Water-soluble pentamethine cyanine dye as well as preparation method and application thereof

Abstract

The invention discloses a water-soluble pentamethine cyanine dye and a preparation method and application thereof, the water-soluble pentamethine cyanine dye has a structure shown in a general formula I: # imgabs0 #. The water-soluble pentamethine cyanine dye has the advantages that a sulfonic group is introduced to an indole benzene ring, a benzyl carboxyl group is introduced to one side of an N site, and different groups are introduced to the other side, so that the water-soluble pentamethine cyanine dye is obtained; the light stability and the water solubility of dye molecules are improved. The pentamethine cyanine dye prepared by the invention shows remarkable advantages in spectral characteristics, has a molar extinction coefficient higher than that of the traditional cyanine dye and has strong light energy absorption capacity, and is beneficial to improving the light energy utilization rate and reducing the dye dosage; the absolute fluorescence quantum yield reaches a high level, the intensity and sensitivity of imaging signals are remarkably enhanced, and a more sensitive and reliable tool is provided for biological imaging and molecular detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent dyes, and particularly to a water-soluble pentamethine cyanine dye, a preparation method thereof, and an application thereof. Background Art

[0002] With the in-depth expansion of the scientific research field, the biofluorescent staining technology has become an indispensable tool for exploring the mysteries of life and analyzing the structure and function of organisms. This technology cleverly bonds fluorescent dye molecules with target biomolecules (such as proteins, antibodies, cell membranes, etc.) through covalent or non-covalent bonds, realizing the precise labeling and efficient detection of specific components in biological samples, and providing strong support for the visualization analysis of intracellular dynamic processes. As an emerging cancer treatment strategy, photodynamic therapy takes a unique approach. Through the energy conversion of dye molecules under light excitation, a series of active factors with a killing effect on cancer cells are induced, thereby inhibiting tumor growth, showing unique advantages that traditional radiotherapy and chemotherapy do not have.

[0003] In the research field of traditional pentamethine cyanine dyes, pentamethine cyanine dyes containing sulfonate substituents can be well used in in vivo and in vitro bio-detection applications because of their high molar extinction coefficient and good water solubility.

[0004] However, the current development of pentamethine cyanine dyes containing sulfonate substituents also faces many challenges, specifically manifested as: solubility problems, poor solubility in non-polar solvents, which hinders the wide application of dyes in biological systems; low fluorescence quantum yield, which limits the application of dyes; poor photostability, which limits the application of dyes in high-resolution imaging processes; and single synthesis method, resulting in few sites where the dyes can be modified. Therefore, near-infrared fluorescent dyes need to be developed to address these problems, overcome the existing defects, and make them have better imaging, labeling, and detection capabilities. Summary of the Invention

[0005] Aiming at the technical problem of insufficient photostability of dyes existing in the prior art, the present invention provides a water-soluble pentamethine cyanine dye, a preparation method thereof, and an application thereof.

[0006] To achieve the above object, the present invention provides a water-soluble pentamethine cyanine dye having the structure of General Formula I:

[0007]

[0008] In General Formula I,

[0009] R is independently selected from one of hydrogen, aryl, substituted aryl, halogen group, carboxyl group, sulfonate group, nitro group, cyano group, alkyl group having 1-18 carbons, carboxyalkyl group having 1-18 carbons, alkylsulfonate group having 1-18 carbons, ester group having 1-18 carbons, alkylsulfonate salt having 1-18 carbons, hydroxyalkyl group having 1-18 carbons, and amide group having 1-18 carbons;

[0010] Y is selected from one of O, S, ester group, and methylene group having 1-18 carbons;

[0011] M is selected from metal ions, NH4 + or H + in one of them.

[0012] Furthermore, R is one of aryl, substituted aryl, and 1,4-diazabicyclo[2.2.2]octyl;

[0013] Y is one of methylene, ethylene, propylene, and ester group;

[0014] M is metal ion Na + .

[0015] To achieve the above object, the present invention also provides a preparation method of the water-soluble pentamethine cyanine dye, including the following steps:

[0016]

[0017] S1. Dissolve compound Y1 and N-alkylation reagent in an organic solvent, and reflux and react for 12-24 h under the protection of an inert gas to obtain a reaction solution. After recrystallization and purification of the reaction solution, compound Y4 is obtained, wherein the molar ratio of compound Y1 to N-alkylation reagent is 1:2-10;

[0018] Dissolve compound Y1 and 4-bromomethylbenzoic acid in an organic solvent, and reflux and react for 12-24 h under the protection of an inert gas to obtain a reaction solution. After recrystallization and purification of the reaction solution, compound Y2 is obtained, wherein the molar ratio of compound Y1 to 4-bromomethylbenzoic acid is 1:2-10;

[0019] The N-alkylation reagent is selected from one of halogen-substituted alkyl, benzyl, benzyl halogen group, benzyl carboxyl group, benzyl sulfonate group, benzyl nitro group, benzyl cyano group, alkyl group having 1-18 carbons, carboxyalkyl group having 1-18 carbons, alkylsulfonate group having 1-18 carbons, ester group having 1-18 carbons, alkylsulfonate salt having 1-18 carbons, hydroxyalkyl group having 1-18 carbons, and amide group having 1-18 carbons;

