A copper-cysteine complex, its preparation method and application
By reacting CuCl2·2H2O with L-cysteine under high temperature and weak alkaline conditions, Cu(I)-cysteine complex was prepared, which solved the problem of poor dispersion of existing Cu-Cy in water and achieved its wider application in the field of biomedicine.
Patent Information
- Application Number
- CN202310488136.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-28
- Publication Date
- 2025-05-27
- Estimated Expiration
- 2043-04-28
AI Technical Summary
The existing copper-cysteine complex Cu-Cy has poor dispersibility in water and is prone to aggregation, which affects its performance as a photosensitizer.
By mixing CuCl2·2H2O with L-cysteine and water, adjusting the pH to weak alkalinity, heating to boiling, and enhancing the reduction ability of the thiol group using high temperature and weak alkalinity conditions, obtaining Cu(I)-cysteine complex, improving its water solubility and free radical generation ability.
Good water solubility and excellent free radical generation ability of the copper-cysteine complex are achieved while maintaining its performance as a photosensitizer.
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Figure CN116535337B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of copper coordination compound composites, and particularly relates to a copper-cysteine complex, a preparation method thereof, and an application thereof. Background Art
[0002] A coordination compound refers to a compound formed by a certain number of ions or molecules (referred to as ligands) that can donate lone pairs of electrons or multiple delocalized electrons and an atom or ion (collectively referred to as the central atom) that has a vacancy for accepting lone pairs of electrons or multiple delocalized electrons, in a certain composition and spatial configuration. As a transition metal element with abundant reserves, low cost, variable oxidation states, and good biocompatibility, copper is a frequent guest in metal complexes. Copper complexes have a wide range of application fields. When used as biomedicines, they can achieve applications such as antibacterial, antitumor, and antioxidant effects. Currently, the copper-cysteamine complex Cu-Cy with copper-sulfur coordination has attracted much attention as a photosensitizer. Cu-Cy can be excited by energies of various wavelengths and generate free radicals, causing damage to tumor cells. Many studies have expanded its application in the field of biomedicine.
[0003] Although the ligand mercaptoethylamine is soluble in water, after copper-sulfur coordination with copper, the complex Cu-Cy becomes insoluble in water due to its cross-linked network molecular structure and exists in the form of nanoparticles. It has poor dispersibility in water and is prone to aggregation in solution, further increasing the particle size. This will also affect its performance as a photosensitizer to a certain extent (Journal of Materials Chemistry C, 2014, 2(21): 4239-4246.). Therefore, synthesizing a new copper-based complex with an unchanged core copper-sulfur coordination structure, modifying the edges of the nanoparticles with hydrophilic groups, maintaining the property of Cu-Cy to generate free radicals, and enhancing its dispersibility in water is of positive significance for exploring the structure-activity relationship of copper-mercapto complexes to generate free radicals and broadening the application of copper-based complexes in the field of biomedicine.
[0004] Since the 1980s, the important biological functions of L-Cys in humans have been reported for the first time. After that, the potential applications of this compound at the industrial level have also increased, mainly in the fields related to pharmaceuticals, medicine, and nutraceuticals. It plays an important role in the food industry (used as a flavoring agent or chelating agent), the pharmaceutical industry (it is part of several drug formulations, such as drugs used to reduce the acetaldehyde level in the mouth), and the cosmetics industry (L-Cys is used as a component in skin or hair care formulations). Based on the special properties of the cysteine molecule itself, such as having multiple sites that can coordinate with metals and having redox properties, it has the potential for metal ion analysis and detection. On the other hand, due to the free amino and carboxyl groups in L-cysteine, it can form zwitterions, thus inducing the assembly of nanoparticles. In summary, cysteine plays an important role in human metabolism, and this special amino acid also has several unique properties (many coordination sites, having redox effects), with broad room for development in the field of coordination chemistry. Currently, there are no reports on copper(I)-cysteine complexes. Summary of the Invention
[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a copper-cysteine complex with good water solubility, its preparation method, and application.
