A method for preparing a phospholipid having a fluorescent property
Fluorescent phospholipid molecules were prepared by click chemistry synthesis, which solved the problems of complex synthesis and large volume in traditional methods. This enabled the application of fluorescent phospholipid molecules in drug carriers and imaging agents, with the effects of biocompatibility and high drug loading.
Patent Information
- Application Number
- CN202310681229.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-09
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2043-06-09
AI Technical Summary
Existing fluorescent phospholipid molecules are complex to synthesize and are large in size, which limits their application in fluorescent labeling and functionalization. Traditional modification methods have problems such as difficulty in controlling reaction conditions and the production of many byproducts.
Fluorescent phospholipids were synthesized using a click chemistry method. The reaction of 3,4-dibromo-1H-pyrrole-2,5-dione with alkyl thiols, followed by a series of reactions with other compounds, was carried out with controlled temperature and purification steps to prepare phospholipid molecules with hydrophilic head groups and hydrophobic tail groups.
A fluorescent phospholipid molecule was successfully synthesized, which can be used as a drug carrier and imaging agent in liposome nanomedicine. It has biocompatibility, biodegradability and high drug loading capacity, and is suitable for targeted delivery of small molecule drugs and imaging diagnosis.
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Figure CN116751227B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of phospholipid synthesis and relates to a method for preparing phospholipids with fluorescent properties. Background Technology
[0002] Phospholipids are important components of biological cell membranes. Phospholipid membranes provide a natural barrier for cells, separating the cell interior from the external environment, providing a stable internal environment, and serving as a platform for cellular metabolism. In natural phospholipids, phosphatidylcholine (PC) from glycerophospholipids is the dominant component.
[0003] Phospholipids can self-assemble into liposomes. These liposomes are biocompatible, biodegradable, low in toxicity, have high drug loading capacity, and controllable release kinetics, making them ideal drug carriers. Liposomes enhance the efficacy and pharmacokinetics of small molecule drugs. Currently, several liposomes based on natural and synthetic phospholipids are commercially available in drug delivery systems. Furthermore, many more liposomal formulations are in different stages of clinical trials. Liposomes have shown broad clinical applications, from diagnostics to therapy. Therapeutic applications of liposomes include small molecule therapy, gene therapy, and immunotherapy. Liposomes loaded with small molecules (anticancer drugs) can passively target and accumulate in tumor tissue. This phenomenon is highly manifested in solid tumors through a process called enhanced penetration and retention effects. In diagnostic applications, ligand-modified liposomes loaded with various imaging agents can target desired tissues for imaging diagnostics. Liposomes have been used in many imaging techniques, such as magnetic resonance imaging, ultrasound, and fluorescence. Liposomes with therapeutic and diagnostic functions contain both therapeutic and imaging agents, showing great potential for therapeutic and diagnostic nanomedicine. Novel organic fluorophores have been widely used in biolabeling, probes, and medical diagnostics. Currently, the complex synthesis and large size of many large planar fluorophores limit their application in fluorescent labeling and functionalization. Among reported organic dyes, maleimide fluorophores are among the smallest, exhibiting high emissivity, large Stokes shift, and ease of modification. The reaction of 3,4-dibromo-1H-pyrrole-2,5-dione with alkyl thiols offers advantages such as high reaction efficiency, mild reaction conditions, and ease of preparation. Traditional fluorescent phospholipid molecules are mostly modified with fluorescent groups on hydrophobic alkyl chains or hydrophilic heads. The modified fluorescent phospholipid molecules are mixed with natural or synthetic phospholipid molecules to form liposomes with fluorescent properties. Summary of the Invention
[0004] In view of this, the present invention provides a method for preparing a phospholipid with fluorescent properties.