[0020] S2. Dissolve compound Y2 and compound S1 in an organic acid and acetic anhydride solvent, heat to 40 - 120 °C under an inert atmosphere and react for 2 - 6 h; after cooling to room temperature, perform recrystallization, and after solid-liquid separation, take the solid phase, dry it, and purify it to obtain compound Y3, wherein the molar ratio of compound Y2 to compound S1 is 1:0.8 - 1.2, and the volume ratio of the organic acid to the acetic anhydride solvent is 1:0.5 - 2;

[0021] S3. Dissolve compound Y3, compound Y4, a base catalyst capable of introducing M, and an acetic anhydride solvent, heat to 40 - 120 °C under an inert atmosphere and react for 2 - 6 h; after cooling to room temperature, perform recrystallization, and after solid-liquid separation, take the solid phase, dry it, and use dichloromethane and methanol as elution solvents to obtain a water-soluble pentamethine cyanine dye after purification, wherein the molar ratio of compound Y3, compound Y4, and the base catalyst is 1:0.8 - 1.2:2 - 10.

[0022] Further, in S1, the organic solvent is selected from at least one of toluene, o-dichlorobenzene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran, and sulfolane.

[0023] Further, in S2, the organic acid is selected from at least one of acetic acid, hydrochloric acid, sulfuric acid, and polyphosphoric acid.

[0024] Further, in S3, the base catalyst capable of introducing M is selected from at least one of sodium carbonate, sodium bicarbonate, sodium acetate, sodium hydroxide, and potassium hydroxide.

[0025] Further, in S1, S2, and S3, the solvent used for recrystallization is selected from at least one of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone, propanol, and isopropanol.

[0026] To achieve the above object, the present invention also provides an application of the water-soluble pentamethine cyanine dye, and the water-soluble pentamethine cyanine dye is applied to cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, DNA sequencing, and preparation of reagents for specific recognition and photodynamic therapy of tumors.

[0027] Further, the fluorescence imaging emission wavelength of the water-soluble pentamethine cyanine dye during application is 600 - 800 nm.

[0028] The present invention has the following beneficial effects:

[0029] 1. The water-soluble pentamethine cyanine dye provided by the present invention shows significant advantages in spectral characteristics. Different water-soluble pentamethine cyanine dyes have a molar extinction coefficient higher than that of traditional pentamethine cyanine dyes in PBS buffer, up to 2.53×10 5 L / mol -1 ·cm -1, indicating its strong ability to absorb light energy, which helps improve the light energy utilization rate and reduce the amount of dye used;

[0030] 2. The absolute fluorescence quantum yield of the water-soluble pentamethine cyanine dye reaches a relatively high level, up to 0.327. A high fluorescence quantum yield means that the dye molecules can be more effectively converted into fluorescence emission after absorbing light energy, thus significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection;

[0031] 3. The water-soluble pentamethine cyanine dye has high photo-stability. By introducing a bulky benzyl group at the indole N position of the dye molecule and introducing different groups on the benzyl group, the photo-stability of the dye molecule is further greatly improved. Among them, the cyanine dye with the indole N position substituted by p-nitrobenzyl has significantly higher photo-stability in water than the linear cyanine dye;

[0032] 4. The water-soluble pentamethine cyanine dye has high structural diversity and modifiability. Through the structure of the indole N position of the cyanine dye in the present invention, the formation of π-π stacking within the dye molecule is successfully achieved, increasing the steric hindrance, thereby hindering the attack of reactive oxygen species on the dye molecule, further inhibiting the photo-bleaching of the dye molecule, and then realizing the regulation of the photo-stability of the dye molecule. This structural diversity and modifiability not only enrich the types of dyes but also provide a broad space for further optimizing their spectral properties and application performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is the high-resolution mass spectrum of Dye 2 disclosed in Example 2 of the present invention;

[0035] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of Dye 2 disclosed in Example 2 of the present invention;

[0036] Figure 3 It is the high-resolution mass spectrum of Dye 3 disclosed in Example 3 of the present invention;

[0037] Figure 4 It is the nuclear magnetic resonance hydrogen spectrum of Dye 3 disclosed in Example 3 of the present invention;

[0038] Figure 5 It is the nuclear magnetic resonance hydrogen spectrum of Dye 4 disclosed in Example 4 of the present invention;

[0039] Figure 6 It is the 1H NMR spectrum of Dye 5 disclosed in Example 5 of the present invention;

[0040] Figure 7 It is the 1H NMR spectrum of Dye 6 disclosed in Example 6 of the present invention;

[0041] Figure 8 It is the absorption spectrum of Dyes 1-4 disclosed in Examples 1-4 of the present invention in PBS buffer;

[0042] Figure 9 It is the fluorescence emission spectrum of Dyes 1-4 disclosed in Examples 1-4 of the present invention in PBS buffer;

[0043] Figure 10 It is the light stability test chart of Dye 2 disclosed in Example 2 of the present invention;

