Oxygen-rhodamine near-infrared fluorescent dye, preparation method and application thereof
The two-step synthesis of oxygen-rhodamine near-infrared fluorescent dyes solves the problem of synthesis complexity in existing technologies, realizes the simple preparation and excellent performance of near-infrared fluorescent dyes with wavelengths exceeding 800 nm, and expands their application in biomedical imaging.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2025-05-12
- Publication Date
- 2026-05-29
Smart Images
Figure CN120718030B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an oxygen-rhodamine near-infrared fluorescent dye, its preparation method, and its application, belonging to the field of dye technology. Background Technology
[0002] In recent years, organic small-molecule fluorescent dyes with wavelengths exceeding 800 nm have attracted widespread attention and become a research hotspot. These near-infrared dyes are highly favored due to their wide applications in anti-counterfeiting, laser printing, and optoelectronic materials. Near-infrared light possesses advantages such as strong tissue penetration, weak tissue scattering, and low tissue absorption, making near-infrared organic small-molecule fluorescent dyes exhibit great potential in the biomedical field, especially in tumor diagnosis and treatment and vascular disease tracing. Therefore, developing near-infrared organic small-molecule fluorescent dyes with wavelengths exceeding 800 nm has become an urgent need in biomedical research. Rhodamine dyes, as classic visible-light organic fluorescent dyes, are widely used in various fluorescent imaging and labeling applications due to their excellent optical properties, such as large molar extinction coefficients and high quantum yields. However, designing near-infrared fluorescent dyes with wavelengths exceeding 800 nm based on the oxygen-rhodamine structure still presents significant challenges. This is mainly due to the complexity of the synthesis methods and the difficulty of the synthesis steps, making the development of such dyes still difficult. Therefore, designing a simple and feasible method for synthesizing near-infrared fluorescent dyes remains a problem that urgently needs to be solved. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a novel near-infrared fluorescent dye based on oxygen-rhodamine, its preparation method and application. The fluorescent dye has a novel structure, superior performance and a simple preparation method.
[0004] To address the above technical problems, this invention first discloses an oxygen-rhodamine near-infrared fluorescent dye, the structural formula of which is shown in formula (a) or formula (c).
[0005]
[0006] Wherein, R1 and R2 are selected from 1 to 2 hydrogens, 1 to 2 alkyl groups, or 1 to 2 alkenyl groups, respectively. When selected from 1 alkyl group or 1 alkenyl group, the remaining C is H. When 2 alkyl groups or 2 alkenyl groups are connected, the two substituents are the same, such as 2 methyl groups or 2 vinyl groups. R5 to R9 are selected from hydrogen, alkyl, methoxy, ethoxy, halogen, carboxyl, sulfonic acid, ethylene glycol, hydroxyl, or amino groups, respectively. n is an integer ≥ 0.
[0007] Furthermore, when R1, R2, and R5 to R9 are selected from alkyl groups, they are straight-chain or branched alkyl groups of C1 to C6; n is an integer from 0 to 4.
[0008] This invention also discloses a method for preparing the aforementioned oxygen-rhodamine near-infrared fluorescent dye, the reaction formulas of which are shown in (I) and (III).
[0009]
[0010] (I)
[0011]
[0012] (III)
[0013] The reaction steps include,
[0014] Step 1: Using the compound of Formula 1 as a starting material, dissolve it in an ether solvent, add alkyl lithium reagent dropwise, and heat at -78°C. o C~0 o The reaction is carried out under C conditions, utilizing the electronic effect of the methoxy group, which acts as an ortho / para directing group, causing the reaction to preferentially occur at the ortho position of the methoxy group. After reacting for 0.5–2 h, an aromatic ester compound is added, and the resulting mixture is then transferred to a reaction vessel at room temperature and reacted for 0.5–4 h to obtain compound of formula 2; the preferred ether solvent is tetrahydrofuran. This step one reaction is carried out in an inert gas atmosphere (such as nitrogen).
[0015] Step 2: Dissolve compound 2 in a solvent, add an acidic reagent, and heat at 70–120 °C. o The reaction proceeds at C for 2–12 h, removing the methyl group from the molecule of compound 2 to obtain compound 3.
[0016] The compounds of formula 3 in reaction formulas (I) and (III) correspond to compounds of formula (a) and (c), respectively.
[0017] Furthermore, in step one, the alkyllithium reagent is n-butyllithium or sec-butyllithium.
[0018] Further, in step one, the molar ratio of the compound of formula 1 to the alkyl lithium reagent is 1:1 to 2.
[0019] Furthermore, in step two, the solvent used is acetic acid, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, or acetone.
[0020] Furthermore, in step two, the mass-to-volume ratio of compound 2 to solvent is 1 g: 3–10 mL.
[0021] Furthermore, in step two, the acidic reagent used is a Lewis acid, inorganic acid, or organic acid, such as aluminum trichloride, boron tribromide, hydrobromic acid, hydrochloric acid, acetic acid, etc.
[0022] Furthermore, in step two, the molar ratio of compound 2 to the acidic reagent is 1:10 to 100.
[0023] The present invention also discloses the application of the aforementioned oxy-rhodamine near-infrared fluorescent dye or the oxy-rhodamine near-infrared fluorescent dye prepared according to the aforementioned method in fluorescence imaging and labeling.
[0024] The near-infrared dye constructed in this invention has the advantage of a simple synthesis method, which can be achieved through a two-step synthesis, greatly simplifying the synthesis process. This method can synthesize novel organic small-molecule fluorescent dyes with absorption / emission wavelengths exceeding 800 nm. The near-infrared dye of this invention exhibits a solvation effect in different solvents, with absorption wavelengths tunable from 820 nm to 880 nm, fluorescence emission wavelengths tunable from 860 nm to 942 nm, and fluorescence emission tailing up to 1200 nm. The near-infrared dye possesses excellent near-infrared fluorescence emission capabilities, making it valuable for potential clinical applications and basic research in the field of biomedical imaging. Attached Figure Description
[0025] Figure 1 The 1H NMR spectrum of the fluorescent dye EO5-D in Example 1 is shown below.
[0026] Figure 2 To test the absorption and emission spectra of EO5-D in bovine serum albumin (BSA) of Example 1;
[0027] Figure 3 To test the adsorption and emission profiles of EO5-D in dichloromethane (DCM) solvent in Example 1;
[0028] Figure 4 To test the adsorption and emission profile of EO5-D in dimethyl sulfoxide (DMSO) solvent in Example 1;
[0029] Figure 5 The adsorption and emission profiles of EO5-D in methanol (MeOH) solvent were tested in Example 1.
[0030] Figure 6 The graph shows the test results of the fluorescence intensity of the fluorescent dye in the centrifuge tube in various embodiments of the present invention. The dye concentration was 50 μM, an InGaAs camera was used, the laser was 880 nm, and a 1200 LP filter material was used. Detailed Implementation
[0031] The present invention will be further explained below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0032] Example 1: Preparation of compound E05-D
[0033]
[0034] The compound of formula 3 in this embodiment corresponds to general formula (a), where R1 and R2 are both dimethyl, R5 is methyl, and R6-R9 are hydrogen.
[0035] The preparation steps are as follows:
[0036] Step 1: Synthesis of Compound 2: Compound 1 (1 g, 3.23 mmol) was added to a dry Shrek flask containing anhydrous THF (10 mL) under a nitrogen atmosphere and stirred for 30 minutes until completely dissolved. The reaction system was cooled to -78°C, and then 2.5 M nBuLi (1.42 mL, 3.55 mmol) was added dropwise while stirring at -78°C for 30 minutes. Then, methyl o-methylbenzoate (0.152 mL, 1.08 mmol) was added, and the mixture was transferred to a reaction vessel at room temperature and reacted for 4 h. After the reaction was complete, the mixture was quenched with water. The reaction system was extracted with ethyl acetate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated. The residue was purified by column chromatography (silica, EtOAc / PE = 1:10, v / v) to obtain compound 2 (0.82 g, 93% yield) as a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ7.70 (s, 1H), 7.64 (s, 2H), 7.54 (s, 1H), 7.37 (s, 1H), 7.25 (d, J = 3.0 Hz,2H), 7.18 (s, 1H), 7.11 – 7.08 (m, 2H), 5.59 (s, 1H), 3.87 (s, 3H), 3.67 (s,3H), 3.33 (t, 4H), 3.30 – 3.24 (m, 4H), 2.37 (s, 3H), 1.83 (t, J = 12.0 Hz,12H), 1.44 (t, J = 15.7, 12.6 Hz, 12H).
[0037] Step 2, Synthesis of Compound EO5-D (Formula 3): In a 25 mL Shrek tube, compound EO5-D (200 mg, 271.36 μmol) was dissolved in acetic acid (2 mL, compound EO5-D mass: solvent volume = 1 g: 10 mL), and 48% hydrobromic acid (1.07 mL, compound EO5-D to HBr molar ratio = 1:35) was added. The reaction was carried out at 120 °C for 5 h. The reaction was then quenched with a saturated Na2CO3 aqueous solution. The aqueous layer was extracted with CH2Cl2 (3 × 50 mL), and the organic layer was dried with Na2SO4, filtered, and concentrated under vacuum. EO5-D (18.29 mg, yield 10%) was purified by column chromatography as a green powder solid. 1 H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 2H), 7.64 (d, J = 7.4 Hz, 1H), 7.59 - 7.53 (m, 2H), 7.41 (s, 2H), 7.35 (s, 2H), 7.30 (d, J = 7.8 Hz, HRMS (ESI) m / z chemical formula: C 48 H 53 N2O + [M] + Calculated value: 673.4152; Measured value: 673.4138.
[0038] The 1H NMR spectrum of the fluorescent dye EO5-D obtained in Example 1 is shown below. Figure 1 As shown.
[0039] Example 2: Preparation of compound E05.
[0040]
[0041] Formula 3 corresponds to the structural formula (a), where R 1、 R2 are all hydrogen, R5 is methyl, and R6-R9 are hydrogen.
[0042] Step 1, Synthesis of Formula 2: Compound 1 (0.8 g, 3.16 mmol) was added to a dry Shrek flask containing anhydrous THF (10 mL) under a nitrogen atmosphere and stirred for 30 minutes to mix thoroughly. The reaction system was cooled to -78°C, and 1.3 M sBuLi (2.67 mL, 3.47 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 minutes. A mixture of methyl o-methylbenzoate (148.15 mmL, 1.05 mmol) was added, and the reaction was allowed to proceed to room temperature for 4 h. After the reaction was completed, the mixture was quenched with water. The reaction system was extracted with ethyl acetate (3 × 20 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The residue was purified by column chromatography (silica) to obtain compound 2 (0.27 g, 40% yield) as a yellow solid.
[0043] Step 2, Synthesis of Compound EO5 (Formula 3): In a 25 mL Shrek tube, compound EO5 (200 mg, 320.09 μmol) was dissolved in acetic acid (2 mL, compound EO5 mass: solvent volume = 1 g: 10 mL), and 48% hydrobromic acid (1.27 mL, compound EO5 to HBr molar ratio = 1:35) was added. The reaction was carried out at 120 °C for 5 h. The reaction was then quenched with a saturated Na2CO3 aqueous solution. The aqueous layer was extracted with CH2Cl2 (3 × 50 mL), and the organic layer was dried with Na2SO4, filtered, and concentrated under vacuum. EO5 (17.98 mg, yield 10%) was purified by column chromatography as a green powder solid.
[0044] Example 3: Preparation of compound E05-R.
[0045]
[0046] The compound in Formula 3 corresponds to structural formula (c), where n is 2, R4 is hydrogen, R5 is methyl, and R6-R9 are hydrogen.
[0047] Synthetic steps of compound 2: Compound 1 (0.8 g, 3.52 mmol) was added to a dry Shrek flask containing anhydrous THF (10 mL) under a nitrogen atmosphere and stirred for 30 minutes to mix thoroughly. The reaction system was cooled to -78°C, and 2.5 M nBuLi (1.55 mL, 3.87 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 minutes. Methyl o-methylbenzoate (165.12 mmL, 1.17 mmol) was added, and the mixture was transferred to a reaction vessel at room temperature and reacted for 4 h. After the reaction was completed, the mixture was quenched with water. The mixture was extracted with ethyl acetate (3 × 20 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The residue was purified by column chromatography (silica, EtOAc / PE = 1:10, v / v) to give compound 2 (0.27 g, 40% yield) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 7.51 (d, J = 8.7 Hz, 1H), 7.42 (d, J =8.8 Hz, 1H), 7.19 – 7.15 (m, 1H), 7.12 (d, J = 3.2 Hz, 2H), 7.06 (s, 1H),7.02 (s, 1H), 6.95 (d, 2H), 6.85 – 6.76 (m, 3H), 6.70 (s, 2H), 5.43 (s, 1H),3.68 (s, 3H), 3.54 (s, 3H), 3.43 – 3.36 (m, 8H), 2.22 (s, 3H), 2.07 – 2.02(m, 8H).
[0048] The synthesis steps of compound EO5-R (Formula 3) were as follows: Compound EO5-R (Formula 2) (200 mg, 349.19 μmol) was dissolved in acetic acid (2 mL, compound EO5-solvent mass: 1 g: 10 mL) in a 25 mL Shrek tube. Hydrobromic acid (48% by mass) (1.38 mL, compound EO5-R to HBr molar ratio 1:35) was added, and the reaction was carried out at 120 °C for 5 h. The reaction was then quenched with a saturated Na2CO3 aqueous solution. The aqueous layer was extracted with CH2Cl2 (3 × 50 mL), and the organic layer was dried with Na2SO4. After filtration, the solution was concentrated under vacuum. Purification by column chromatography yielded EO5-R (17.79 mg, yield 10%) as a green powder.
[0049] If the compound of formula 1 used in each of Examples 1-3 of this invention is not readily available, it can be obtained by purchasing existing compounds from the market and preparing them using existing conventional preparation methods for later use.
[0050] The luminescence properties of the fluorescent dye EO5-D provided in Example 1 of this invention were tested using a UV-Vis spectrophotometer and a fluorescence spectrometer, and the resulting UV-Vis absorption and fluorescence spectra were obtained.
[0051] The absorption and emission spectra of this substance in bovine serum albumin (BSA) were tested as follows: Figure 2 As shown, the test results show absorption emission at 860 / 889 nm and fluorescence emission tailing to 1100 nm.
[0052] Its absorption and emission in dichloromethane (DCM) solvent were measured at 846 / 886 nm, respectively, with fluorescence emission tailing to 1100 nm. Figure 3 As shown.
[0053] Its absorption and emission in dimethyl sulfoxide (DMSO) solvent were measured at 858 / 910 nm, respectively, with fluorescence emission tailing to 1100 nm. Figure 4 As shown.
[0054] Its absorption and emission in methanol (MeOH) solvent were measured at 840 / 884 nm, respectively, with fluorescence emission tailing to 1100 nm. Figure 5 As shown.
[0055] All of the above indicate that the fluorescent dye EO5-D of the present invention has an ultraviolet absorption peak at around 840 nm and a fluorescence emission wavelength of around 900 nm, indicating that it has the effect of achieving near-infrared light absorption and emission.
[0056] Comparative testing of fluorescence intensity of different dyes in this invention embodiment: The fluorescence intensity of the EO5 series in centrifuge tubes was measured at a dye concentration of 50 μM, using an InGaAs camera, an 880 nm laser, and a 1200 LP filter. At the same concentration, the fluorescence intensity of the EO5 series was significantly higher than that of ICG (indocyanine green fluorescent dye). Figure 6 As shown.
Claims
1. An oxygen-rhodamine near-infrared fluorescent dye, characterized in that: Its structural formula is shown in equation (a) or equation (c). Wherein, R1 and R2 are selected from 1 to 2 hydrogens, 1 to 2 alkyl groups, or 1 to 2 alkenyl groups, respectively; R5 to R9 are selected from hydrogen, alkyl, methoxy, ethoxy, halogen, carboxyl, sulfonic acid, ethylene glycol, hydroxyl, or amino groups, respectively; when R1, R2, and R5 to R9 are selected from alkyl groups, they are straight-chain or branched alkyl groups of C1 to C6; n is an integer from 0 to 4.
2. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 1, characterized in that: The reaction formulas for this method are shown in (I) and (III). （Ⅰ） （Ⅲ） The reaction steps include, Step 1: Using the compound of Formula 1 as a starting material, dissolve it in an ether solvent, add alkyl lithium reagent dropwise, and heat at -78°C. o C~0 o After reacting for 0.5–2 h under C conditions, aromatic ester compounds are added, and the resulting mixture is then transferred to a reaction vessel at room temperature and reacted for 0.5–4 h to obtain compound 2. Step 2: Dissolve compound 2 in a solvent, add an acidic reagent, and heat at 70–120 °C. o The reaction was carried out at C for 2–12 h to obtain compound of formula 3. In step two, the acidic reagent used is either an inorganic acid or an organic acid.
3. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 2, characterized in that: In step one, the alkyllithium reagent is n-butyllithium or sec-butyllithium.
4. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 2 or 3, characterized in that: In step one, the molar ratio of the compound of formula 1 to the alkyl lithium reagent is 1:1 to 2.
5. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 2, characterized in that: In step two, the solvent used is acetic acid, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, or acetone.
6. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 2 or 5, characterized in that: In step two, the mass-to-volume ratio of compound 2 to solvent is 1 g: 3 to 10 mL.
7. The method for preparing the oxygen-rhodamine near-infrared fluorescent dye according to claim 2, characterized in that: In step two, the molar ratio of compound 2 to acidic reagent is 1:10 to 100.
8. The application of an oxy-rhodamine near-infrared fluorescent dye according to claim 1 or an oxy-rhodamine near-infrared fluorescent dye obtained by the preparation method according to any one of claims 2 to 7 in the preparation of reagents for fluorescence imaging and labeling.