[0006] In the present invention, CuCl 2 ·2H 2 O is mixed with L-cysteine and water, the pH is adjusted to weakly alkaline, and it is heated to boiling. Under high temperature and weakly alkaline conditions, the reducing ability of the thiol group on cysteine is enhanced, and part of the divalent copper is oxidized to monovalent copper, which then coordinates with the monovalent copper to obtain a Cu(I)-cysteine complex.
[0007] The purpose of the present invention is achieved through the following preparation method:
[0008] A preparation method of a copper-cysteine complex, comprising the following steps:
[0009] Mix a soluble copper salt, L-cysteine, and deionized water, stir, adjust the pH, heat to boiling and reflux to obtain a copper-cysteine complex.
[0010] Preferably, the temperature of the stirring is room temperature, and the time is 2-10 min;
[0011] Preferably, the soluble copper salt is CuCl 2 ·2H 2 O;
[0012] Preferably, the molar volume ratio of the soluble copper salt to deionized water is 1 mmol: 30-80 ml.
[0013] Preferably, the molar ratio of the soluble copper salt to L-cysteine is 1:3 to 1:8.
[0014] Preferably, adjusting the pH means adjusting the pH to 7 to 11;
[0015] Preferably, the solution used for adjusting the pH is a NaOH solution;
[0016] More preferably, the concentration of the NaOH solution is 0.1 to 2 mol / L.
[0017] Preferably, the time of boiling under reflux is 40 to 90 min.
[0018] Preferably, the heating rate is 1 to 10 °C / min.
[0019] Preferably, the reaction solution after boiling under reflux is filtered, the filtrate is collected, dialyzed, rotary evaporated, and dried to obtain a copper-cysteine complex.
[0020] More preferably, the dialysis is carried out using a dialysis bag with a molecular weight cut-off of 500 to 3500 Da;
[0021] More preferably, the dialysis time is 24 to 96 h, and the water is changed every 12 - 24 h;
[0022] More preferably, the drying temperature is 50 - 80 °C, and the drying time is 12 to 24 h; the drying is vacuum drying. Even more preferably, the drying temperature is 60 °C.
[0023] The copper-cysteine complex prepared by the above preparation method.
[0024] Use of the above copper-cysteine complex in the preparation of a photosensitizer.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] (1) The raw materials of the present invention are inexpensive, the reaction steps are simple, and the reaction conditions are easy to achieve.
[0027] (2) The copper-cysteine complex prepared by the present invention has excellent luminescence properties, good water dispersibility, and excellent free radical generation ability. Description of the Drawings
[0028] Figure 1 It is the macroscopic morphology diagram and solution color development diagram of the copper-cysteine complex prepared in Example 1 of the present invention.
[0029] Figure 2 It is the transmission electron microscope diagram of the solid of the copper-cysteine complex prepared in Example 1 of the present invention.
[0030] Figure 3 This is the particle size diagram of the aqueous solution of the copper-cysteine complex prepared in Example 1 of the present invention.
[0031] Figure 4 This is the high-resolution mass spectrum diagram of the copper-cysteine complex prepared in Example 1 of the present invention.
[0032] Figure 5 This is the infrared spectrum diagram of the copper-cysteine complex Cu-Cys (left) and the ligand L-cysteine (right) prepared in Example 1 of the present invention.
[0033] Figure 6 This is the emission spectrum diagram for detecting the free radical generation ability of the copper-cysteine complex Cu-Cys prepared in Example 1 of the present invention by the DCFH method. Detailed implementation manners
[0034] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto.
[0035] In the examples of the present invention, those not specified under specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Raw materials, reagents, etc. without indicating the manufacturer can be obtained as conventional products through commercial purchase.
