Near-infrared water-soluble cyanine fluorescent dye and preparation method thereof

Abstract

The invention relates to the technical field of organic compounds and preparation thereof, in particular to a near-infrared water-soluble cyanine fluorescent dye and a preparation method thereof. The molecular performance is optimized through structural regulation and control, so that the defects of the current pentamethine cyanine dye in practical application can be overcome, and the pentamethine cyanine dye has important guiding significance for developing cyanine dyes with more diversified functions.

Description

Technical Field

[0001] This invention relates to the technical field of organic compounds and their preparation, and more specifically, to a near-infrared water-soluble cyanine fluorescent dye and its preparation method. Background Technology

[0002] Fluorescent dyes are substances that, after absorbing light of a certain wavelength, emit light of a wavelength longer than the absorbed wavelength. With technological advancements, fluorescent dyes, as functional pigments, have been widely applied in various fields, particularly in life sciences, clinical medical diagnostics, and immunoassay, where their research has garnered significant global attention. Currently, commercially available fluorescent dyes such as phenanthridines, acridines, imidazoles, and cyanine family dyes play crucial roles in genomics, nucleic acid quantification, and cell analysis. However, the absorption and emission wavelengths of most fluorescent dyes are in the ultraviolet-visible region, which leads to problems such as tissue damage, poor tissue penetration, autofluorescence interference, and reduced detection efficiency when applied to biological sample detection.

[0003] Classical fluorescence imaging techniques typically use the visible light range of 400–650 nm for excitation and emission. This is susceptible to interference, absorption, and scattering from the autofluorescence of biological tissues, resulting in low tissue penetration and limited clarity, failing to fully reflect physiological and pathological information. Recent advancements in near-infrared imaging technology have offered new opportunities to overcome this problem. Because near-infrared I region fluorescent probes emit wavelengths in the 700–1000 nm range, biological tissues exhibit low self-absorption, scattering, and autofluorescence within this range, background fluorescence interference can be minimized, thus improving sensitivity. Fluorescence detection technology, with its pollution-free, fast-response, high-sensitivity, and ease-of-operation characteristics, is widely used in ion detection, molecular recognition, cell labeling, and photodynamic therapy. Especially in the context of the deepening development of life sciences, fluorescence technology demonstrates its unparalleled advantages in analyzing and detecting trace substances in life processes.

[0004] Currently developed fluorescent probes mainly include porphyrins, coumarins, rhodamines, borodipyrrole methylene difluoropolymers (BODIPY), and cyanine dyes. Among them, cyanine dyes connect electron donors (D) and acceptors (A) through polymethyl cyanide conjugated chains. Their absorption and emission spectra are affected by the length of the methyl cyanide chain and the substituents, achieving full coverage in the visible and near-infrared regions, thus finding wide application in bioanalysis. In particular, pentamethyl cyanine dyes have maximum absorption and emission wavelengths in the biological blank window region around 800 nm, effectively avoiding interference from biological background fluorescence during bioanalysis and improving detection accuracy.

[0005] Indocyanine dyes possess characteristics such as high stability, large molar extinction coefficient, wide tunable absorption wavelength range, and strong structural modifiability, and have been applied in fields such as optical disc storage media, nonlinear optical materials, and solar cells. Their applications are particularly widespread in biomedical fields such as biomolecular labeling, photodynamic therapy (PDT), DNA sequencing, and pH probes. Since most biomolecules, such as DNA, are water-soluble, the application of indocyanine dyes in the biomedical field requires not only good spectral properties but also good water solubility. However, most indocyanine dyes have poor water solubility, thus limiting their application in biomedicine. To improve this, hydrophilic groups, such as carboxyl, hydroxyl, and sulfonic acid groups, can be introduced into the cyanine dye molecule to enhance its water solubility. This paper demonstrates that introducing two sulfonic acid groups into the indole ring significantly enhances water solubility. The fluorescence emission wavelength can be altered by changing the length of the methylene chain. Amershan Pharmacia Biotech was the first to apply cyanine dyes with different fluorescence emission wavelengths to the dual-color fluorescence detection of gene chips; currently, commercially available complete sets of instruments and reagents are available, but they are expensive. Cyanide dyes have become the preferred fluorescent labels for gene chip assays, but due to the numerous side reactions in cyanide dye synthesis and the similar polarity of the byproducts, the separation and purification of these products is quite difficult. Water-soluble cyanide dyes have high molecular polarity, making separation and purification even more challenging. This invention solves the separation difficulties at their source through the design of a simple chemical reaction. Summary of the Invention

