Film-embedded conjugated oligomeric electrolyte based on natural product, and preparation method and application of film-embedded conjugated oligomeric electrolyte

Abstract

The invention discloses a film-embedded conjugated oligomeric electrolyte based on a natural product, and the structure of the film-embedded conjugated oligomeric electrolyte takes a natural polyphenol compound as a conjugated skeleton part, so that the overall biocompatibility of molecules is improved, and the toxicity is reduced; alkyl chains with different lengths or numbers are used as side chain parts to adjust the compatibility with a cell membrane and promote the membrane embedding and retention of molecules, and the alkyl chains can disturb the phospholipid arrangement of the cell membrane and increase the membrane permeability after the molecules are embedded into the membrane; a positively charged group is used as an end group part, and a negatively charged cell membrane is anchored through electrostatic attraction, so that the local molecular concentration is improved. The cell membrane is modified by the embedded membrane conjugated oligomeric electrolyte derived from the natural product, so that the permeability of the cell membrane is improved, the cytotoxicity is reduced, and the catalytic efficiency of cells is improved. The cell membrane is modified by the embedded membrane conjugated oligomeric electrolyte derived from the natural product, so that the permeability of the cell membrane is improved, the cytotoxicity is reduced, and the catalytic efficiency of cells is improved.

Description

Technical Field

[0001] The present invention relates to the field of whole-cell catalysis technology, and in particular to a natural product-based membrane-embedded conjugated oligoelectrolyte, a preparation method and an application thereof. Background Art

[0002] Whole-cell catalysis refers to the process of converting substrates into specific products under the action of living or dormant / dead cells. It offers low catalytic costs and eliminates steps such as cell lysis and protein concentration required by traditional enzyme catalysis. The advantages of whole-cell catalysis are particularly evident when complex transformations involving coenzymes are involved. Therefore, it exhibits unique advantages and great application prospects in fields such as pharmaceuticals, industrial synthesis, and green renewable energy.

[0003] During whole-cell catalysis, cell membrane permeability is a key factor influencing catalytic efficiency. Reports indicate that free enzyme catalysis is 10–100 times faster than cellular catalysis. The amphiphilic nature of lipopolysaccharide molecules creates a significant barrier to both hydrophilic and hydrophobic molecules. Therefore, it is necessary to develop better methods to increase the rate of material transfer across the cell membrane and reduce unnecessary energy loss during catalysis.

[0004] The traditional cationic surfactant cetyltrimethylammonium bromide (CTAB) is commonly used to improve cell membrane permeability. While it is highly effective and inexpensive, it can easily lead to excessive cell membrane damage, resulting in cell lysis and precluding reuse. Furthermore, CTAB often contaminates the product, making further removal difficult. Therefore, developing a highly effective, low-toxic, and easily removable method for improving cell membrane permeability is of great significance.

[0005] Membrane-intercalating conjugated oligoelectrolytes are a class of molecular systems with a hydrophobic, linear conjugated backbone flanked by flexible side chains terminated with polar groups. Reference 1 (Guillermo C. Bazan, et al. Membrane-Intercalating Conjugated Oligoelectrolytes: Impact on Bioelectrochemical Systems. Adv. Mater . 2015, 27, 2958-2973.) discloses the effects of membrane-embedded conjugated oligoelectrolytes on bioelectrochemistry. The distribution of hydrophobic and hydrophilic groups in membrane-embedded conjugated oligoelectrolyte molecules is similar to that in phospholipid bilayers. Positively charged membrane-embedded conjugated oligoelectrolyte molecules can spontaneously embed into cell membranes through electrostatic and hydrophobic interactions, and the distance between the two charge segments can be used to regulate the permeability of the cell membrane.

[0006] Reference 2 (Wang Bing, et al. Conjugated Oligoelectrolytes: Materials for Acceleration of Whole Cell Biocatalysis. Chem. Mater. 2018, 30, 5836-5840, doi: 10.1021 / acs.chemmater.8b02848.) reported conjugated oligomers (COEs) that increase membrane permeability. However, the synthesis of previously reported COEs is complex and costly, making them unsuitable for large-scale production. The existence of linear, modifiable conjugated backbones in natural products can greatly simplify molecular synthesis processes, reduce synthesis costs, and enhance biocompatibility. Furthermore, natural products are more biodegradable and less polluting, aligning with the development of green chemistry and enabling greener, more efficient, and safer regulation of whole-cell catalysis. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a membrane-embedded conjugated oligoelectrolyte based on natural products, which can modify cell membranes, improve the permeability of cell membranes while having low toxicity, thereby improving the catalytic efficiency of cells.

[0008] A membrane-intercalated conjugated oligoelectrolyte based on natural products, the structure of which is shown below: Wherein, n=0 or 1, R1~R6 are each independently selected from H or , at least one of R1~R3 ​​is , at least one of R4~R6 is , k is selected from any integer from 3 to 11, ” is the connection location.

[0009] In the present invention, the structure uses a natural polyphenol compound as the conjugated backbone, reducing material costs while improving the overall biocompatibility of the molecule and lowering toxicity. Alkyl chains of varying lengths or numbers are used as side chains to adjust compatibility with cell membranes, promoting molecular insertion and retention. Once inserted, the alkyl chains disrupt the arrangement of phospholipids in the cell membrane, increasing membrane permeability. Positively charged groups are used as terminal groups, anchoring the negatively charged cell membrane through electrostatic attraction, thereby increasing local molecular concentration. Modifying the cell membrane with a membrane-inserting conjugated oligoelectrolyte derived from a natural product improves cell membrane permeability, while also reducing cytotoxicity and enhancing cellular catalytic efficiency.

[0010] Preferably, the structure of the natural product-based membrane-embedded conjugated oligoelectrolyte is selected from any of the following structures: 、 、 .

[0011] The present invention also provides a method for preparing the above-mentioned membrane-embedded conjugated oligoelectrolyte based on natural products, comprising the following steps: (1) After a nucleophilic substitution reaction between a natural polyphenol compound and a halogenated alkane, an intermediate compound with a side chain halogenated alkyl is obtained; (2) The side chain halogenated alkylated intermediate compound obtained in step (1) is subjected to a quaternization reaction with trimethylamine to obtain a natural product-based membrane-embedded conjugated oligoelectrolyte.

[0012] The specific preparation method is as follows: Wherein, n=0 or 1, at least one R on the two benzene rings is not H, and k is selected from any integer from 3 to 11. ” is the connection location.

[0013] Preferably, the natural polyphenol compound is resveratrol, oxidized resveratrol or pterostilbene.

[0014] In the present invention, the natural polyphenol compound can be a stilbene polyphenol compound, that is, a structure in which two or three benzene rings are connected by vinyl groups and the terminal benzene ring has a hydroxyl substituent.

[0015] Preferably, the structure of the halogenated alkane is , where k is selected from any integer between 3 and 11.

[0016] In the present invention, the bromine at both ends can undergo a nucleophilic substitution reaction with phenol and a quaternization reaction with trimethylamine, thereby introducing a side chain alkyl group.

[0017] Preferably, the side chain halogenated alkylated intermediate compound is first reacted with a tetrahydrofuran solution of trimethylamine overnight, and then a methanol solution of trimethylamine is added to continue the reaction.

[0018] In the present invention, trimethylamine needs to be added twice, namely, a tetrahydrofuran solution of trimethylamine and a methanol solution of trimethylamine. The quaternary ammonium salt compound formed by the reaction is insoluble in tetrahydrofuran and will precipitate. After the methanol solution of trimethylamine is added, the precipitated compound will dissolve and the reaction will continue.

[0019] The present invention also provides the use of the natural product-based membrane-embedded conjugated oligoelectrolyte in whole-cell catalysis.

[0020] Preferably, the natural product-based membrane-embedded conjugated oligoelectrolyte increases the membrane permeability of Escherichia coli and promotes the transport of membrane contents through the cell membrane.

[0021] In the present invention, the slope of the absorbance curve of the product is calculated to represent the whole-cell catalytic rate, and compared with cetyltrimethylammonium bromide (CTAB). The membrane-embedded conjugated oligoelectrolyte based on natural products increases the membrane permeability of Escherichia coli and promotes the transport of membrane contents through the cell membrane.

[0022] Preferably, the effective concentration of the natural product-based membrane-embedded conjugated oligoelectrolyte for Escherichia coli is 2-64 μM.

[0023] In the present invention, when the natural product-based membrane-embedded conjugated oligoelectrolyte performs whole-cell catalysis in Escherichia coli, the effective concentration is 2-64 μM, the survival rate of Escherichia coli is above 99%, and there is no obvious toxicity.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention prepares natural product-based membrane-inserting conjugated oligoelectrolytes using natural polyphenol compounds as conjugated backbones, alkyl chains of varying lengths or numbers as side chains, and positively charged groups as end groups. The natural product-derived membrane-inserting conjugated oligoelectrolytes modify cell membranes, increasing their permeability while reducing cytotoxicity and improving cellular catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of Re-C10 prepared in Example 1.

[0026] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of Re-C8 prepared in Example 1.

[0027] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of Re-C12 prepared in Example 1.

[0028] Figure 4 This is a statistical diagram of the whole-cell catalytic efficiency of Re-C10 prepared in Example 1.

[0029] Figure 5 This is a graph of the whole-cell biocatalytic efficiency of Re-C10 prepared in Example 1 at 0, 2, 4, 8, 16, and 32 μM.

[0030] Figure 6 The graph shows the effect of Re-C10 at 0, 2, 4, 8, 16, 32, and 64 μM on the growth curve of Escherichia coli. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited to the following examples.

[0032] The raw materials used in the present invention are all commercially available.

[0033] Example 1: Synthesis of Re-C10 (1) Weigh 1,2-dibromodecane (21 g, 70 mmol) and resveratrol (1.6 g, 7 mmol) into a flask, add 30 mL of acetone, blow under N2 atmosphere for 30 minutes, stir at 80°C for 12 hours to carry out nucleophilic substitution reaction. After the reaction is completed, the reaction system is separated by gradient elution to obtain 1.1 g of a side chain alkylated intermediate compound; (2) The side chain alkylated intermediate compound obtained in step (1) was dissolved in 5 mL of anhydrous tetrahydrofuran, and 1 mL of trimethylamine tetrahydrofuran solution was added. The mixture was stirred at 40 °C for 24 hours to carry out quaternization reaction. 1 mL of trimethylamine methanol solution was added and the mixture was stirred at 40 °C for 24 hours. The solvent was removed from the reaction system and redissolved with pure water. The solution was filtered with a 0.22 μm hydrophilic PVDF syringe filter and freeze-dried to obtain 1.36 g of a natural product-based membrane-embedded conjugated oligoelectrolyte, named Re-C10, whose hydrogen spectrum is shown as follows: Figure 1 shown.

[0034] Example 2: Synthesis of Re-C8 The preparation method is the same as that of Example 1, except that the raw material used is 1,8-dibromooctane, named Re-C8, whose hydrogen spectrum is as follows Figure 2 shown.

[0035] Example 3: Synthesis of Re-C12 The preparation method is the same as that of Example 1, except that the raw material used is 1,12-dibromododecane, named Re-C12, whose hydrogen spectrum is as follows Figure 3 shown.

[0036] Sample analysis 1. Enhancement of Escherichia coli ( E. coli ) of whole-cell catalysis (1) Comparison of the effects of Re-C10 and CTAB Prepared 1 OD (OD600 nm = 1) after induction with 2% lactose E. coli ,Will E. coliAfter centrifugation, the supernatant was discarded and equal volumes of 1 / 3× PBS buffer solution, 8 μM Re-C10, and 8 μM CTAB were added. The mixture was mixed and incubated in a 37°C constant temperature incubator for 30 min. The supernatant was discarded and the pellet was washed twice with 1× PBS buffer solution. The pellet was then resuspended in 1× PBS buffer solution to 0.2 OD (OD600 nm = 0.2). 100 μL of each aliquot was placed in a 96-well plate and 100 μL of 10 μM o-nitrophenyl-β-D-galactopyranoside (ONPG) was added to each well for detection. E. coli The leakage of intracellular β-galactosidase was monitored by measuring the absorbance of the sample well at 420 nm every 20 seconds using a multifunctional microplate reader for a total of 20 minutes. The slope value obtained by linear fitting the curve of time and absorbance at 420 nm was used as the E. coli The index of membrane permeability, which in turn reflects the whole-cell biocatalytic efficiency, is shown in the following example. Figure 4 shown.

[0037] Figure 4 This is a statistical diagram of the whole-cell catalytic efficiency of Re-C10 prepared in Example 1. As shown in the figure, The larger the slope value, the E. coli The higher the membrane permeability, the faster the whole cell catalytic efficiency. E. coli The rate of ONPG hydrolysis was faster than that of 1×PBS buffer solution group and CTAB group, which proved that Re-C10 could enhance E. coli membrane permeability, improving E. coli The whole-cell catalytic efficiency is higher than that of the traditional cationic surfactant CTAB.

[0038] (2) Minimum effective concentration of Re-C10 Prepared 1 OD (OD600 nm = 1) after induction with 2% lactose E. coli ,Will E. coli After centrifugation, the supernatant was discarded and an equal volume of 1 / 3× PBS buffer solution and Re-C10 at concentrations of 2, 4, 8, 16, and 32 μM were added. The mixture was mixed and incubated in a 37°C constant temperature incubator for 30 min. The supernatant was then centrifuged and discarded. The pellet was washed twice with 1× PBS buffer solution and then resuspended in 1× PBS buffer solution to 0.2 OD (OD600 nm = 0.2). 100 μL of each aliquot was placed in a 96-well plate and 100 μL of 10 μM o-nitrophenyl-β-D-pyranogalactopyranoside (ONPG) was added to each well for detection. E. coliThe leakage of intracellular β-galactosidase was monitored using a multifunctional microplate reader at 420 nm every 20 seconds for a total of 20 minutes. The slope of the curve obtained by linear fitting the time-to-420 nm absorption value can be used as an indicator of E. coli membrane permeability, thereby reflecting the whole-cell biocatalytic efficiency. The results are shown in Figure 2. Figure 5 shown.

[0039] Figure 5 The whole-cell biocatalytic efficiency of Re-C10 at 0, 2, 4, 8, 16, and 32 μM prepared in Example 1 is shown. As shown in the figure, Re-C10 can hydrolyze ONPG at 2 μM, and the hydrolysis rate accelerates with increasing concentration, that is, the whole-cell biocatalytic efficiency improves.

[0040] 2. Cytotoxicity Re-C10 was prepared at final concentrations of 0, 2, 4, 8, 16, 32, and 64 μM, and 1 OD E. coli . Add equal volumes of liquid LB medium to the 96-well plate. E. coli Mix well and incubate in a 37°C constant temperature incubator for 0-3.5 h. Measure the absorbance at 600 nm with a microplate reader to calculate the effect of different concentrations of Re-C10 on the E. coli The toxicity of Figure 6 shown.

[0041] Figure 6 The toxicity test graph of Re-C10 to Escherichia coli at 0, 2, 4, 8, 16, 32, and 64 μM is shown in the figure. When the concentration of Re-C10 is below 64 μM, E. coli The growth curve of the control group showed no significant difference, indicating that Re-C10 did not affect the growth of E. coli It has no obvious toxicity.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A membrane-embedded conjugated oligoelectrolyte based on natural products, characterized in that: The structure is as follows: Wherein, n=0 or 1, R1~R6 are each independently selected from H or , at least one of R1~R3 ​​is , at least one of R4~R6 is , k is selected from any integer between 3 and 11, ” is the connection location.

2. The natural product-based membrane-embedded conjugated oligoelectrolyte according to claim 1, characterized in that The structure of the natural product-based membrane-embedded conjugated oligoelectrolyte is selected from any of the following structures: 、 、 。 3. The method for preparing a membrane-embedded conjugated oligoelectrolyte based on natural products according to claim 1 or 2, characterized in that: The following steps are involved: (1) reacting natural polyphenol compounds with halogenated alkanes to obtain side chain halogenated alkylated intermediate compounds; (2) The side chain halogenated alkylated intermediate compound obtained in step (1) is subjected to a quaternization reaction with trimethylamine to obtain a natural product-based membrane-embedded conjugated oligoelectrolyte.

4. The method for preparing a membrane-embedded conjugated oligoelectrolyte based on natural products according to claim 3, characterized in that: The natural polyphenol compound is resveratrol, oxidized resveratrol or pterostilbene.

5. The method for preparing a membrane-embedded conjugated oligoelectrolyte based on natural products according to claim 3, characterized in that: The structure of the halogenated alkane is , where k is selected from any integer between 3 and 11.

6. The method for preparing a membrane-embedded conjugated oligoelectrolyte based on natural products according to claim 3, characterized in that: The side chain halogenated alkylated intermediate compound is first reacted with a tetrahydrofuran solution of trimethylamine overnight, and then a methanol solution of trimethylamine is added to continue the reaction.

7. