058]

[0059] The nucleic acid probe molecule represented by general formula I will be described in detail below with reference to the embodiments.

[0060] Example 1

[0061] Synthesize using R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate II, R4 as methyl, Y1 - For I - The method for developing nucleic acid probe molecules includes the following steps;

[0062] (1) Synthesis of intermediates shown in general formula VI

[0063] 4-Methylpyridine (53.69 mmol) was added to a two-necked flask containing 20 mL of acetonitrile solution, and iodomethane (64.77 mmol) was added. The mixture was refluxed and stirred overnight at 80 °C. After the reaction was completed, the reaction solution was cooled to room temperature, and then ethyl acetate was added to precipitate the solid. The solid was filtered to obtain a pale yellow solid, which was named intermediate VI, with a yield of approximately 99%.

[0064] (2) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0065] Phosphorus oxychloride (1 mol) was added to a two-necked flask containing anhydrous DMF (2 mol), and stirred for 30 min under nitrogen protection at 0 °C. Then, N,N-dihydroxyethylaniline (0.33 mol) was dissolved in anhydrous DMF (150 mL) and added to the above system. The mixture was stirred at 85-90 °C for 2.5 h, and then cooled to room temperature. The cooled reaction solution was poured into an ice-water mixture, and an appropriate amount of NaOH was added to adjust the pH to neutral. A yellow precipitate formed. The precipitate was filtered, dried, and a yellow solid, 4-N,N(2-chloroethyl)aminobenzaldehyde, was obtained, with a yield of approximately 58%.

[0066] (3) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0067] Under nitrogen protection, sodium metal (8 mmol) was dissolved in a round-bottom flask containing anhydrous ethanol (50 mL), ethanethiol (7 mmol) was added, and the mixture was stirred at room temperature for 1 h. Then, 4-N,N(2-chloroethyl)aminobenzaldehyde (3 mmol) prepared in step (2) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by rotary evaporation, and water and dichloromethane were added for extraction. The extract was dried over anhydrous magnesium sulfate, concentrated by vacuum rotary evaporation, and purified by column chromatography to obtain a red oily substance with a yield of approximately 58.5%.

[0068] (4) Synthesis of target product N1

[0069] Intermediate VI (1 mmol) obtained in step (1) and 4-N,N-bis(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (3) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C for 5 h, and then the reaction was stopped. The reaction solution was cooled to room temperature, evaporated to dryness, and purified by column chromatography to obtain a brownish-red solid with a yield of approximately 92%. This solid was named compound N1, and its structural formula is shown below:

[0070]

[0071] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ8.70(d,J=6.4Hz,1H),8.06(d,J=6.4Hz,1H),7.91(d,J=16.1Hz,1H),7.61(d,J=8.6Hz,1H),7.18(d,J=16.1Hz,1H),6 .77(d,J=8.6Hz,1H),4.19(s,2H),3.80–3.50(m,2H),2.73(t,J=7.2Hz,2H),2.62(q,J=7.3Hz,2H),1.21(t,J=7.2Hz,3H).HRMS(ESI):m / z calcd for C 22 H 31 N2S2 + ([MI)) + ):387.1923,found:387.1917.

[0072] Example 2

[0073] Synthesize using R1 as a hydrogen atom, R2 as N,N-di(2-chloroethyl)amino, R3 as intermediate II, where R4 is phenyl, and Y1 - For I - The method for developing nucleic acid probe molecules includes the following steps:

[0074] (1) Synthesis of intermediates shown in general formula VIII

[0075] 4-Methylpyridine (1.2 mmol) and 1-chloro-2,4-dinitrobenzene (1 mmol) were added to a two-necked flask containing 30 mL of ethanol solution. The mixture was refluxed at 80 °C and stirred overnight. After the reaction was completed, the reaction solution was concentrated by rotary evaporation. Then, diethyl ether was added to precipitate the solid. The precipitate was obtained by filtration and was a brown viscous solid, namely intermediate VIII, with a yield of about 50%.

[0076] (2) Synthesis of 4-methyl-1-phenylpyridine

[0077] The intermediate VIII (1 mmol) obtained in step (1) and aniline (1.2 mmol) were added to a two-necked flask containing 20 mL of ethanol solution. The mixture was refluxed at 80 °C and stirred overnight. After the reaction was completed, the reaction solution was concentrated by rotary evaporation, and then ether was added to precipitate the product. The precipitate was obtained by filtration and was a brownish-black solid. The crude product was purified by column chromatography to obtain a black solid, namely 4-methyl-1-phenylpyridine, with a yield of approximately 80%.

[0078] (3) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0079] Same as step (2) in Example 1.

[0080] (4) Synthesis of the target product N2

[0081] The 4-methyl-1-phenylpyridine (1 mmol) obtained in step (2) and the 4-N,N-di(2-chloroethyl)aminobenzaldehyde (1.2 mmol) obtained in step (3) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C for 5 h, and then the reaction was stopped. The reaction solution was cooled to room temperature, evaporated to dryness, and purified by column chromatography to obtain a brownish-red solid with a yield of approximately 88.3%. This solid was named compound N2, and its structural formula is shown below:

[0082]

[0083] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ9.08(d,J=7.0Hz,1H),8.24(d,J=7.0Hz,1H),8.14(d,J=16.1Hz,1H),7.92–7.82(m,1H),7.78–7 .62(m,2H),7.38(d,J=16.1Hz,1H),6.92(d,J=9.0Hz,1H),3.90–3.83(m,2H),3.80(t,J=6.9Hz,2H), HRMS(ESI):m / z calcd for C 23 H 23 Cl2N2 + ([M-Cl)) + ):397.1233,found:397.1228.