[0044] Figure 11 It is the light stability test chart of Dye 3 disclosed in Example 3 of the present invention;

[0045] Figure 12 It is the light stability test chart of Dye 4 disclosed in Example 4 of the present invention;

[0046] Figure 13 It is the light stability test chart of Dye 5 disclosed in Example 5 of the present invention;

[0047] Figure 14 It is the light stability test chart of Dye 6 disclosed in Example 6 of the present invention;

[0048] Figure 15 It is the molecular structure and light stability test chart of the comparative molecule of the present invention, a commercially available water-soluble pentamethine cyanine dye. Detailed implementation manners

[0049] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0050] Unless otherwise specified, the terms used herein have the following meanings. The term "halogen" used in the present invention includes fluorine, chlorine, bromine and iodine; "alkyl" includes straight-chain alkyl and branched-chain alkyl; "room temperature" refers to the temperature range of 20-25 °C.

[0051] Preparation examples: Preparation of raw materials and intermediates

[0052] Preparation Example 1: Preparation of Compound Y2:

[0053]

[0054] Potassium 2,3,3-trimethylindole-5-sulfonate (Compound Y1, 2.00 g, 7.21 mmol) and 4-(bromomethyl)benzoic acid (3.10 g, 14.42 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of diethyl ether, and at this time, a solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Compound Y2 as a light pink solid powder (1.95 g, 5.22 mmol), with a yield of 72.4%.

[0055] Preparation Example 2: Preparation of Compound Y3:

[0056]

[0057] Compound Y2 (2.50 g, 6.69 mmol) and Compound S1 (1.73 g, 6.69 mmol) were added to a 50 mL two-necked round-bottom flask, and then 20 mL of a mixed solvent of acetic acid:acetic anhydride with a volume ratio of 1:1 was added. The mixture was heated to 100 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate. The crude product was purified by C18 reverse silica gel column chromatography using a 10%-50% aqueous methanol solution as the elution solvent to obtain Y3 (2.80 g, 5.14 mmol), with a yield of 76.8%.

[0058] Examples

[0059] Example 1:

[0060] Preparation of Water-Soluble Pentamethine Cyanine Dye 1:

[0061] S1: Preparation of Compound Y4.1

[0062]

[0063] Potassium 2,3,3-trimethylindole-5-sulfonate (Compound Y1, 2.00 g, 7.21 mmol) and benzyl bromide (2.47 g, 14.42 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of diethyl ether, and at this time, a solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Compound Y4.1 as a light pink solid powder (1.80 g, 5.46 mmol), with a yield of 75.8%.

[0064] S2: Preparation of Water-Soluble Pentamethine Cyanine Dye 1

[0065]

[0066] Add Y4.1 (200 mg, 0.607 mmol), Y3 prepared in Preparation Example 2 (331 mg, 0.607 mmol), and sodium acetate (249 mg, 3.04 mmol) into a 50 mL two-necked round-bottom flask, then add 10 mL of acetic anhydride solvent, and heat to 50 °C under a nitrogen atmosphere for reaction for 4 h; after cooling to room temperature, add the reaction solution into 50 mL of diethyl ether, filter, wash the filter cake with ethyl acetate, and dry; use a 10%-50% methanol aqueous solution as the elution solvent, and purify the crude product by C18 reverse silica gel column chromatography to obtain water-soluble pentamethine cyanine dye 1 (103 mg, 0.135 mmol), with a yield of 22.3%. 1 H NMR (600 MHz, Methanol-d4) δ 8.38 (td, J = 13.0, 5.1 Hz, 2H), 8.05–8.01 (m, 2H), 7.94 (d, J = 1.7 Hz, 2H), 7.86–7.79 (m, 4H), 7.34–7.29 (m, 5H), 7.27 (d, J = 8.4 Hz, 1H), 6.49 (t, J = 12.4 Hz, 1H), 6.29 (t, J = 13.3 Hz, 2H), 5.43 (d, J = 17.4 Hz, 4H), 1.78 (d, J = 6.2 Hz, 12H).

[0067] Example 2:

[0068] Preparation of Water-Soluble Pentamethine Cyanine Dye 2:

[0069] S1: Preparation of Compound Y4.2

[0070]

[0071] Add compound Y1 2,3,3-trimethylindole-5-sulfonate potassium (2.00 g, 7.21 mmol) and p-nitrobenzyl bromide (3.12 g, 14.42 mmol) into a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and reflux the reaction for 12 h under a nitrogen atmosphere; after cooling to room temperature, dissolve the remaining solid in the reaction solution with a small amount of methanol and slowly drop it into 150 mL of diethyl ether, and at this time, the solid precipitates; then filter, wash, and dry to obtain Y4.2 as a light pink solid powder (1.70 g, 3.42 mmol), with a yield of 63.0%.