[0005] This invention specifically provides the following technical solution: a method for preparing phospholipids with fluorescent properties, comprising the following steps:
[0006] 1) Dissolve 3,4-dibromo-1H-pyrrole-2,5-dione in anhydrous ethanol, add sodium bicarbonate, and add alkyl thiols dropwise while stirring to carry out the reaction at a temperature of 20℃-50℃. The molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiols is 1:2 to 1:5. After the reaction is completed, the product is purified by column chromatography to obtain product a.
[0007] 2) Dissolve product a obtained in step 1) and 3-bromo-1-propanol in acetone, add potassium carbonate, heat to reflux and react at a temperature of 20℃~60℃. The molar ratio of product a to 3-bromo-1-propanol is 1:1~1:5. Purify the product by column chromatography to obtain product b.
[0008] 3) Dissolve product b obtained in step 2) in excess triethylamine in tetrahydrofuran, lower the temperature of the reaction solution to 0°C, add 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, and react at 25°C. The molar ratio of product b to 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane is 1:1 to 1:5. No purification is required to obtain product c.
[0009] 4) Dissolve product c obtained in step 3) in acetonitrile, add trimethylamine / acetonitrile solution, seal, and react at 50℃~80℃. The molar ratio of product b to trimethylamine is 1:1~1:5. Purify the product by column chromatography to obtain the final product.
[0010] Further, the alkyl thiol mentioned in step 1) is tetradecyl thiol, hexadecyl thiol, or oleyl thiol.
[0011] Further, in step 1), during purification, a gradient elution is performed using a mixed solvent of n-hexane / ethyl acetate with a volume ratio of 20:1 to 10:1.
[0012] Furthermore, during purification in step 2), a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 5:1 is used for elution.
[0013] Furthermore, in step 4), during purification, a mixed solvent of dichloromethane / methanol / water with a volume ratio of 65:25:4 is used for elution.
[0014] Furthermore, when the alkyl thiol in step 1) is tetradecyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:3; when the alkyl thiol in step 1) is hexadecyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:3; when the alkyl thiol in step 1) is oleyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:3.
[0015] Furthermore, the reaction temperature in step 1) is 50°C.
[0016] Furthermore, the reaction temperature in step 2) is 55°C.
[0017] Furthermore, the reaction temperature in step 4) is 60°C.
[0018] The beneficial effects of this invention are as follows:
[0019] 1. The synthesized phospholipid molecules have a hydrophilic head group and a hydrophobic tail group, requiring alkyl thiols and 3,4-dibromodiphenyl ethers during synthesis.
[0020] -1H-pyrrole-2,5-dione reacts first, then the hydrophilic phosphocholine head group is synthesized; otherwise, the organic amine reacts with the bromine site of 3,4-dibromo-1H-pyrrole-2,5-dione that reacts first.
[0021] 2. Step 1) Add an excess of alkyl thiol and react it completely with 3,4-dibromo-1H-pyrrole-2,5-dione to generate a product with two hydrophobic chains. If too little alkyl thiol is added, a single-chain byproduct will be generated, which is not conducive to subsequent purification.
[0022] 3. A suitable temperature should be selected for the reaction system. High temperatures are beneficial for the reaction, but excessively high temperatures will increase the pressure in the reaction vessel, posing a danger. In step 1), a temperature of 50℃ is suitable for the reaction system. (Step 3)
[0023] When adding 2-chloro-2-oxo-1,3,2-dioxophosphoric cyclopentane, the reaction system temperature should be lowered to 0°C to prevent a rapid rise in the reaction system temperature caused by the exothermic reaction and to prevent dangers during the reaction process.
[0024] 4. The synthesized final phospholipid product has fluorescent properties and can be used as a chemical drug carrier, nucleic acid drug carrier, and imaging agent, showing great promise in liposome nanomedicine. Attached Figure Description
[0025] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:
[0026] Figure 1 These are the chemical structural formulas of three phospholipids with fluorescent properties.
[0027] Figure 2 This is a synthetic route diagram for phospholipids with fluorescent properties.
[0028] Figure 3 This is a graph showing the 1H NMR spectrum analysis results of DMMPC.
[0029] Figure 4 This is a graph showing the mass spectrometry analysis results of DMMPC.
[0030] Figure 5 The image shows the 1H NMR spectrum analysis results for DPMPC.
[0031] Figure 6 This is a graph showing the mass spectrometry analysis results of DPMPC.
[0032] Figure 7 This is a graph showing the 1H NMR spectrum analysis results from DOMPC.
[0033] Figure 8 This is a graph showing the mass spectrometry analysis results of DOMPC.
[0034] Figure 9 The image shows the results of UV-Vis absorption and fluorescence emission analysis of DMMPC.
[0035] Figure 10 The image shows the UV-Vis absorption and fluorescence emission analysis results of DPMPC.
[0036] Figure 11 The image shows the results of UV-Vis absorption and fluorescence emission analysis of DOMPC.
[0037] Figure 12 An optical microscope image of a giant vesicle of DMMPC.
[0038] Figure 13 An optical microscope image of a giant vesicle of DPMPC.
[0039] Figure 14 An optical microscope image of a giant vesicle of DOMPC. Detailed Implementation
[0040] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
[0041] like Figure 1 and Figure 2 As shown, this invention utilizes click chemistry to synthesize three types of luminescent phospholipid molecules. Figure 1 These are the chemical structural formulas of three luminescent phospholipid molecules. Figure 2 This is the synthetic route.
[0042] Example 1
[0043] The synthesis of DMMPC, a phospholipid with fluorescent properties, follows these steps:
[0044] 1) In a round-bottom flask, add 2 g (7.85 mmol, 1 eq) of 3,4-dibromo-1H-pyrrole-2,5-dione and 3.2963 g (39.24 mmol, 5 eq) of sodium bicarbonate dissolved in 100 mL of anhydrous ethanol. Add dropwise a solution of tetradecyl mercaptan (5.43 g, 23.54 mmol, 3 eq) in ethanol with stirring. React overnight at 50 °C. Monitor the reaction progress by thin-layer chromatography (TLC) and terminate the reaction when it is complete. Filter to remove the solid, and remove the solvent from the reaction solution using a rotary evaporator. Separate and purify the product using column chromatography, eluting with n-hexane / ethyl acetate (20:1–10:1). Collect the solution containing the product and remove the solvent using a rotary evaporator to obtain 3.62 g of a yellow solid, designated as compound 2-1, with the following structural formula: Yield: 83.6%.
[0045] 2) Compound 2-1 (3 g, 5.42 mmol, 1.0 eq), potassium carbonate (1.50 g, 10.83 mmol, 2.0 eq), and 3-bromo-1-propanol (2.26 g, 16.26 mmol, 3.0 eq) dissolved in 40 mL of acetone were added to a round-bottom flask. The mixture was heated to reflux at 55 °C. The reaction progress was monitored by TLC, and the reaction was terminated when complete. The solid was removed by filtration, and the solvent was removed from the reaction solution using a rotary evaporator. The product was purified by column chromatography, eluting with n-hexane / ethyl acetate (5:1). The solution containing the product was collected, and the solvent was removed by rotary evaporation to obtain 2.50 g of a yellow solid, designated as compound 3-1, with the following structural formula: Yield: 75.3%.
[0046] 3) Compound 3-1 (2 g, 3.27 mmol, 1.0 eq) and triethylamine (1.65 g, 16.34 mmol, 5.0 eq) dissolved in 50 mL of tetrahydrofuran were added to a round-bottom flask. The flask was cooled to 0 °C in an ice-water bath. 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (0.93 g, 6.54 mmol, 2.0 eq) was added, and the reaction was carried out at 25 °C. The reaction progress was monitored by TLC, and the reaction was terminated when it was complete. The solvent in the reaction solution was removed by rotary evaporation without further purification to obtain compound 4-1, with the following structural formula: Direct investment next step.
[0047] 4) Compound 4-1 (2.34 g, 3.26 mmol) dissolved in acetonitrile was added to a sealed tube, followed by the addition of 2 M trimethylamine / acetonitrile solution (4.90 mL, 9.80 mmol, 3.0 eq). The reaction was carried out in the sealed tube and heated to 60 °C for 24 hours. After the reaction was stopped, the solvent was removed from the reaction solution using a rotary evaporator. The crude product was purified by column chromatography using dichloromethane / methanol / water (65:25:4) as elution. The solution containing the product was collected, and the solvent was removed using a rotary evaporator to give 1.23 g of a yellow solid, designated DMMPC, with a yield of 48.6%.
[0048] Figure 3 This is a graph showing the 1H NMR spectrum analysis results for DMMPC. From... Figure 3 The results of the 1H NMR spectrum analysis of DMMPC are as follows: 1 H NMR(400MHz,Chloroform-d)δ4.35(s,2H),3.87(t,J=6.2Hz,4H),3.58(t,J=7.4Hz,2H),3.41(s,9H),3.23(t,J=7.4Hz,4H) ,1.85(h,J=6.2Hz,2H),1.62(p,J=7.4Hz,4H),1.39(dq,J=11.3,6.4,5.0Hz,4H),1.28–1.19(m,40H),0.87(t,J=6.7Hz,6H).
[0049] Figure 4 This is a mass spectrometry analysis result of DMMPC. From... Figure 4 The mass spectrometry analysis results of DMMPC can be obtained as C 40 H 77 N₂O₆PS₂[M+Na] + 799.53
[0050] Example 2
[0051] The synthesis of DPMPC, a phospholipid with fluorescent properties, follows these steps:
[0052] 1) In a bottom-fired flask, add 3,4-dibromo-1H-pyrrole-2,5-dione (2 g, 7.85 mmol, 1.0 eq) and sodium bicarbonate (3.3 g, 39.24 mmol, 5.0 eq) dissolved in 100 ml of anhydrous ethanol. While stirring, add hexadecyl mercaptan (6.09 g, 23.54 mmol, 1.0 eq) dropwise.
[0053] A solution of 3.0 mmol (eq) in ethanol was prepared. The reaction was carried out overnight at 50°C. The reaction was monitored by TLC, and the reaction was terminated when the reaction was complete. The solid was removed by filtration, and the solvent was removed from the reaction solution using a rotary evaporator. The product was purified by column chromatography, eluting with a mixture of n-hexane / ethyl acetate (20:1 to 10:1), and the solvent was removed by rotary evaporation to give 4.02 g of a yellow solid, designated as compound 2-2, with the following structural formula: Yield: 84.02%.
[0054] 2) Add compound 2-2 (3 g, 4.92 mmol, 1.0 eq) and potassium carbonate (1.36 g, 9.84 mmol, 2.0 eq) to a round-bottom flask.
[0055] 3-Bromo-1-propanol (2.05 g, 14.76 mmol, 3.0 eq) was dissolved in 40 mL of acetone and refluxed at 55 °C. The reaction was monitored by TLC, and terminated when complete. The solid was removed by filtration, and the solvent was removed from the reaction solution using a rotary evaporator. The product was purified by column chromatography, eluting with a 5:1 mixture of n-hexane and ethyl acetate, and the solvent was removed by rotary evaporation to give 2.42 g of a yellow solid, designated as compound 3-2, with the following structural formula:
[0056] Yield: 73.6%.
[0057] 3) Add compound 3-2 (2 g, 2.99 mmol, 1.0 eq) and triethylamine (1.51 g, 14.97 mmol, 5.0 eq) to a round-bottom flask.
[0058] Dissolve 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (0.85 g, 5.99 mmol, 2.0 eq) in 50 mL of tetrahydrofuran. Cool the system to 0 °C using an ice-water bath. Add 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (0.85 g, 5.99 mmol, 2.0 eq). Initiate the reaction at 25 °C. Monitor the reaction progress by TLC. Terminate the reaction when complete. Remove the solvent from the reaction solution using a rotary evaporator. No purification is required. This compound is designated as compound 4-2, with the structural formula [structural formula would be inserted here]. Direct investment next step.
[0059] 4) Compound 4-2 (2.31 g, 2.98 mmol) dissolved in acetonitrile was added to a sealed tube, followed by the addition of 2 M trimethylamine / acetonitrile solution (4.49 mL, 8.97 mmol, 3 eq). The reaction was carried out in the sealed tube and heated to 60 °C for 24 h, after which the reaction was stopped. The mixture was cooled to room temperature, and the solvent was removed using a rotary evaporator. The product was purified by column chromatography, eluting with dichloromethane / methanol / water (65:25:4). The solution containing the product was collected, and the solvent was removed using a rotary evaporator to give 1.28 g of a yellow solid, designated DPMPC, with a yield of 51.3%.
[0060] Figure 5 This is the 1H NMR spectrum analysis result for DPMPC. From... Figure 5 The 1H NMR spectrum analysis results of DPMPC can be obtained as follows: 1 HNMR(400MHz,Chloroform-d)δ4.32(s,2H),3.92–3.77(m,4H),3.57(t,J=7.6Hz,2H),3.37(s,9H),3.22(t ,J=7.3Hz,4H),1.84(s,2H),1.63(q,J=7.3Hz,4H),1.39(t,J=7.6Hz,4H),1.25(s,48H),0.90–0.85(m,6H).
[0061] Figure 6 This is a graph showing the mass spectrometry analysis results of DPMPC. From... Figure 6 The mass spectrometry analysis results of DMMPC can be obtained as C 44 H 85 N₂O₆PS₂[M+Na] + 855.64
[0062] Example 3
[0063] The synthesis of the fluorescent phospholipid DOMPC follows these steps:
[0064] 1) In a round-bottom flask, add 2 g (7.85 mmol, 1.0 eq) of 3,4-dibromo-1H-pyrrole-2,5-dione and 3.3 g (39.2 mmol, 5.0 eq) of sodium bicarbonate dissolved in 100 mL of anhydrous ethanol. Add dropwise an ethanol solution of oleoresinol (6.70 g, 23.54 mmol, 3.0 eq) with stirring. React overnight at 50 °C. Monitor the reaction progress by TLC and terminate the reaction when it is complete. Filter to remove the solid, and remove the solvent from the reaction solution using a rotary evaporator. Separate and purify the product using column chromatography, eluting with a gradient of n-hexane / ethyl acetate (20:1–10:1). Collect the solution containing the product and remove the solvent using a rotary evaporator. 4.24 g of a yellow viscous solid is obtained, designated as compound 2-3, with the following structural formula: Yield: 81.7%
[0065] 2) Compound 2-3 (3 g, 4.53 mmol, 1.0 eq), potassium carbonate (1.25 g, 9.06 mmol, 2.0 eq), and 3-bromo-1-propanol (1.89 g, 13.59 mmol, 3.0 eq) dissolved in 40 mL of acetone were added to a round-bottom flask. The mixture was refluxed at 55 °C. The reaction was monitored by TLC, and the reaction was terminated when complete. The solid was removed by filtration, and the solvent was removed from the reaction solution using a rotary evaporator. The product was purified by column chromatography, eluting with a 5:1 mixture of n-hexane and ethyl acetate. The solution containing the product was collected, and the solvent was removed by rotary evaporation to obtain 2.46 g of a yellow viscous solid, designated as compound 3-3, with the structural formula [structural formula missing]. Yield: 75.9%.
[0066] 3) In a round-bottom flask, add compound 3-3 (2 g, 2.78 mmol, 1.0 eq) and triethylamine (1.41 g, 13.88 mmol, 5.0 eq) dissolved in 50 mL of tetrahydrofuran. Cool to 0°C in an ice-water bath. Add 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (0.79 g, 5.56 mmol, 2.0 eq), and react at 25°C. Monitor the reaction progress by TLC. Terminate the reaction when it is complete. Remove the solvent from the reaction solution using a rotary evaporator. No purification is required. This compound is designated as 4-3, with the following structural formula: Direct investment next step.
[0067] 4) Compound 4-3 (2.15 g, 2.6 mmol) dissolved in acetonitrile was added to a sealed tube, followed by the addition of 2 M trimethylamine / acetonitrile solution (4.17 mL, 8.34 mmol, 3.0 eq). The reaction was carried out in the sealed tube and heated to 60 °C for 24 h, after which the reaction was stopped. The mixture was cooled to room temperature, and the solvent was removed using a rotary evaporator. The product was purified by column chromatography, eluting with a dichloromethane / methanol / water (65:25:4) mixture. The solution containing the product was collected, and the solvent was removed using a rotary evaporator to give 1.24 g of a yellow viscous solid, designated DOMPC, with a yield of 50.4%.
[0068] Figure 7 This is the result of the 1H NMR spectrum analysis by DOMPC. From... Figure 7 The 1H NMR spectroscopy results of DOMPC were obtained as follows: 1HNMR(400MHz,Chloroform-d)δ5.40–5.30(m,4H),4.43(s,2H),3.97–3.82(m,4H),3.58(t,J=7.5Hz,2H),3.38(s,9H),3.23(t,J=7.4Hz ,4H),2.00(q,J=6.5Hz,6H),1.87(s,2H),1.63(q,J=7.3Hz,4H),1.38(t,J=7.3Hz,4H),1.33–1.23(m,40H),0.89(dd,J=6.5,2.2Hz,6H).
[0069] Figure 8 This is a graph showing the mass spectrometry analysis results of DOMPC. From... Figure 8 The mass spectrometry analysis results of DMMPC can be obtained as C 48 H 89 N₂O₆PS₂[M+Na] + 907.70
[0070] Test Example 1: Determination of Absorption and Fluorescence Emission Spectra of Phospholipids with Fluorescent Properties
[0071] DMMPC, DPMPC, and DOMPC were dissolved in methanol, and the absorption spectra of the resulting methanol solutions were measured using a UV-Vis spectrophotometer. Figure 9 , Figure 10 , Figure 11 The absorption curve was obtained. The fluorescence emission spectrum of the solution was measured using a fluorescence spectrometer. Figure 9 , Figure 10 , Figure 11 The emission curves of DMMPC, DPMPC, and DOMPC molecules are shown. The absorption in methanol is 340-500 nm, with the maximum absorption wavelength being 410 nm.
[0072] Figure 9 This is a graph showing the fluorescence excitation and emission analysis results of DMMPC. From... Figure 9 As can be seen, the maximum absorption wavelength of DMMPC is 410nm, and the maximum fluorescence emission wavelength is 570nm. Therefore, fluorescent phospholipids have the characteristic of emitting green fluorescence.
[0073] Figure 10 This is a graph showing the fluorescence excitation and emission analysis results of DPMPC. From... Figure 10 As can be seen, the maximum absorption wavelength of DPMPC is 410nm, and the maximum fluorescence emission wavelength is 580nm. Therefore, fluorescent phospholipids have the characteristic of emitting green fluorescence.
[0074] Figure 11 This is a graph showing the fluorescence excitation and emission analysis results of DOMPC. From... Figure 11 As can be seen, the maximum absorption wavelength of DOMPC is 410 nm, and the maximum fluorescence emission wavelength is 580 nm. Therefore, fluorescent phospholipids have the characteristic of emitting green fluorescence. Example 2: Optical microscopy test of phospholipid self-assembly forming giant vesicles.