[0036] Preparation of copper-cysteine metal complex
[0037] Dissolve CuCl 2 ·2H 2 O in deionized water, add L-cysteine to obtain an acidic solution, stir vigorously for 2 - 10 min, adjust the pH to 7 - 11 with NaOH solution. At this time, the solution is yellow, heat to boiling and reflux for 40 - 90 min, the solution color changes to brown, stop heating, and the reaction solution emits blue fluorescence under the irradiation of a 365 nm ultraviolet lamp. Filter the reaction solution, collect the filtrate, dialyze the filtrate for 48 - 96 h, change the water every 24 h, after dialysis, spin-dry the solution, and place it in a vacuum drying oven at 60 °C for 12 - 24 h to obtain a brown solid powder.
[0038] The equivalent ratio of CuCl 2 ·2H 2 O to L-cysteine is 1:3 - 1:8; the concentration of the NaOH solution is 0.1 - 1 mol / L; the dialysis uses a dialysis bag with a molecular weight cut-off of 500 - 3500 Da.
[0039] Example 1
[0040] Dissolve 174 mg of CuCl 2 ·2H 2O (1 mmol) was dissolved in 50 ml of deionized water, 611 mg of L-cysteine (5 mmol) was added, and the mixture was vigorously stirred for 5 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to reflux for 40 min, and the color of the reaction solution turned brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0041] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h while keeping stirring. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 23%.
[0042] Example 2
[0043] 174 mg of CuCl 2 ·2H 2 O (1 mmol) was dissolved in 50 ml of deionized water, 611 mg of L-cysteine (5 mmol) was added, and the mixture was vigorously stirred for 2 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to reflux for 40 min, and the color of the reaction solution turned brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0044] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h while keeping stirring. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 9%.
[0045] Example 3
[0046] 174 mg of CuCl 2 ·2H 2 O (1 mmol) was dissolved in 50 ml of deionized water, 611 mg of L-cysteine (5 mmol) was added, and the mixture was vigorously stirred for 10 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to reflux for 40 min, and the color of the reaction solution turned brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0047] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h while keeping stirring. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 23%.
[0048] Example 4
[0049] 174 mg of CuCl 2·2H 2 1 mmol of CuCl₂·2H₂O was dissolved in 50 ml of deionized water, 367 mg of L-cysteine (3 mmol) was added, and the mixture was vigorously stirred for 5 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to boiling under reflux for 40 min, and the color of the reaction solution changed to brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0050] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h, and stirring was maintained. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 12%.
[0051] Example 5
[0052] 174 mg of CuCl 2 ·2H 2 1 mmol of CuCl₂·2H₂O was dissolved in 50 ml of deionized water, 733 mg of L-cysteine (6 mmol) was added, and the mixture was vigorously stirred for 5 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to boiling under reflux for 40 min, and the color of the reaction solution changed to brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0053] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h, and stirring was maintained. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 20%.
[0054] Example 6
[0055] 174 mg of CuCl 2 ·2H 2 1 mmol of CuCl₂·2H₂O was dissolved in 50 ml of deionized water, 978 mg of L-cysteine (8 mmol) was added, and the mixture was vigorously stirred for 5 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. It was heated to boiling under reflux for 40 min, and the color of the reaction solution changed to brown. Heating was stopped, and the reaction solution emitted blue fluorescence under irradiation with a 365 nm ultraviolet lamp.
[0056] The reaction solution was filtered, the filtrate was collected and dialyzed with a dialysis bag (MW = 500) for three days, the water was changed every 24 h, and stirring was maintained. After 3 days, the sample solution was rotary evaporated and placed in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 16%.
[0057] Example 7
[0058] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 611 mg of L-cysteine (5 mmol), stir vigorously for 5 min, adjust the pH to 7 with 1 mol / L NaOH solution. At this time, the reaction solution is yellow. Heat it to boiling under reflux for 40 min. The color of the reaction solution turns brown. Stop heating. The reaction solution emits blue fluorescence under irradiation of a 365 nm ultraviolet lamp.
[0059] Filter the reaction solution, collect the filtrate and dialyze it with a dialysis bag (MW = 500) for three days, change the water every 24 h, keep stirring. After 3 days, rotary evaporate the sample solution and place it in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 20.8%.
[0060] Example 8
[0061] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 611 mg of L-cysteine (5 mmol), stir vigorously for 5 min, adjust the pH to 11 with 1 mol / L NaOH solution. At this time, the reaction solution is yellow. Heat it to boiling under reflux for 40 min. The color of the reaction solution turns brown. Stop heating. The reaction solution emits blue fluorescence under irradiation of a 365 nm ultraviolet lamp.
[0062] Filter the reaction solution, collect the filtrate and dialyze it with a dialysis bag (MW = 500) for three days, change the water every 24 h, keep stirring. After 3 days, rotary evaporate the sample solution and place it in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 19.7%.
[0063] Example 9
[0064] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 611 mg of L-cysteine (5 mmol), stir vigorously for 5 min, adjust the pH to 8 with 1 mol / L NaOH solution. At this time, the reaction solution is yellow. Heat it to boiling under reflux for 90 min. The color of the reaction solution turns brown. Stop heating. The reaction solution emits blue fluorescence under irradiation of a 365 nm ultraviolet lamp.
[0065] Filter the reaction solution, collect the filtrate and dialyze it with a dialysis bag (MW = 500) for three days, change the water every 24 h, keep stirring. After 3 days, rotary evaporate the sample solution and place it in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 23%.
[0066] Example 10
[0067] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 611 mg of L-cysteine (5 mmol), stir vigorously for 5 min, adjust the pH to 8 with 1 mol / L NaOH solution. At this time, the reaction solution is yellow. Heat it to boiling under reflux for 40 min. The color of the reaction solution turns brown. Stop heating. The reaction solution emits blue fluorescence under irradiation of a 365-nm ultraviolet lamp.
[0068] Filter the reaction solution, collect the filtrate and dialyze it with a dialysis bag (MW = 3500) for three days, change the water every 24 h, keep stirring. After 3 days, rotary evaporate the sample solution and place it in a vacuum drying oven at 60 °C for 12 h to obtain a black-brown solid powder with a yield of 9%.
[0069] Comparative Example 1
[0070] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 122 mg of L-cysteine (1 mmol), stir vigorously for 5 min, adjust the pH to 8 with 1 mol / L NaOH solution. At this time, the reaction solution is light yellow. Heat it to boiling under reflux for 40 min. The color of the reaction solution turns light blue. Stop heating. A large amount of blue precipitate is obtained, and no product that emits blue fluorescence under irradiation of a 365-nm ultraviolet lamp can be obtained.
[0071] Comparative Example 2
[0072] Dissolve 174 mg of CuCl 2 ·2H 2 O (1 mmol) in 50 ml of deionized water, add 244 mg of L-cysteine (2 mmol), stir vigorously for 5 min, adjust the pH to 8 with 1 mol / L NaOH solution. At this time, the reaction solution is yellow. Heat it to boiling under reflux for 40 min. The color of the reaction solution turns blue. Stop heating. A large amount of blue precipitate is obtained, and no product that emits blue fluorescence under irradiation of a 365-nm ultraviolet lamp can be obtained.
[0073] Comparative Example 3
[0074] Dissolve 174 mg of CuCl 2 ·2H 2O (1 mmol) was dissolved in 50 ml of deionized water, 367 mg of L-cysteine (3 mmol) was added, and the mixture was vigorously stirred for 5 min. The pH was adjusted to 8 with 1 mol / L NaOH solution. At this time, the reaction solution was yellow. Stirring was continued for 40 min, and the color of the reaction solution gradually changed to grayish brown. Stirring was stopped to obtain a grayish brown precipitate, and the product that emits blue fluorescence under irradiation with a 365 nm ultraviolet lamp was not obtained.
[0075] Characterization of Copper-Cysteine Complex
[0076] 1. Macroscopic and Microscopic Morphologies
[0077] Figure 1 are the macroscopic morphology diagram and solution color development diagram of the copper-cysteine complex prepared in Example 1 of the present invention (the left figure is the morphology of Cu-Cys solid, and the right figure is the comparison diagram of Cu-Cys aqueous solution under irradiation with a 365 nm ultraviolet lamp and natural light). It can be seen from the figure that the morphology of Cu-Cys in the solid state is a blackish brown solid, which is amorphous. The Cu-Cys aqueous solution is yellowish brown and emits blue fluorescence under a 365 nm ultraviolet lamp.