[0072] S2: Preparation of Water-Soluble Pentamethine Cyanine Dye 2

[0073]

[0074] Y4.2 (200 mg, 0.534 mmol), Y3 prepared in Preparation Example 2 (291 mg, 0.534 mmol), and sodium acetate (219 mg, 2.67 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, 10 mL of acetic anhydride solvent was added, and the mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by C18 reverse silica gel column chromatography using a 10%-50% aqueous methanol solution as the eluent to obtain water-soluble pentamethine cyanine dye 2 (120 mg, 0.135 mmol) with a yield of 27.9%. 1 1H NMR (600 MHz, Methanol-d4) δ 8.34 (q, J = 12.0, 11.3 Hz, 2H), 8.24 (t, J = 9.4 Hz, 2H), 7.97 (dd, J = 23.5, 10.3 Hz, 4H), 7.84 (tt, J = 16.5, 8.3 Hz, 2H), 7.48 (dd, J = 12.4, 8.1 Hz, 2H), 7.28 (dd, J = 44.6, 8.8 Hz, 4H), 6.50 (t, J = 12.5 Hz, 1H), 6.30 (dq, J = 43.3, 9.0, 4.9 Hz, 2H), 5.49 (dd, J = 18.5, 10.6 Hz, 4H), 1.82 (s, 12H).

[0075] Example 3:

[0076] Preparation of water-soluble pentamethine cyanine dye 3:

[0077] S1: Preparation of compound Y4.3

[0078]

[0079] Potassium 2,3,3-trimethylindole-5-sulfonate (2.00 g, 7.21 mmol) and potassium p-bromomethylbenzenesulfonate (4.17 g, 14.42 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere; after cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 ml of diethyl ether, and at this time, the solid precipitated; then it was filtered, washed, and dried to obtain Y4.3 as a light pink solid powder (2.35 g, 5.25 mmol) with a yield of 72.8%.

[0080] S2: Preparation of water-soluble pentamethine cyanine dye 3

[0081]

[0082] Y4.3 (200 mg, 0.447 mmol), Y3 prepared in Preparation Example 2 (243 mg, 0.447 mmol), and sodium acetate (183 mg, 2.23 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, 10 mL of acetic anhydride solvent was added, and the mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by C18 reverse silica gel column chromatography using a 10% - 50% aqueous methanol solution as the elution solvent to obtain the water-soluble pentamethine cyanine dye 3 (115 mg, 0.133 mmol) with a yield of 29.8%. 1 H NMR (600 MHz, Methanol-d4) δ 8.31 (td, J = 13.0, 5.1 Hz, 2H), 8.01–7.97 (m, 2H), 7.94 (d, J = 1.7 Hz, 2H), 7.86–7.79 (m, 4H), 7.34–7.29 (m, 4H), 7.27 (d, J = 8.4 Hz, 1H), 6.49 (t, J = 12.4 Hz, 1H), 6.29 (t, J = 13.3 Hz, 2H), 5.43 (d, J = 17.4 Hz, 4H), 1.81 (d, J = 6.2 Hz, 12H).

[0083] Example 4:

[0084] Preparation of water-soluble pentamethine cyanine dye 4:

[0085] S1: Preparation of alkylating agent

[0086]

[0087] p-Nitrophenethyl alcohol (2.00 g, 12.0 mmol) and trifluoromethanesulfonic anhydride (4.05 g,, 14.36 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of dichloromethane, and the mixture was reacted at -50 °C for 15 min under a nitrogen atmosphere. After warming to room temperature, the reaction solution was extracted with water and dichloromethane, and the organic phase was evaporated to dryness. The crude product was purified by normal-phase silica gel column chromatography using petroleum ether:ethyl acetate = 30:1 - 5:1 as the elution solvent to obtain the alkylating agent as a pale yellow oily liquid (2.26 g, 7.55 mmol) with a yield of 63.1%.

[0088] S2: Preparation of compound Y4.4

[0089]

[0090] The compound Y1 potassium 2,3,3-trimethylindole-5-sulfonate (1.00 g, 3.61 mmol) and p-nitrophenethyl trifluoromethanesulfonate (2.16 g, 7.22 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the reaction was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly dropped into 150 mL of ether, and at this time, the solid precipitated; then it was filtered, washed, and dried to obtain Y4.4 as a light pink solid powder (960 mg, 2.47 mmol), with a yield of 68.5%.

[0091] S3: Preparation of water-soluble pentamethine cyanine dye 4

[0092]

[0093] Y4.4 (200 mg, 0.515 mmol), Y3 prepared in Preparation Example 2 (280 mg, 0.515 mmol), and sodium acetate (211 mg, 2.57 mmol) were added to a 50 mL two-necked round-bottom flask, and then 10 mL of acetic anhydride solvent was added. The reaction was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried; using a 10%-50% methanol aqueous solution as the elution solvent, the crude product was purified by C18 reverse silica gel column chromatography to obtain water-soluble pentamethine cyanine dye 4 (103 mg, 0.126 mmol), with a yield of 24.4%. 1 H NMR (600 MHz, DMSO-d6) δ 8.36–8.22 (m, 2H), 8.13–8.09 (m, 2H), 7.96–7.92 (m, 2H), 7.85 (d, J = 1.6 Hz, 1H), 7.81 (d, J = 1.6 Hz, 1H), 7.63–7.54 (m, 4H), 7.35 (dd, J = 8.3, 4.5 Hz, 3H), 7.25 (d, J = 8.3 Hz, 1H), 6.39 (t, J = 12.3 Hz, 1H), 6.23 (dd, J = 13.7, 6.5 Hz, 2H), 5.47 (s, 2H), 4.43 (t, J = 7.2 Hz, 2H), 3.18 (dd, J = 8.5, 5.8 Hz, 3H), 1.74 (s, 6H), 1.62 (s, 6H).

[0094] Example 5:

[0095] Preparation of water-soluble pentamethine cyanine dye 5

[0096] S1: Preparation of alkylating agent

[0097]

[0098] p-Nitrophenethyl alcohol (2.00 g, 12.0 mmol), 6-bromohexanoic acid (2.80 g, 14.4 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (6.82 g, 18.0 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of dichloromethane, and the reaction was carried out at -50 °C for 15 min under a nitrogen atmosphere. After warming to room temperature, the reaction mixture was extracted with water and dichloromethane, and the organic phase was evaporated to dryness. The crude product was purified by normal-phase silica gel column chromatography using petroleum ether:ethyl acetate = 30:1 - 5:1 as the elution solvent to obtain the alkylating reagent as a pale yellow oily liquid (3.25 g, 9.44 mmol), with a yield of 78.9%.

[0099] S2: Preparation of Compound Y4.5

[0100]

[0101] Potassium 2,3,3-trimethylindole-5-sulfonate (1.00 g, 3.61 mmol) and p-nitrophenethyl 6-bromohexanoate (2.49 g, 7.22 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the reaction was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction mixture was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y4.5 as a light pink solid powder (1.20 g, 2.39 mmol), with a yield of 66.1%.

[0102] S3: Preparation of Water-Soluble Pentamethine Cyanine Dye 5

[0103]

[0104] Y4.5 (200 mg, 0.398 mmol), Y3 prepared in Preparation Example 2 (217 mg, 0.398 mmol), and sodium acetate (163 mg, 1.99 mmol) were added to a 50 mL two-necked round-bottom flask, and then 10 mL of acetic anhydride solvent was added. The reaction was heated to 50 °C for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by C18 reverse-phase silica gel column chromatography using a 10% - 50% methanol aqueous solution as the elution solvent to obtain water-soluble pentamethine cyanine dye 5 (130 mg, 0.126 mmol), with a yield of 35.0%. 11H NMR (600 MHz, DMSO-d6) δ 8.35 (dt, J = 30.4, 13.0 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.85 (dd, J = 10.3, 1.6 Hz, 2H), 7.66 (dd, J = 8.2, 1.6 Hz, 1H), 7.59 (dd, J = 8.2, 1.6 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.3 Hz, 1H), 6.47 (t, J = 12.3 Hz, 1H), 6.39 (d, J = 14.0 Hz, 1H), 6.18 (d, J = 13.5 Hz, 1H), 5.44 (s, 2H), 4.24 (t, J = 6.5 Hz, 2H), 4.09 (t, J = 7.3 Hz, 2H), 2.98 (t, J = 6.5 Hz, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.74 (s, 6H), 1.68 (s, 6H), 1.64 (q, J = 7.4 Hz, 2H), 1.49 (p, J = 7.5 Hz, 2H), 1.30 (p, J = 7.8 Hz, 2H).

[0105] Example 6:

[0106] Preparation of water-soluble pentamethine cyanine dye 6

[0107] S1: Preparation of compound Y2.6

[0108]

[0109] Potassium 2,3,3-trimethylindole-5-sulfonate (2.00 g, 7.21 mmol) and 1,3-diiodopropane (10.67 g, 36.05 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 ml of ether, and the solid precipitated at this time. Subsequently, it was filtered, washed, and dried. The obtained light pink solid powder was added to a 100 mL two-necked round-bottom flask containing 10 mL of sulfolane, and then triethylenediamine (971 mg, 8.65 mmol) was added. The mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of ether, and the solid precipitated at this time. Subsequently, it was filtered, washed, and dried to obtain 960 mg (1.85 mmol) of the purple-red solid powder of Y4.6, with a yield of 25.6%.

[0110] S2: Preparation of Water-Soluble Pentamethine Cyanine Dye 6

[0111]

[0112] Y4.6 (200 mg, 0.385 mmol), Y3 prepared in Preparation Example 2 (210 mg, 0.385 mmol), and sodium acetate (158 mg, 1.93 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, 10 mL of acetic anhydride solvent was added, and the mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by C18 reverse silica gel column chromatography using a 10%-50% methanol aqueous solution as the elution solvent to obtain water-soluble pentamethine cyanine dye 6 (106 mg, 0.132 mmol) with a yield of 34.4%. 1 H NMR (400 MHz, Deuterium Oxide) δ 7.89 (s, 2H), 7.63 (d, J = 24.7 Hz, 6H), 7.11 (s, 4H), 6.38 (s, 1H), 6.09 (s, 2H), 5.20 (s, 2H), 4.02 (s, 2H), 3.28 (s, 2H), 3.21 (s, 6H), 3.00 (s, 6H), 2.11 (s, 2H), 1.45 (d, J = 14.1 Hz, 12H).