[0075] 20 μl of dichloromethane solutions of DMMPC, DPMPC, and DOMPC (2 mg / ml) were respectively added dropwise to ITO glass plates, allowing the solutions to spread evenly. Two phospholipid-coated ITO glass plates were prepared using this method and dried under vacuum for 20 minutes. Double-sided conductive copper foil tape was used to adhere the phospholipid-coated ITO glass plates to the surface. The two phospholipid-coated ITO glass plates were placed with their faces facing a silicone gasket, forming a sandwich structure. A 200 mM sucrose solution was injected into the central cavity. An arbitrary waveform generator was connected and energized at 1 kHz and 5 V for 2 hours. After energizing, the solution was removed. The solution was observed under an optical microscope to obtain… Figure 12 , Figure 13 , Figure 14 .
[0076] Figure 12 This is an optical microscope image of a DMMPC giant vesicle. The microscope image shows that DMMPC assembles into a giant vesicle, with a diameter of 10-20 μm.
[0077] Figure 13 This is an optical microscope image of a DPMPC giant vesicle. The microscope image shows that DPMPCs assemble into giant vesicles, with diameters ranging from 10 to 20 μm.
[0078] Figure 14 This is an optical microscope image of giant DOMPC vesicles. The microscope image shows that DOMPC assembles into giant vesicles, with diameters ranging from 10 to 20 μm.
[0079] This test demonstrates that DMMPC, DPMPC, and DOMPC, as amphiphilic molecules, possess the properties of natural phospholipids and can assemble into giant vesicles. These structures hold promise for applications in cell biomimetic research. Furthermore, vesicles formed by DMMPC, DPMPC, and DOMPC can serve as encapsulation agents for imaging agents and drug delivery, showing broad application prospects in the biomedical field.
[0080] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for preparing a phospholipid with fluorescent properties, characterized in that, The steps are as follows: 1) Dissolve 3,4-dibromo-1H-pyrrole-2,5-dione in anhydrous ethanol, add sodium bicarbonate, and add alkyl thiols dropwise while stirring to carry out the reaction at a temperature of 20℃-50℃. The molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiols is 1:2 to 1:
5. After the reaction is completed, the product is purified by column chromatography to obtain product a. 2) Dissolve product a obtained in step 1) and 3-bromo-1-propanol in acetone, add potassium carbonate, heat under reflux at a temperature of 20℃~60℃, and the molar ratio of product a to 3-bromo-1-propanol is 1:1~1:
5. Purify the product by column chromatography to obtain product b. 3) Dissolve product b obtained in step 2) in excess triethylamine in tetrahydrofuran, lower the temperature of the reaction solution to 0°C, add 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, and react at 25°C. The molar ratio of product b to 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane is 1:1 to 1:
5. No purification is required to obtain product c. 4) Dissolve product c obtained in step 3) in acetonitrile, add trimethylamine / acetonitrile solution, seal, and react at 50℃~80℃. The molar ratio of product b to trimethylamine is 1:1~1:
5. Purify the product by column chromatography to obtain the final product. The alkyl thiol is tetradecyl thiol, hexadecyl thiol, or oleyl thiol; When the alkyl thiol mentioned in step 1) is tetradecyl thiol, the final product is... ; When the alkyl thiol mentioned in step 1) is hexadecyl thiol, the final product is... When the alkyl thiol mentioned in step 1) is an oleothiol, the final product is... .
2. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, Step 1) During purification, a gradient elution is performed using a mixed solvent of n-hexane / ethyl acetate with a volume ratio of 20:1 to 10:
1.
3. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, Step 2) During purification, a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 5:1 is used for elution.
4. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, Step 4) During purification, a mixed solvent of dichloromethane / methanol / water with a volume ratio of 65:25:4 is used for elution.
5. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, When the alkyl thiol in step 1) is tetradecyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:3; when the alkyl thiol in step 1) is hexadecyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:3; when the alkyl thiol in step 1) is oleyl thiol, the molar ratio of 3,4-dibromo-1H-pyrrole-2,5-dione to alkyl thiol is 1:
3.
6. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, The reaction temperature for step 1) is 50°C.
7. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, The reaction temperature for step 2) is 55℃.
8. The method for preparing a phospholipid with fluorescent properties according to claim 1, characterized in that, The reaction temperature for step 4) is 60℃.