[0006] This invention provides a near-infrared water-soluble cyanine fluorescent dye and its preparation method. By optimizing molecular properties through structural regulation, it can not only make up for the shortcomings of the current pentamethine cyanine dye in practical applications, but also has important guiding significance for the development of cyanine dyes with more diverse functions.

[0007] In a first aspect, the present invention provides a near-infrared water-soluble cyanine fluorescent dye having the following structural formula:

[0008]

[0009] In the formula, n = 1, 2, 3, 4, 5, 6;

[0010] R1 is selected from SO3 - CH2SO3 - CH2CH2SO3 - One of them;

[0011] R2 is selected from one of CH3, CH2CH3, and CH2CH2CH3.

[0012] Preferably, the near-infrared water-soluble cyanine fluorescent dye is pentamethine near-infrared fluorescent dye.

[0013] Preferably, n=3, R1=SO3 - R2 = CH2CH3, the synthetic route of the pentamethyl cyanine near-infrared fluorescent dye is as follows:

[0014]

[0015] Secondly, the present invention provides a method for preparing a near-infrared water-soluble cyanine fluorescent dye, comprising the following steps:

[0016] (1) Weigh 4-hydrazylbenzenesulfonic acid into a flask, add acetic acid, and then add tert-butylmethyl ketone dropwise to the solution. Stir the reaction at 120-150℃. After the reaction is complete, cool down to room temperature and dropwise into ethyl acetate. A yellow precipitate is precipitated. Filter and wash with ethyl acetate. After vacuum drying, a yellow solid 2 is obtained.

[0017] (2) Weigh yellow solid 2 into a flask, add acetonitrile, then add iodoethane dropwise to the solution, heat to reflux and stir to react. When the reaction is complete, cool to room temperature and a pink precipitate is formed. Filter and wash with acetonitrile, and dry under vacuum to obtain red solid 3.

[0018] (3) Weigh the red solid 3 into a flask, add n-butanol, then add 2-(4-methoxyphenyl)malondialdehyde dropwise to the solution, heat to reflux and stir to react, when the reaction is complete, cool to room temperature, remove solvent by evaporation, add water, extract with dichloromethane, dry, remove solvent by evaporation, and separate by column chromatography to obtain blue solid product 4.

[0019] (4) Weigh solid product 4 into a flask, add dichloromethane, cool down, and then add boron tribromide and dichloromethane solution dropwise to the solution. After the addition is complete, raise the temperature to room temperature and react. When the reaction is complete, cool down and add water for extraction, dry, remove the solvent by evaporation, and separate by column chromatography to obtain blue solid product 5.

[0020] (5) Weigh the blue solid product 5 into a flask, add DMF, then add potassium carbonate to the solution and stir, add bromo-ethylene glycol-tert-butyl propionate, heat the reaction, and when the reaction is complete, cool down and add water to extract, dry, remove the solvent by evaporation, and separate by column chromatography to obtain the blue solid product 6.

[0021] (6) Weigh the blue solid product 6 into a flask, add dichloromethane, cool down, then add trifluoroacetic acid dropwise to the solution and stir. Heat to room temperature and react until the reaction is complete. Remove the solvent by evaporation and separate by column chromatography to obtain the near-infrared water-soluble cyanine fluorescent dye.

[0022] Thirdly, the present invention provides an application of a near-infrared water-soluble cyanine fluorescent dye, wherein the near-infrared water-soluble cyanine fluorescent dye is prepared into a probe, and the probe is used for applications in cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, and DNA sequencing.