[0113] Comparative Example 1

[0114] The comparative molecule is the commercial molecule 2-[5-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadienyl]-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium inner salt, CAS: 146368-11-8.

[0115]

[0116] Tests, Results and Analysis

[0117] (1) Identification of the Molecular Structure of Water-Soluble Pentamethine Cyanine Dye

[0118] The compounds water-soluble pentamethine cyanine dye 2 and water-soluble pentamethine cyanine dye 3 were identified by high-precision and high-sensitivity high-resolution mass spectrometry analysis technology. The results are as Figure 1 and Figure 3 shown, as Figure 1The high-resolution mass spectrometry analysis results of water-soluble pentamethine cyanine dye 2 are shown. A clear and sharp main peak is presented in the figure. The position of this peak highly coincides with the molecular weight obtained from the theoretical calculation of water-soluble pentamethine cyanine dye 2, and no obvious impurity peaks or fragment peaks are observed. This fully demonstrates the purity of water-soluble pentamethine cyanine dye 2 and the correctness of its structure, marking the successful preparation of compound 2. Meanwhile, the distribution of characteristic peaks in the mass spectrometry map coincides with the functional groups and connection modes in the expected structure, further verifying the specified structure of water-soluble pentamethine cyanine dye 2. Similarly, Figure 3 The high-resolution mass spectrometry analysis results of water-soluble pentamethine cyanine dye 3 are shown. Similar to water-soluble pentamethine cyanine dye 1, the mass spectrometry map of water-soluble pentamethine cyanine dye 3 also presents a main peak, whose position precisely corresponds to the theoretical molecular weight of water-soluble pentamethine cyanine dye 3, and the map is clear and interference-free, reflecting the high purity and structural accuracy of water-soluble pentamethine cyanine dye 3.

[0119] Compounds water-soluble pentamethine cyanine dye 2, water-soluble pentamethine cyanine dye 3, water-soluble pentamethine cyanine dye 4, water-soluble pentamethine cyanine dye 5, and water-soluble pentamethine cyanine dye 6 were identified using nuclear magnetic resonance spectroscopy analysis technology, and the results are as Figure 2 、 Figure 4 、 Figure 5 、 Figure 6 and Figure 7 shown. Figure 2 As shown by the nuclear magnetic resonance hydrogen spectrum analysis results of water-soluble pentamethine cyanine dye 2 in, each peak in the nuclear magnetic spectrum can find its attribution in the molecule, and the total number of hydrogen atoms can correspond, and no obvious impurity peaks are observed. This further demonstrates the purity of water-soluble pentamethine cyanine dye 1 and the correctness of its structure. Similarly, Figure 4 、 Figure 5 、 Figure 6 and Figure 7 respectively show the nuclear magnetic resonance hydrogen spectrum analysis results of water-soluble pentamethine cyanine dye 3, water-soluble pentamethine cyanine dye 4, water-soluble pentamethine cyanine dye 5, and water-soluble pentamethine cyanine dye 6, demonstrating the purity of the prepared water-soluble pentamethine cyanine dyes and the correctness of their structures.

[0120] (2) Tests on the ultraviolet-visible absorption spectrum, fluorescence spectrum, and photophysical properties of fluorescent dye compounds

[0121] (2.1) Dye preparation method

[0122] A balance with a precision of one ten-thousandth was used to accurately weigh the dye that had undergone strict vacuum drying treatment. Subsequently, the weighed dye was dissolved in dimethyl sulfoxide (DMSO) to prepare a dye stock solution with a concentration of 3 mmol / L. This stock solution was carefully aliquoted into brown sample bottles to prevent light from interfering with its stability and stored in a 4°C refrigerator to ensure chemical stability and activity during long-term storage. Before performing ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy tests, test solutions need to be prepared. The specific steps are as follows: Use a high-precision micropipette to accurately measure 3 μL of the dye stock solution and slowly inject it into a quartz cuvette containing 3 mL of a predetermined solvent (such as water). Gently shake to ensure uniform mixing of the solution. This operation dilutes the dye concentration to 3 μmol / L, which is suitable for subsequent spectral analysis.

[0123] (2.2) Broad-spectrum conditions and photophysical property test conditions

[0124] All spectral tests were performed in a strictly controlled constant temperature environment of 25°C to eliminate the potential impact of temperature fluctuations on the experimental results and ensure the repeatability and accuracy of the obtained data. During the photophysical property test, the accurately prepared dye stock solution was used as the test solution. According to the principle of Beer's law, by measuring the absorbance at a specific wavelength and combining with the molar extinction coefficient calculation formula, the molar extinction coefficient of each dye was calculated; using a high-precision absolute fluorescence quantum yield meter (Hamamatsu, model C11347), the absolute fluorescence quantum yield of each dye sample was measured under the same conditions to comprehensively evaluate its fluorescence emission efficiency.

[0125] (2.3) Result analysis

[0126] Figure 8 And Figure 9 Are the absorption spectra and fluorescence emission spectra of water-soluble pentamethine cyanine dye 1, water-soluble pentamethine cyanine dye 2, water-soluble pentamethine cyanine dye 3, and water-soluble pentamethine cyanine dye 4 in PBS buffer, respectively. The results show that these dyes have similar maximum absorption peaks and fluorescence emission peaks, which are located at about 646 nm and 668 nm, respectively.

[0127] It should be particularly noted that even after specific activation treatment, the maximum emission wavelength of the water-soluble pentamethine cyanine dye did not change significantly, indicating good structural stability. Through targeted chemical modification, the spectral properties of such dyes, including absorption and emission wavelengths, can be finely tuned, providing broad possibilities for their applications in cutting-edge scientific and technological fields such as near-infrared biofluorescence imaging and protein labeling detection.

[0128] The photophysical properties of water-soluble pentamethine cyanine dyes 1 - water-soluble pentamethine cyanine dyes 6 prepared in Examples 1 - 6 and the water-soluble dye of the commercial pentamethine cyanine dye in Comparative Example 1 were tested, and the test results are shown in Table 1;

[0129] Table 1 Photophysical property parameters of water-soluble pentamethine cyanine dyes 1-12

[0130]

[0131] Table 1 details the key photophysical property parameters of water-soluble pentamethine cyanine dye 1 to water-soluble pentamethine cyanine dye 6, including absorption wavelength, emission wavelength, molar extinction coefficient (ε), and absolute fluorescence quantum yield

[0132] Figure 8 Among them, the absorption spectra of water-soluble pentamethine cyanine dyes 1 to 6 in aqueous solution are narrow and steep in shape, without obvious aggregation peaks, and the molar extinction coefficients in Table 1 do not show a significant decrease compared with the reference molecules, indicating that the prepared water-soluble pentamethine cyanine dye molecules can be well dispersed in aqueous solution and can be well dissolved in water.

[0133] The molar extinction coefficients of water-soluble pentamethine cyanine dyes 1 to 6 are all significantly higher than those of traditional water-soluble pentamethine cyanine dyes, and can reach up to 2.53×10 5 L / mol -1 ·cm -1 (for water-soluble pentamethine cyanine dye 2). A high molar extinction coefficient means that the dye molecules have a stronger ability to absorb light, can achieve effective light energy capture and conversion at a lower concentration, thereby improving the light energy utilization rate, reducing the amount of dye used, and reducing potential biological toxicity. All the water-soluble pentamethine cyanine dyes prepared in this scheme show relatively high fluorescence quantum yields, and can reach up to 0.327 (for water-soluble pentamethine cyanine dye 3). A relatively high fluorescence quantum yield means that the dye molecules can be more effectively converted into fluorescence emission after absorbing light energy, thereby enhancing the intensity and sensitivity of the imaging signal and improving the imaging quality. The above data not only reveal the diversity and superiority of this type of dye in spectral characteristics, but also deeply reflect the remarkable achievements of the present invention in solving the problems of the prior art.

[0134] (3) Photostability test of water-soluble pentamethine cyanine dyes

[0135] (3.1) Test method for dye photostability

[0136] Use a high-precision micropipette to accurately measure 4 μL of the dye mother liquor, and slowly inject it into a quartz cuvette containing 3 mL of a mixed solvent of PBS buffer and methanol with an equal volume ratio. Gently shake to ensure uniform mixing of the solution. This operation dilutes the dyes prepared in Examples 2-6 and the commercial pentamethine dye to a concentration of 3 μmol / L respectively, and irradiates them under a 660 nm LED lamp. Adjust the distance between the quartz cuvette and the lamp so that the light power is 20 mW / cm2 The absorption spectrum of the dye was measured every 10 min.

[0137] Table 2 Photostability results of water-soluble pentamethine cyanine dyes prepared in Examples 2-6 and commercial pentamethine cyanine dyes

[0138]

[0139] Figures 10 - 14 They are the photostability test results of the prepared water-soluble pentamethine cyanine dye 2, water-soluble pentamethine cyanine dye 3, water-soluble pentamethine cyanine dye 4, water-soluble pentamethine cyanine dye 5 and water-soluble pentamethine cyanine dye 6 respectively. Figure 15 For the comparison molecule, the molecular structure and photostability test results of the commercial water-soluble pentamethine cyanine dye are shown. Figures 10 - 15 Combined with the results in Table 2, it can be seen that after the water-soluble pentamethine cyanine dye 2 was irradiated with light at a light power of 20 mW / cm² at 660 nm for 1 h, its maximum absorption only decreased by about 3.0%, while the maximum absorption of the commercial pentamethine cyanine dye in Comparative Example 1 decreased by 59.3% under the same conditions. This shows that the introduction of the benzyl structure in this water-soluble pentamethine cyanine dye can greatly improve the photostability of the dye, and the introduction of the p-nitrobenzyl group can more significantly improve the photostability of the dye, thereby improving the imaging effect of the dye. 2 In summary, by introducing sulfonate ions on the indole benzene ring and different groups at the N position, precise regulation of the water solubility and photostability of the dye molecules was achieved. The pentamethine cyanine dyes prepared in the present invention show significant advantages in spectral characteristics. The molar extinction coefficient is higher than that of traditional cyanine dyes, and the ability to absorb light energy is strong, which helps to improve the light energy utilization rate and reduce the amount of dye used; the absolute fluorescence quantum yield reaches a relatively high level, significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection.