[0084] Example 3

[0085] The synthesis uses R1 as a hydrogen atom, R2 as a dimethylamino atom, R3 as intermediate II, R4 as a phenyl atom, and Y1 as an intermediate. - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0086] (1) Synthesis of intermediates shown in general formula VIII

[0087] Same as step (1) in Example 2.

[0088] (2) Synthesis of 4-methyl-1-phenylpyridine

[0089] Same as step (2) in Example 2.

[0090] (3) Synthesis of target product N3

[0091] The 4-methyl-1-phenylpyridine (1 mmol) and p-dimethylaminobenzaldehyde (1 mmol) obtained in step (2) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed at 80 °C and stirred for 4 h before the reaction was stopped. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated by rotary evaporation. After purification by column chromatography, a red solid was obtained with a yield of about 60%, which was named compound N3. Its structural formula is shown below:

[0092]

[0093] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ9.04(d,J=5.8Hz,1H),8.20(d,J=5.8Hz,1H),8.13(d,J=16.0Hz,1H),7.86(d,J=7.5Hz,1H),7.6 9(dd,J=20.4,7.5Hz,2H),7.32(d,J=15.9Hz,1H),6.82(d,J=7.8Hz,1H),3.05(d,J=0.9Hz,3H), HRMS(ESI):m / zcalcd for C 21 H 21 N2 + ([M-Cl)) + ):301.1699,found:301.1692.

[0094] Example 4

[0095] The synthesis was carried out with R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate II, and R4 as a phenyl group, Y1 - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0096] (1) Synthesis of intermediates shown in general formula VIII

[0097] Same as step (1) in Example 2.

[0098] (2) Synthesis of 4-methyl-1-phenylpyridine

[0099] Same as step (2) in Example 2.

[0100] (3) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0101] Same as step (2) in Example 1.

[0102] (4) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0103] Same as step (3) in Example 1.

[0104] (5) Synthesis of the target product N4

[0105] The 4-methyl-1-phenylpyridine (1 mmol) obtained in step (2) and the 4-N,N-di(3-thiopentyl)aminobenzaldehyde (1 mmol) obtained in step (4) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed at 80 °C and stirred for 4 h before the reaction was stopped. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated by rotary evaporation. After purification by column chromatography, a red solid was obtained with a yield of approximately 94.5%, which was named compound N4. Its structural formula is shown below:

[0106]

[0107] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ9.04(d,J=6.8Hz,1H),8.20(d,J=6.8Hz,1H),8.12(d,J=16.0Hz,1H),7.86(d,J=7.0Hz,1H),7.73(d,J=7.7Hz,1H),7.66(d,J=8.8Hz ,1H),7.32(d,J=16.0Hz,1H),6.81(d,J=8.8Hz,1H),3.67–3.60(m,2H),2.7 8–2.70(m,2H),2.63(q,J=7.4Hz,2H),1.22(t,J=7.4Hz,4H),HRMS(ESI):m / z calcdfor C 27 H 33 N2S2 + ([M-Cl)) + ):449.2080,found:449.2075.

[0108] Example 5

[0109] The synthesis uses R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate II, where R4 is biphenyl, and Y1 - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0110] (1) Synthesis of intermediates shown in general formula VIII

[0111] Same as step (1) in Example 2.

[0112] (2) Synthesis of 4-methyl-1-biphenylpyridine

[0113] Intermediate VIII (1 mmol) obtained in step (1) and 4-aminobiphenyl (1.2 mmol) were added to a two-necked flask containing 20 mL of ethanol solution. The mixture was refluxed at 80 °C and stirred overnight. After the reaction was completed, the reaction solution was concentrated by rotary evaporation, and then a large amount of diethyl ether was added to precipitate the product. The precipitate was obtained by filtration and was a brownish-black solid crude product. The crude product was purified by column chromatography to obtain a black solid, namely 4-methyl-1-biphenylpyridine, with a yield of approximately 33.2%.

[0114] (3) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0115] Same as step (2) in Example 1.

[0116] (4) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0117] Same as step (3) in Example 1.

[0118] (5) Synthesis of target product N5

[0119] The 4-methyl-1-biphenylpyridine (1 mmol) obtained in step (2) and the 4-N,N-di(3-thiopentyl)aminobenzaldehyde (1 mmol) obtained in step (4) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed at 80 °C and stirred for 4 h before the reaction was stopped. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated by rotary evaporation. After purification by column chromatography, a red solid was obtained with a yield of approximately 93.27%, which was named compound N5, and its structural formula is shown below:

[0120]

[0121] The structure was confirmed to be correct by NMR: 1H NMR (400MHz, DMSO) δ9.09(d,J=7.0Hz,1H),8.22(d,J=7.0Hz,1H),8.13(d,J=16.0Hz,1H),8. 02(d,J=8.8Hz,1H),7.95(d,J=8.7Hz,1H),7.82–7.76(m,1H),7.67(d,J=8.9Hz,1H),7.55(t ,J=7.5Hz,1H),7.47(t,J=7.3Hz,1H),7.33(d,J=16.0Hz,1H),6.80(d,J=9.0Hz,1H),3.69–3 .61(m,2H),2.78–2.70(m,2H),2.63(q,J=7.4Hz,2H),1.22(t,J=7.4Hz,3H), HRMS(ESI):m / z calcd for C 33 H 37 N2S2 + ([M-Cl)) + ):525.2393,found:525.2383.

[0122] Example 6

[0123] The synthesis uses R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate III, where X is a sulfur atom, R5 is a methyl atom, R6 is a methyl atom, R7 is a hydrogen atom, and Y2... - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0124] (1) Synthesis of 2,3,6-trimethylbenzothiazole

[0125] 2,6-Dimethylbenzothiazole (1 mmol) and iodomethane (3 mmol) were added to a two-necked flask containing acetonitrile solution (5 mL). The mixture was refluxed overnight at 100 °C under nitrogen protection. After the reaction was completed, the reaction solution was evaporated to dryness, and ethyl acetate was added to precipitate the solid. The precipitate was filtered and dried to obtain a white powdery solid with a yield of about 45%.

[0126] (2) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0127] Same as step (2) in Example 1.

[0128] (3) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0129] Same as step (3) in Example 1.

[0130] (4) Synthesis of target product N6

[0131] The 2,3,6-trimethylbenzothiazole (1 mmol) obtained in step (1) and the 4-N,N-bis(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (3) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 94.2%, which was named compound N6. Its structural formula is shown below:

[0132]

[0133] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ8.11(s,1H),8.02(t,J=11.5Hz,2H),7.90(d,J=8.9Hz,2H),7.68–7.58(m,2H),6.83(d,J=9.0Hz, 2H),4.22(s,3H),3.73–3.64(m,4H),2.80–2.71(m,4H),2.63(q,J=7.4Hz,4H),1.22(t,J=7.4Hz,7H), HRMS(ESI):m / z calcd for C 25 H 33 N2S3 + ([MI)) + ):457.1800,found:457.1797.

[0134] Example 7

[0135] The synthesis uses R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate III, where X is a sulfur atom, R5 is methyl, R6 is phenyl, R7 is a hydrogen atom, and Y2... - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0136] (1) Synthesis of 2,3-dimethylnaphthothiazole

[0137] 2-Methylnaphthothiazole (1 mmol) and iodomethane (3 mmol) were added to a two-necked flask containing acetonitrile solution (5 mL). The mixture was refluxed overnight at 100 °C under nitrogen protection. After the reaction was completed, the reaction solution was evaporated to dryness, and ethyl acetate was added to precipitate the solid. The precipitate was filtered and dried to obtain a white powdery solid with a yield of about 88%.

[0138] (2) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0139] Same as step (2) in Example 1.

[0140] (3) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0141] Same as step (3) in Example 1.

[0142] (4) Synthesis of target product N7

[0143] The 2,3-dimethylnaphthothiazole (1 mmol) obtained in step (1) and the 4-N,N-di(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (3) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 95%, which was named compound N7. Its structural formula is shown below:

[0144]

[0145] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ8.35(d,J=9.2Hz,1H),8.23(dd,J=13.8,8.6Hz,3H),8.12(d ,J=15.3Hz,1H),7.91(d,J=8.8Hz,2H),7.86(d,J=7.5Hz,1H),7.79(t,J=7.5Hz,1 H),7.71(d,J=15.4Hz,1H),6.83(d,J=8.8Hz,2H),4.35(s,3H),3.81–3.52(m,4H) ,2.84–2.70(m,4H),2.63(q,J=7.4Hz,4H),1.22(t,J=7.3Hz,6H), HRMS(ESI):m / z calcd for C 28 H 33 N2S3 + ([MI)) + ):493.1800,found:493.1791.

[0146] Example 8

[0147] The synthesis was carried out with R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate II, and R4 as 4-aminophenyl, Y1 - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0148] (1) Synthesis of intermediates shown in general formula VIII

[0149] Same as step (1) in Example 2.

[0150] (2) Synthesis of 4-methyl-1-(4-aminophenyl)pyridine

[0151] The intermediate VIII (1 mmol) obtained in step (1) and p-phenylenediamine (1.2 mmol) were added to a two-necked flask containing 20 mL of ethanol solution. The mixture was refluxed at 80 °C and stirred overnight. After the reaction was completed, the reaction solution was concentrated by rotary evaporation. Then, a large amount of diethyl ether was added to precipitate the product, which was then filtered to obtain a brownish-black solid crude product. The crude product was purified by column chromatography to obtain a black solid, namely 4-methyl-1-biphenylpyridine, with a yield of approximately 58.2%.

[0152] (3) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0153] Same as step (2) in Example 1.

[0154] (4) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0155] Same as step (3) in Example 1.

[0156] (5) Synthesis of target product N8

[0157] The 4-methyl-1-(4-aminophenyl)pyridine (1 mmol) obtained in step (2) and the 4-N,N-bis(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (4) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 82.5%, which was named compound N8. Its structural formula is shown below:

[0158]

[0159] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ8.90(d,J=6.8Hz,1H),8.11(d,J=6.9Hz,1H),8.01(d,J=16.0Hz,1H),7.63(d,J=8.8Hz,1H),7.46(d,J=8.8Hz,1H),7.27(d,J=16.0 Hz,1H),6.77(dd,J=11.3,8.9Hz,2H),5.86(s,1H),3.67–3.57(m,2H),2.79 –2.69(m,2H),2.62(q,J=7.4Hz,2H),1.21(t,J=7.4Hz,4H), HRMS(ESI):m / z calcd forC 27 H 34 N3S2+ ([M-Cl)) + ):464.2189,found:464.2189.

[0160] Example 9

[0161] A method for synthesizing a mitochondrial-nuclear cascade nucleic acid probe molecule with R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate III, where X is a sulfur atom, R5 is a hydrogen atom, R6 is a hydrogen atom, and R7 is a cyano group, includes the following steps:

[0162] (1) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0163] Same as step (2) in Example 1.

[0164] (2) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0165] Same as step (3) in Example 1.

[0166] (3) Synthesis of target product N9

[0167] 2-Cyanobenzothiazole (1 mmol) and 4-N,N-bis(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (2) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 40%, which was named compound N9, and its structural formula is shown below:

[0168]

[0169] The structure was confirmed to be correct by NMR: 1 H NMR (600MHz, CDCl3) δ8.24(s,1H),8.07(d,J=8.2Hz,1H),8.01(d,J=8.9Hz,2H),7.88(d,J=7.9Hz,1H),7.51(t,J=7.7Hz,1H),7.40(t,J =7.3Hz,1H),6.73(d,J=9.0Hz,2H),3.69–3.60(m,4H),2.81–2.73(m,4H),2.63(q,J=7.4Hz,4H),1.31(t,J=7.4Hz,7H), HRMS(ESI):m / z calcd for C 24 H 27 N3S3([M)) + ):453.1367,found:454.1443.

[0170] Example 10

[0171] The synthesis was carried out with R1 as a hydrogen atom, R2 as N,N-di(3-thiopentyl)amino, R3 as intermediate III, where X is C(CH3)2, R5 is methyl, R6 is a hydrogen atom, R7 is a hydrogen atom, and Y2... - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0172] (1) Synthesis of 1,2,3,3-tetramethylbenzoindole

[0173] 2,3,3-trimethylbenzoindole (1 mmol) and iodomethane (3 mmol) were added to a two-necked flask containing acetonitrile solution (5 mL). The mixture was refluxed overnight at 100 °C under nitrogen protection. After the reaction was completed, the reaction solution was dried by rotary evaporation, and ethyl acetate was added to precipitate the solid. The precipitate was filtered and dried to obtain a pink powder solid with a yield of about 95%.

[0174] (2) Synthesis of 4-N,N(2-chloroethyl)aminobenzaldehyde

[0175] Same as step (2) in Example 1.

[0176] (3) Synthesis of 4-N,N-bis(3-thiopentyl)aminobenzaldehyde

[0177] Same as step (2) in Example 1.

[0178] (4) Synthesis of the target product N10

[0179] The 1,2,3,3-tetramethylbenzoindole (1 mmol) obtained in step (1) and the 4-N,N-bis(3-thiopentyl)aminobenzaldehyde (1.2 mmol) obtained in step (3) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 80%. This solid was named compound N10, and its structural formula is shown below:

[0180]

[0181] The structure was confirmed to be correct by NMR: 1H NMR (400MHz, DMSO) δ8.31(d,J=15.7Hz,1H),8.09(d,J=8.7Hz,2H),7.79(d,J=7.4Hz, 1H),7.73(d,J=7.9Hz,1H),7.57(t,J=7.7Hz,1H),7.50(t,J=7.4Hz,1H),7.30(d,J=15 .8Hz,1H),6.89(d,J=8.9Hz,2H),4.00(d,J=6.5Hz,3H),3.74(t,J=7.3Hz,4H),2.82–2 .73(m,4H),2.64(q,J=7.4Hz,4H),1.75(s,6H),1.22(t,J=7.4Hz,7H), HRMS(ESI):m / z calcd for C 27 H 27 N2S2 + ([MI)) + ):453.2393,found:454.2378.

[0182] Example 11

[0183] The synthesis uses R1 as a hydrogen atom, R2 as a dimethylamino group, R3 as intermediate III, where X is a sulfur atom, R5 is a methyl group, R6 is a methyl group, R7 is a hydrogen atom, and Y2 is a hydrogen atom. - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0184] (1) Synthesis of 2,3,6-trimethylbenzothiazole

[0185] Same as step (1) in Example 6.

[0186] (2) Synthesis of target product N11

[0187] The 2,3,6-trimethylbenzothiazole (1 mmol) and 4-dimethylaminobenzaldehyde (1.2 mmol) obtained in step (1) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 85%. This solid was named compound N11, and its structural formula is shown below:

[0188]

[0189] The structure was confirmed to be correct by NMR: 1H NMR (400MHz, DMSO) δ8.09 (s, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.61 (d, J = 10.2Hz, 1H), 6.84 (s, 1H), 4.21 (s, 2H), 3.10 (s, 4H), 1.23 (s, 3H), HRMS (ESI): m / z calcd for C 19 H 21 N2S + ([MI)) + ):309.1420,found:309.1415.

[0190] Example 12

[0191] The synthesis uses R1 as a hydrogen atom, R2 as a dimethylamino group, R3 as intermediate III, where X is a sulfur atom, R5 is a methyl group, R6 is a phenyl group, R7 is a hydrogen atom, and Y2 is a hydrogen atom. - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0192] (1) Synthesis of 2,3-dimethylnaphthothiazole

[0193] Same as step (1) in Example 7.

[0194] (2) Synthesis of the target product N12

[0195] The 2,3-dimethylnaphthothiazole (1 mmol) and 4-dimethylaminobenzaldehyde (1.2 mmol) obtained in step (1) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed and stirred at 80 °C to obtain a red solid with a yield of approximately 88%. This solid was named compound N12, and its structural formula is shown below:

[0196]

[0197] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ8.34(d,J=9.1Hz,1H),8.24(d,J=8.1Hz,1H),8.22–8.15(m,2H),8.10(d,J=15.3Hz,1H),7.87(dd,J=12. 7,8.1Hz,3H),7.78(t,J=7.5Hz,1H),7.68(d,J=15.3Hz,1H),6.80(d,J=8.9Hz,2H),4.34(s,3H),3.07(s,6H), HRMS(ESI):m / z calcd for C 22 H 21N2S + ([MI)) + ):345.1420,found:345.1413.

[0198] Example 13

[0199] The synthesis uses R1 as a hydrogen atom, R2 as a morpholino group, R3 as intermediate II, where R4 is a biphenyl group, and Y1... - For I - A method for mitochondrial-nuclear cascade nucleic acid probe molecules includes the following steps:

[0200] (1) Synthesis of intermediates shown in general formula VIII

[0201] Same as step (1) in Example 5.

[0202] (2) Synthesis of 4-methyl-1-biphenylpyridine

[0203] Same as step (2) in Example 5.

[0204] (3) Synthesis of target product N13

[0205] The 4-methyl-1-biphenylpyridine (1 mmol) and 4-morpholinobenzaldehyde (1 mmol) obtained in step (2) were added to a two-necked flask containing 5 mL of methanol solution. After adding 2 drops of piperidine, the mixture was refluxed at 80 °C and stirred for 4 h before the reaction was stopped. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated by rotary evaporation. After purification by column chromatography, a red solid was obtained with a yield of approximately 89%, which was named compound N13. Its structural formula is shown below:

[0206]

[0207] The structure was confirmed to be correct by NMR: 1 H NMR (400MHz, DMSO) δ9.14(d,J=6.8Hz,1H),8.26(d,J=6.8Hz,1H),8.14(d,J=16.1Hz ,1H),8.04(d,J=8.6Hz,1H),7.96(d,J=8.6Hz,1H),7.81(d,J=7.4Hz,1H),7.70(d,J =8.8Hz,1H),7.55(t,J=7.5Hz,1H),7.48(d,J=7.3Hz,1H),7.42(d,J=16.1Hz,1H),7 .07(d,J=8.8Hz,1H),3.79–3.72(m,2H),3.31(m,J=4.8Hz,3H), HRMS(ESI):m / zcalcd for C 22 H 21 N2S+ ([MI)) + ):419.2118,found:419.2121.

[0208] Performance testing

[0209] Using the commercially available DNA dye propidium iodide (PI) as a control, the compounds prepared in Examples 1-13 above were subjected to the following performance tests:

[0210] The commercially available DNA dye propidium iodide (PI) was purchased from Solarbio. Propidium iodide (PI) has good selectivity for DNA, but its synthesis is complex. In addition, the dye has a short maximum absorption wavelength (352 nm), which requires high-performance lasers. Furthermore, the dye is only localized to the cell nucleus and cannot image DNA in mitochondria.

[0211] Test Example 1: Determination of Absorption and Emission Wavelengths

[0212] Test method: The compounds obtained in Examples 1-13 above were dissolved in DMSO to prepare a 10 mM dye stock solution. The prepared dye stock solution was then added dropwise to a quartz dish containing 3 mL of PBS solution to prepare a 4 μM test solution. The absorption (excitation) spectrum and fluorescence (emission) spectrum were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively. The test results are shown in [reference]. Figure 1 .

[0213] from Figure 1 As can be seen, the excitation wavelengths of the compounds prepared in Examples 1-13 are in the blue-green range (400-550 nm), and the emission wavelengths are in the range of 550-650 nm. Among them, the Stokes shifts of Examples 1, 2, 3, 4, 5, 8, 9, and 13 exceed 100 nm, exhibiting the advantage of a large Stokes shift. The absorption and emission wavelengths of the commercial DNA dye PI are 535 nm and 615 nm, respectively, with a Stokes shift of less than 100 nm. The Stokes shifts of Examples 1, 2, 3, 4, 5, 8, 9, and 13 are all greater than those of the commercial DNA dye PI, demonstrating the advantage of high contrast.

[0214] Test Example 2: Nucleic Acid Selectivity Test

[0215] Test method: The dye stock solution prepared using the nucleic acid probes prepared in Examples 1-13 above was added to a quartz dish containing 3 mL of PBS solution to prepare a 4 μM test solution. Calf thymus DNA and yeast RNA were dissolved separately in PBS solution to prepare 1 mg / mL stock solutions. These stock solutions were then added to the quartz dishes to bring the DNA and RNA concentrations in the test solutions to 140 μg / mL. The fluorescence (emission) spectra were measured using a fluorescence spectrophotometer. The test results are shown in [reference needed]. Figure 2 .

[0216] from Figure 2 It can be seen that the compounds in Examples 1-13 all respond to DNA and RNA. Among them, the responses of Examples 1, 9, and 10 to DNA and RNA are relatively weak, and the fluorescence intensity does not change much before and after the addition of DNA and RNA. Examples 6, 7, 11, and 12 all have obvious responses to DNA and RNA, and the fluorescence intensity is significantly enhanced before and after the addition of DNA and RNA. Examples 2, 3, 4, 5, 8, and 13 have obvious fluorescence responses to DNA, but relatively poor fluorescence responses to RNA.

[0217] Test Example 3

[0218] HepG2 live cells were stained using the mitochondrial-nuclear cascade nucleic acid probe molecules prepared in Examples 1-13 above. HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. After confluence, the cells were digested with trypsin and cultured in confocal microscopy dishes. When the cell density reached approximately 70-80%, the cells were washed three times with PBS, and 2 mL of DMEM medium was added. A prepared dye stock solution (2 μM) was then added, and the cells were incubated for 10 min. Observations were then performed using a confocal laser scanning microscope under both dark and light conditions. The results showed… Figure 3 .

[0219] from Figure 3 As can be seen, Examples 3, 4, 5, 6, 7, and 8 all showed the localization effects of different organelles under dark conditions and under light conditions. Among them, Examples 3, 4, 5, and 8 only responded to DNA, so they showed the sub-organelle imaging effect of locating mitochondria under dark conditions and locating the cell nucleus under light conditions. Examples 6 and 7 responded to both DNA and RNA, so they showed the sub-organelle imaging effect of locating mitochondria under dark conditions and locating the nucleolus under light conditions.

[0220] Test Example 4

[0221] The mitochondrial-nuclear cascade nucleic acid probe molecules prepared in Examples 1-13 above were used to stain HepG2 live cells. HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. After confluence, the cells were digested with trypsin and cultured in confocal microscopy dishes. When the cell density reached approximately 70-80%, the cells were washed three times with PBS, and 2 mL of DMEM medium was added. A prepared dye stock solution (2 μM) was then added, and the cells were incubated for 10 min. The fluorescence migration of subcellular organelles under light conditions was observed using a confocal laser scanning microscope. The results showed… Figure 4-7 .

[0222] from Figure 4-7 It can be seen that, under illumination, Examples 3, 4, 5, and 8 exhibit cascade imaging effects from mitochondria to the cell nucleus; Examples 6 and 7 exhibit cascade imaging effects from mitochondria to the nucleolus; the subcellular organelle migration rates of Examples 3-8 are different, with Example 4 showing the best subcellular organelle migration effect.

[0223] Based on the above experiments, it can be seen that Examples 1-5, 8, 9, and 13 exhibit larger Stokes shifts, which can overcome background interference; Examples 2-5, 8, and 13 show better selectivity for DNA; in HepG2 live cell staining, Examples 3-8 demonstrate unique mitochondrial-nuclear cascade nucleic acid imaging effects, capturing fluorescence signals in both mitochondria and the nucleus. Therefore, Example 4 is the optimal example. To demonstrate that the compound prepared in this application can be used to distinguish between apoptosis and ferroptosis, compound N4 prepared in Example 4 was further tested as an example:

[0224] (1) Photophysical properties

[0225] Compound N4 was dissolved in DMSO to prepare a 10 mM dye stock solution. This stock solution was then added dropwise to quartz dishes containing 3 mL of PBS solution to achieve compound concentrations of 4, 8, 12, and 16 μM. Absorption and fluorescence emission spectra were recorded. The results are shown below. Figure 8 .

[0226] from Figure 8 As can be seen, the maximum absorption wavelength of compound N4 is 486 nm, the maximum emission wavelength is 635 nm, and it has a large Stokes shift (149 nm). Furthermore, as the concentration increases, the absorption and emission wavelengths of compound N4 do not show obvious red shifts or blue shifts, indicating that the absorption and emission wavelengths of compound N4 are stable.

[0227] Add 3 mL of different solvents (PBS, THF, DCM, EtOH, DMSO) to a quartz dish, and add a certain amount of N4 dye stock solution to the quartz dish. The dye concentration is 4 μM. Measure the UV absorption and fluorescence emission spectra. See [link to results]. Figure 9 .

[0228] from Figure 9 It can be seen that the absorption and emission wavelengths of compound N4 vary slightly in different polar solvents, but the Stokes shift is relatively large. The shortest absorption wavelength is 486 nm in PBS solution, while the longest absorption wavelength is 550 nm in dichloromethane, which has low polarity. The emission wavelength does not change significantly in different solvents. The fluorescence intensity is the highest in dichloromethane with low polarity, and the fluorescence intensity is weak in highly polar solvents (EtOH, DMSO, PBS).

[0229] (2) Nucleic acid titration experiment of compound N4

[0230] Compound N4 was prepared at a concentration of 4 μM in a cuvette containing 3 mL of PBS buffer (pH = 7.4). DNA and RNA solutions of 0–120 μg / mL were gradually added, with a concentration gradient of 20 μg / mL. The test results were shown in… Figure 10 and Figure 11 .

[0231] from Figure 10 As can be seen, the maximum absorption wavelength of compound N4 did not change significantly before and after the addition of DNA and RNA, and the maximum absorption wavelength was 486 nm. As the amount of DNA and RNA added increased, the maximum absorption peak decreased slightly, indicating that compound N4 had a weak binding to DNA and RNA and that its molecular conformation did not change significantly.

[0232] from Figure 11 It can be seen that, compared with RNA, compound N4 has a better response to DNA. Before and after binding to DNA, the absorption wavelength of compound N4 did not change significantly, with a maximum absorption wavelength of 486 nm. The fluorescence signal was significantly enhanced, while the maximum emission wavelength did not change significantly, remaining at 635 nm. Furthermore, the fluorescence enhancement to DNA was 7.66 times higher than that to RNA.

[0233] (3) Selectivity test of compound N4

[0234] Compound N4 was prepared at a concentration of 4 μM in a 3 mL cuvette. Then, 15 μM of DNA, RNA, 100 μM of protein (BSA, HSA, NADPH), 100 μM of sugar (Glucose), 100 μM of peptide (GSH), 100 μM of amino acids (L-Asp, L-Glu, L-Ser, L-Cys), and anions and cations (Cu) were added. + Cu 2+ Ag + ,Zn + HSO3 - ,·OH, 1 O2 - The fluorescence intensity was tested and recorded separately, and the detection results are as follows: Figure 12 .

[0235] Depend on Figure 12 The results showed that the fluorescence response of compound N4 to DNA was much higher than that to other substrates. Therefore, compound N4 has excellent selectivity for DNA and can resist the interference of other substrates on its fluorescence signal. This experiment proves that compound N4 has good specificity for nucleic acids and produces fluorescence signals only in the presence of DNA and RNA.

[0236] (4) HepG2 live cell staining experiment with compound N4

[0237] Compound N4 at a concentration of 2 μM and commercial mitochondrial dye Mito-Tracker Deep Red (MTDR) at a concentration of 100 nM were prepared and added to pre-incubated HepG2 cells (cell culture density 10). 5 In culture dishes containing 70-80% cell / mL cells / mL of culture medium, the cells were incubated at 37°C and 5% CO2 for 30 min. After staining, the cells were rinsed with PBS for 1 min each time, followed by the addition of serum-free culture medium. Representative areas were selected and imaged using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of compound N4 was 488 nm, and the receiving band was 600-650 nm. The excitation wavelength of Mito-Tracker Deep Red was 630 nm, and the receiving band was 650-700 nm. The mitochondrial colocalization effect was tested, and the results were shown in [data missing]. Figure 13 middle.

[0238] from Figure 13As can be seen, compound N4 is mainly localized in mitochondria, and the cell structure remains intact after the addition of both dyes. Compound N4 can penetrate the cell membrane of living cells and can be observed while the cells are alive. The co-localization results of Mito-Tracker Deep Red and compound N4 show that compound N4 mainly stains mitochondria under dark conditions, with a co-localization coefficient of 0.964.

[0239] (5) Nucleic acid digestion experiment of compound N4

[0240] When the HepG2 cell density in the culture dish reached 70-80%, the culture medium was removed, and the cells were washed with PBS for 1 min × 3. Then, ice-cold ethanol was added to fix the cells, and the cells were stored at -20°C for 10 min. After removal, the cells were washed with PBS for 1 min × 3, and quantitative amounts of DNase and RNase were added to different dishes for nucleic acid digestion, followed by incubation for several hours. A 2 μM solution of compound N4 and a 2 mg / mL solution of Hoechst 33342 were added to the digested HepG2 cells for 30 min and 5 min respectively, followed by washing with PBS for 1 min × 3. Serum-free culture medium was then added, and representative areas were imaged using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of compound N4 was 488 nm, and the receiving band was 600-650 nm. The excitation wavelength of Hoechst 33342 was 405 nm, and the receiving band was 440-480 nm. The test results showed... Figure 14 middle.

[0241] from Figure 14 The results showed that the fluorescence of cells treated with DNase disappeared, while the fluorescence of cells treated with RNase did not decrease significantly, indicating that both compound N4 and Hoechst 33342 mainly stained the DNA in cells. At the same time, the fluorescence of compound N4 and Hoechst 33342 did not show obvious overlap, indicating that compound N4 hardly stained the DNA in the cell nucleus.

[0242] (6) Mitochondrial-nuclear cascade imaging experiment of compound N4

[0243] A 2 μM solution of compound N4 and a 100 nM solution of the commercial mitochondrial dye Mito-Tracker DeepRed were added to pre-incubated HepG2 cells (cell culture density 10⁻¹²). 5In a culture dish containing 70-80% coverage of cells / mL, incubate for 25 min at 37°C and 5% CO2. Then add 2 mg / mL Hoechst 33342 and incubate for another 5 min under the same conditions. Wash with PBS for 1 min each time, then add serum-free medium and incubate at 450 nm and 50 mW / cm². 2 Under external light source illumination, images were captured and recorded on average once per second using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of compound N4 was 488 nm, and the receiving band was 600-650 nm; the excitation wavelength of Hoechst 33342 was 405 nm, and the receiving band was 440-480 nm; the excitation wavelength of Mito-Tracker Deep Red was 630 nm, and the receiving band was 650-700 nm. The results are shown in... Figure 15 middle.

[0244] from Figure 15 The results showed that under excitation at a wavelength of 488 nm, compound N4 could rapidly migrate from mitochondria to the nucleus. The fluorescence in mitochondria gradually decreased, while the fluorescence in the nucleus gradually increased. The fluorescence migration effect was significant, enabling simultaneous observation of mitochondria and the nucleus.

[0245] (7) Assay for apoptosis and ferroptosis monitoring of compound N4

[0246] HepG2 cells were cultured in two dishes until the cell density reached 70-80% coverage of the bottom. One dish was incubated with H2O2 (0.4 mM) for 30 min to induce apoptosis, while the other dish was incubated with erastin (10 μM) for 12 h to induce ferroptosis. After adding H2O2 and erastin, respectively, 2 μM of compound N4 was added 15 min later. Imaging was performed using an OLYMPUS FV1000 laser confocal microscope. The excitation wavelength of compound N4 was 488 nm, and the receiving wavelength was 600-650 nm. The results showed... Figure 16 middle.

[0247] from Figure 16 The results showed that after H2O2 treatment, the fluorescence of compound N4 decreased in the mitochondria and gradually increased in the nucleus, indicating that when cells undergo apoptosis, compound N4 can migrate from the mitochondria to the nucleus, and the migration process can be observed in real time. Before and after erastin treatment, the fluorescence of compound N4 did not change significantly, and it remained in the mitochondria. Therefore, compound N4 can be used to distinguish between apoptosis and ferroptosis.

[0248] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 class of nucleic acid probes with large Stokes shifts, characterized in that, Its general structural formula is shown in I: Wherein, R1 is H; R2 is selected from N,N-di(2-chloroethyl)amino or N,N-di(3-thiopentyl)amino; R3 is selected from structural formula II; R4 is selected from phenyl, 4-biphenyl or 4-aminophenyl; Y1 - Selected from halide anions, ClO4 - PF6 - CH3COO - or OTs - One of them.

2. The nucleic acid probe with a large Stokes shift according to claim 1, characterized in that, R2 is selected from N,N-di(3-thiopentyl)amino, and in structural formula II, R4 is selected from 4-biphenyl or 4-aminophenyl.

3. The nucleic acid probe with a large Stokes shift according to claim 1, characterized in that, The nucleic acid probe molecule is selected from any one of the following structural formulas: N4, N5, or N8. 。 4. The method for preparing the large Stokes shift nucleic acid probe according to claim 2, characterized in that, The reaction includes the following steps: Compound IV containing R2 substitution and Compound VI containing R4 substitution react under the catalysis of a catalyst to obtain the corresponding compound shown in Formula I. The reaction time is 4-10 h and the reaction temperature is 80-100 °C. ； The molar ratio of compound IV to compound VI is 1:1.0-1.

2.

5. The preparation method according to claim 4, characterized in that, The compound of formula VI is prepared by the following method: (1) 4-methylpyridine and 1-chloro-2,4-dinitrobenzene were reacted in a reaction solvent at a molar ratio of 1:1-2 to prepare the compound shown in formula VIII; (2) The compound of formula VIII obtained in step (1) reacts with the para-amino-substituted aromatic compound in a reaction solvent at a molar ratio of 1:1-5 to obtain the compound of formula VI. 。 6. The preparation method according to claim 4, characterized in that, The catalyst is selected from piperidine, sodium carbonate, or sodium bicarbonate.

7. The preparation method according to claim 5, characterized in that, In step (1), the reaction conditions are: reaction temperature of 70-90℃, reaction time of 6-24 h, and reaction solvent of methanol or ethanol. In step (2), the reaction conditions are: reaction temperature of 70-90℃, reaction time of 6-24 h, and reaction solvent of methanol or ethanol.

8. The application of the large Stokes shift nucleic acid probe according to any one of claims 1-2, characterized in that, The nucleic acid probe is used for mitochondrial-nuclear cascade nucleic acid imaging for non-disease diagnosis and treatment purposes under light induction.

9. The application according to claim 8, characterized in that, The nucleic acid probe migrates from the mitochondria to the cell nucleus under the drive of a wavelength of 468-515 nm.

Images