[0023] In summary, the present invention has the following beneficial effects:

[0024] This invention uses substituted phenylmalondialdehyde and modified indole as molecular building blocks, and systematically regulates the electrical properties, steric hindrance, and water solubility of these building blocks through molecular engineering strategies to prepare near-infrared fluorescent probes. These probes possess numerous advantages, including long fluorescence emission wavelengths and large Stokes shifts, effectively avoiding interference from biological background fluorescence. In nucleic acid labeling, near-infrared fluorescent probes exhibit significant advantages such as high signal-to-noise ratio, good selectivity, and good biocompatibility.

[0025] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the scope of protection of the present invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments. It should be noted that: unless otherwise specified, the conditions in the following embodiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0027] Example

[0028] A method for preparing pentamethyl cyanine near-infrared fluorescent dye specifically includes the following steps:

[0029] (1) Weigh 10 g of 4-hydrazinobenzenesulfonic acid (53.13 mmol) into a 250 mL flask, add 100 mL of acetic acid, and then add 5 g of tert-butylmethyl ketone (58.45 mmol) dropwise to the solution. The mixture was stirred at 120 °C and the reaction was monitored by TLC. After the reaction was complete, the temperature was lowered to room temperature, and the reaction solution was added dropwise to 100 mL of ethyl acetate. A yellow precipitate was precipitated, filtered, and washed with ethyl acetate. The filter cake was dried under vacuum to obtain 11.4 g of yellow solid 2, with a yield of 90%.

[0030] LCMS(ESI):m / z:[MH] - C 11 H 13 NO3S,calcd for 239.06; found,239. 1 H NMR (600MHz, DMSO-d6); δ7.61(d,J=1.6Hz,1H),7.58–7.49(m,1H),7.33(d,J=7.9Hz,1H),2.21(s,3H),1.24(s,6H).

[0031] (2) Weigh 10 g of compound 2 (33.67 mmol) into a 500 mL flask, add 200 mL of acetonitrile, and then add 32.6 g of iodoethane (208.95 mmol) dropwise to the solution. The mixture was heated to reflux and stirred for 24 hours. HPLC monitoring showed that the reaction was complete. The mixture was cooled to room temperature, and a pink precipitate formed. This precipitate was filtered and washed with acetonitrile. The filter cake was dried under vacuum to obtain 10.7 g of pure red solid 3, with a yield of 95%. LCMS (ESI): m / z: [MH] - C 13 H 19 NO3S,calcd for 269; found 269, 1 H NMR (600MHz, DMSO-d6) δ8.03(d,J=1.6Hz,1H),7.90(d,J=8.3Hz,1H),7.83(dd,J=8.3 ,1.6Hz,1H),4.47(q,J=7.3Hz,2H),2.81(s,3H),1.54(s,6H),1.43(t,J=7.3Hz,3H).

[0032] (3) Weigh 9 g of compound 3 (41.79 mmol) into a 250 mL flask, add 100 mL of n-butanol, and then add 3 g of 2-(4-methoxyphenyl)malondialdehyde (16.84 mmol) dropwise to the solution. Heat to reflux and stir for 12 hours. Monitor the reaction by HPLC, which shows that the reaction is complete. Cool to room temperature, evaporate to dryness to remove the solvent, add water, extract with dichloromethane, dry, evaporate to dryness to remove the solvent, and separate by column chromatography to obtain 9.1 g of blue solid product 4, with a yield of 80%.

[0033] LCMS(ESI):m / z:[MH] - C 36 H 41 N2O7S2,calcd for 677; found 676. 1 H NMR (600MHz, DMSO-d6): δ8.48(d,J=14.0Hz,2H),7.84(d,J=1.6Hz,2H),7.62(dd,J=8.3,1.6Hz,2H),7.30(d,J=8.3Hz,2H),7.27–7 .16(m,2H),7.12(d,J=8.4Hz,2H),5.71(d,J=14.0Hz,2H),3.85(s,4H),3.80(d,J=7.4Hz,3H),1.75(s,12H),1.10(t,J=7.2Hz,6H).

[0034] (4) Weigh 9 g of compound 4 (13.28 mmol) into a 500 mL flask, add 200 mL of dichloromethane, cool to 0 °C, and then add dropwise 6.6 g of boron tribromide (16.84 mmol) dichloromethane solution. After the addition is complete, raise the temperature to room temperature and react for 2 hours. HPLC monitoring shows that the reaction is complete. Cool to about 10 °C, add water for extraction, dry, remove solvent by evaporation, and separate by column chromatography to obtain 8 g of blue solid product 5, with a yield of 90%.

[0035] LCMS(ESI):m / z:[MH] - C 35 H 39 N2O7S2,calcd for 663; found 663. 1 H NMR (600MHz, DMSO-d6) δ9.66(s,1H),8.46(d,J=14.0Hz,2H),7.83(d,J=1.6Hz,2H),7.62(dd,J=8.1,1.6Hz,2H),7.30(d,J=8.3Hz,2 H), 7.10 (d, J = 8.0Hz, 2H), 6.94 (d, J = 8.0Hz, 2H), 5.74 (d, J = 14.0Hz, 2H), 3.80 (q, J = 7.3Hz, 4H), 1.75 (s, 12H), 1.10 (t, J = 7.2Hz, 6H).

[0036] (5) Weigh 5g of compound 5 (7.53mmol) into a 250mL flask, add 100mL of DMF, then add 3.12g of potassium carbonate to the solution and stir for half an hour. Add 4.5g of bromo-ethylene glycol-tert-butyl propionate, raise the temperature to 80℃ and react for 24 hours. HPLC monitoring showed that the reaction was complete. Cool to about room temperature, add water for extraction, dry, remove solvent by evaporation, and separate by column chromatography to obtain 5.5g of blue solid product 6, with a yield of 83%.

[0037] LCMS(ESI):m / z:[MH] - C 46 H 59 N2O 11 S2,calcd for 879;found 880. 1H NMR(600MHz,DMSO-d6)δ8.58(d,J=14.0Hz,2H),7.84(d,J=1.6Hz,2H),7.62(dd,J=8 .3,1.6Hz,2H),7.30(d,J=8.3Hz,2H),7.27–7.16(m,2H),7.12(d,J=8.4Hz,2H),5.71 (d,J=14.0Hz,2H),3.85(s,4H),3.80(d,J=7.4Hz,3H),3.70–3.64(m,6H),3.55(t,J= 6.0Hz, 4H), 3.04 (t, J = 6.0Hz, 2H). 1.75 (s, 12H), 1.45 (s, 9H), 1.10 (t, J = 7.2Hz, 6H).

[0038] (6) Weigh 5g of compound 6 (7.53mmol) into a 250mL flask, add 100mL of dichloromethane, cool to 0℃, then add 6.6g of trifluoroacetic acid dropwise to the solution and stir for half an hour. Then raise the temperature to room temperature and react for 6 hours. HPLC monitoring showed that the reaction was complete. Remove the solvent by evaporation and separate by column chromatography to obtain 4g of blue solid product 7, with a yield of 85%.

[0039] LCMS(ESI):m / z:[MH] - C 42 H 51 N2O 11 S2,calcd for 823; found 823. 1 H NMR (600MHz, DMSO-d6) δ8.58(d,J=14.0Hz,2H),7.84(d,J=1.6Hz,2H),7.62(dd,J =8.3,1.6Hz,2H),7.30(d,J=8.3Hz,2H),7.27–7.16(m,2H),7.12(d,J=8.4Hz,2H) ,5.71(d,J=14.0Hz,2H),3.85(s,4H),3.80(d,J=7.4Hz,3H),3.70–3.64(m,6H),3 .55(t,J=6.0Hz,4H),3.04(t,J=6.0Hz,2H).1.75(s,12H),1.10(t,J=7.2Hz,6H).

[0040] (7) Weigh 5g of the blue solid product into a flask, add DMF, then add potassium carbonate to the solution and stir. Add tert-butyl 3-bromopropionate, heat to reflux and react until complete. Cool down and add water for extraction, dry, remove solvent by evaporation, and separate by column chromatography to obtain the blue solid product. Put the blue solid product into a 250mL flask, add 100mL of dichloromethane, cool to 0℃, then add 6.6g of trifluoroacetic acid dropwise to the solution and stir for half an hour. Heat to room temperature and react for 6 hours. HPLC monitoring of the reaction showed that the reaction was complete. Remove solvent by evaporation and separate by column chromatography to obtain the final blue solid product with a yield of 80%.