[0140] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

[0141] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A water-soluble pentamethine cyanine dye, characterized in that, It has a structure of General Formula I: In General Formula I, R is selected from one of hydrogen, aryl, substituted aryl, halogen group, carboxyl group, sulfonate group, nitro group, cyano group, alkyl group having 1 - 18 carbons, carboxyalkyl group having 1 - 18 carbons, alkylsulfonate group having 1 - 18 carbons, ester group having 1 - 18 carbons, alkylsulfonate salt having 1 - 18 carbons, hydroxyalkyl group having 1 - 18 carbons, amide group having 1 - 18 carbons; Y is selected from one of O, S, ester group, alkylene group having 1 - 18 carbons; M is selected from one of metal ions, NH4 + or H + in the following.

2. The water - soluble pentamethine cyanine dye according to claim 1, characterized in that, R is selected from one of aryl, substituted aryl, 1,4 - diazabicyclo[2.2.2]octyl group; Y is selected from one of methylene, ethylene, propylene, ester group.

3. A method for preparing the water-soluble pentamethine dye according to claim 1, characterized in that, It includes the following steps: S1. Dissolve compound Y1 and N - alkylating reagent in an organic solvent, and reflux the reaction for 12 - 24 h under the protection of an inert gas to obtain a reaction solution. After the reaction solution is recrystallized and purified, compound Y4 is obtained, wherein the molar ratio of compound Y1 to N - alkylating reagent is 1:2 - 10; Dissolve compound Y1 and 4 - bromomethylbenzoic acid in an organic solvent, and reflux the reaction for 12 - 24 h under the protection of an inert gas to obtain a reaction solution. After the reaction solution is recrystallized and purified, compound Y2 is obtained, wherein the molar ratio of compound Y1 to 4 - bromomethylbenzoic acid is 1:2 - 10; The N - alkylating reagent is selected from one of halogen - substituted alkyl, benzyl, benzyl halogen group, benzyl carboxyl group, benzyl sulfonate group, benzyl nitro group, benzyl cyano group, alkyl group having 1 - 18 carbons, carboxyalkyl group having 1 - 18 carbons, alkylsulfonate group having 1 - 18 carbons, ester group having 1 - 18 carbons, alkylsulfonate salt having 1 - 18 carbons, hydroxyalkyl group having 1 - 18 carbons, amide group having 1 - 18 carbons; S2. Dissolve compound Y2 and compound S1 in an organic acid and acetic anhydride solvent, heat to 40 - 120 °C under an inert atmosphere, and react for 2 - 6 h; after cooling to room temperature, recrystallize, separate the solid and liquid, and take the solid phase. After drying and purification, compound Y3 is obtained, wherein the molar ratio of compound Y2 to compound S1 is 1:0.8 - 1.2, and the volume ratio of the organic acid to acetic anhydride solvent is 1:0.5 - 2; S3. Dissolve compound Y3, compound Y4, a base catalyst capable of introducing M and acetic anhydride solvent, heat to 40 - 120 °C under an inert atmosphere, and react for 2 - 6 h; after cooling to room temperature, recrystallize, separate the solid and liquid, and take the solid phase. After drying, use dichloromethane and methanol as elution solvents, and after purification, a water - soluble pentamethine cyanine dye is obtained, wherein the molar ratio of compound Y3, compound Y4 and the base catalyst is 1:0.8 - 1.2:2 - 10.

4. The preparation method of the water-soluble pentamethine cyanine dye according to claim 3, characterized in that, In S1, the organic solvent is selected from at least one of toluene, o - dichlorobenzene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran, sulfolane.

5. The preparation method of the water-soluble pentamethine cyanine dye according to claim 3, characterized in that, In S2, the organic acid is selected from at least one of acetic acid, hydrochloric acid, sulfuric acid, polyphosphoric acid.

6. The preparation method of the water-soluble pentamethine cyanine dye according to claim 3, wherein In S3, the base catalyst capable of introducing M is selected from at least one of sodium carbonate, sodium bicarbonate, sodium acetate, sodium hydroxide, and potassium hydroxide.

7. The preparation method of the water-soluble pentamethine cyanine dye according to claim 3, characterized in that, In S1, S2, and S3, the solvent used for recrystallization is selected from at least one of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone, propanol, and isopropanol.

8. Use of the water-soluble pentamethine cyanine dye according to claim 1, characterized in that, The water-soluble pentamethine cyanine dye is applied to cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, DNA sequencing, and preparation of reagents for specific recognition and photodynamic therapy of tumors.

9. Use of the water-soluble pentamethine cyanine dye according to claim 8, characterized in that, When the water-soluble pentamethine cyanine dye is applied, the fluorescence imaging emission wavelength is 600–800 nm.

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