[0078] Figure 2 is the scanning electron microscope image of the copper-cysteine complex solid prepared in Example 1 of the present invention. It can be seen from the figure that the particle size of the Cu-Cys solid is about 500 nm.
[0079] Figure 3 is the particle size diagram of the aqueous solution of the copper-cysteine complex prepared in Example 1 of the present invention. It can be seen from the figure that the particle size distribution in the Cu-Cys aqueous solution is below 200 nm.
[0080] 2. High-Resolution Liquid Mass Spectrometry
[0081] The copper-cysteine complex is soluble in water, slightly soluble in methanol, and insoluble in organic solvents such as dioxane, acetone, ethyl acetate, dichloroethane, n-hexane, acetonitrile, and tetrahydrofuran.
[0082] Using ultrapure water as the mobile phase, it was determined that the structure of the copper-cysteine complex is nanoparticles formed by the metal center and the ligand L-cysteine linked through Cu-S.
[0083] The structural schematic diagram of the copper-cysteine complex prepared in the present invention is as follows:
[0084]
[0085] Figure 4 is the high-resolution mass spectrum of the copper-cysteine complex prepared in Example 1 of the present invention. From the test results in the figure, it can be determined that the structure of the copper-cysteine complex is the above schematic diagram structure.
[0086] 3. Fourier transform infrared spectrum
[0087] Figure 5 It is the infrared spectrum of the copper-cysteine complex Cu-Cys (left) and the ligand L-cysteine (right) prepared in Example 1 of the present invention; the complex Cu-Cys and the ligand L-Cys were subjected to infrared testing, and by comparing the two, it was confirmed that the S-H bond disappeared.
[0088] 4. Free radical generation ability of copper-cysteine complex
[0089] Detected by the DCFH method, DCFH can be converted into the fluorescent molecule DCF in the presence of free radicals, and the free radical generation ability of the sample is characterized based on the change in fluorescence at 525 nm.
[0090] Figure 6 It is the emission spectrum of detecting the free radical generation ability of the copper-cysteine complex Cu-Cys prepared in Example 1 of the present invention under natural light by the DCFH method; among them, a is the complex Cu-Cys and b is the blank control. It can be seen from the figure that Cu-Cys retains the ability to generate free radicals of Cu-Cy.
[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. A preparation method of a copper-cysteine complex, characterized in that, it comprises the following steps: Mix a soluble copper salt, L-cysteine and deionized water, stir, adjust the pH, raise the temperature to boiling and reflux to obtain a copper-cysteine complex; the molar ratio of the soluble copper salt to L-cysteine is 1:3 to 1:8, the adjustment of the pH is to adjust the pH to 7 to 11, and the time of the boiling reflux is 40 to 90 min.
2. The preparation method of the copper-cysteine complex according to claim 1, characterized in that, the temperature of the stirring is room temperature and the time is 2 to 10 min; The soluble copper salt is CuCl 2 ·2H 2 O; the molar volume ratio of the soluble copper salt to deionized water is 1 mmol: 30 to 80 ml.
3. The preparation method of the copper-cysteine complex according to claim 1, characterized in that, the heating rate is 1 to 10 °C / min; the solution used for adjusting the pH is a NaOH solution; the concentration of the NaOH solution is 0.1 to 2 mol / L.
4. The preparation method of the copper-cysteine complex according to claim 1, characterized in that, the reaction solution after the boiling reflux is filtered, the filtrate is collected, dialyzed, spin-dried, and dried to obtain a copper-cysteine complex.
5. The preparation method of the copper-cysteine complex according to claim 4, characterized in that, the dialysis uses a dialysis bag with a molecular weight cut-off of 500 to 3500 Da; the dialysis time is 24 to 96 h, and the water is changed every 12 - 24 h; the drying temperature is 50 to 80 °C and the time is 12 to 24 h; the drying is vacuum drying.