[0041] n=1, R1=SO3 - R2=CH2CH3

[0042]

[0043] LCMS(ESI):m / z:[MH] - C38H43N2O9S2,calcd for 735; found 735. 1 H NMR (600MHz, DMSO-d6) δ8.58(d,J=14.0Hz,2H),7.84(d,J=1.6Hz,2H),7.62(dd,J=8.3,1.6Hz,2H),7.30(d,J=8.3Hz,2H),7.27–7.16(m,2H), 7.12(d,J=8.4Hz,2H),5.71(d,J=14.0Hz,2H),3.85(s,4H),3.55(t,J=6.0Hz,2H),3.04(t,J=6.0Hz,2H).1.75(s,12H),1.10(t,J=7.2Hz,6H).

[0044] The above description is merely an exemplary embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A near-infrared water-soluble cyanine fluorescent dye, characterized in that, It has the following structural formula: In the formula, n = 1, 2, 3, 4, 5, 6; R1 is selected from SO3 - CH2SO3 - CH2CH2SO3 - One of them; R2 is selected from one of CH3, CH2CH3, and CH2CH2CH3.

2. The near-infrared water-soluble cyanine fluorescent dye according to claim 1, characterized in that, The near-infrared water-soluble cyanine fluorescent dye is pentamethine near-infrared fluorescent dye.

3. The near-infrared water-soluble cyanine fluorescent dye according to claim 2, characterized in that, With n=3 and R1=SO3 - R2 = CH2CH3, the synthetic route of the pentamethyl cyanine near-infrared fluorescent dye is as follows:

4. The method for preparing the near-infrared water-soluble cyanine fluorescent dye according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Weigh 4-hydrazylbenzenesulfonic acid into a flask, add acetic acid, and then add tert-butylmethyl ketone dropwise to the solution. Stir the reaction at 120-150℃. After the reaction is complete, cool down to room temperature and dropwise into ethyl acetate. A yellow precipitate is precipitated. Filter and wash with ethyl acetate. After vacuum drying, a yellow solid 2 is obtained. (2) Weigh yellow solid 2 into a flask, add acetonitrile, then add iodoethane dropwise to the solution, heat to reflux and stir to react. When the reaction is complete, cool to room temperature and a pink precipitate is formed. Filter and wash with acetonitrile, and dry under vacuum to obtain red solid 3. (3) Weigh the red solid 3 into a flask, add n-butanol, then add 2-(4-methoxyphenyl)malondialdehyde dropwise to the solution, heat to reflux and stir to react, when the reaction is complete, cool to room temperature, remove solvent by evaporation, add water, extract with dichloromethane, dry, remove solvent by evaporation, and separate by column chromatography to obtain blue solid product 4. (4) Weigh solid product 4 into a flask, add dichloromethane, cool down, and then add boron tribromide and dichloromethane solution dropwise to the solution. After the addition is complete, raise the temperature to room temperature and react. When the reaction is complete, cool down and add water for extraction, dry, remove the solvent by evaporation, and separate by column chromatography to obtain blue solid product 5. (5) Weigh the blue solid product 5 into a flask, add DMF, then add potassium carbonate to the solution and stir, add bromo-ethylene glycol-tert-butyl propionate, heat the reaction, and when the reaction is complete, cool down and add water to extract, dry, remove the solvent by evaporation, and separate by column chromatography to obtain the blue solid product 6. (6) Weigh the blue solid product 6 into a flask, add dichloromethane, cool down, then add trifluoroacetic acid dropwise to the solution and stir. Heat to room temperature and react until the reaction is complete. Remove the solvent by evaporation and separate by column chromatography to obtain the near-infrared water-soluble cyanine fluorescent dye.

5. An application of a near-infrared water-soluble cyanine fluorescent dye, characterized in that, The near-infrared water-soluble cyanine fluorescent dye according to any one of claims 1-4 is used to prepare a probe, which is used for applications in cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, and DNA sequencing.

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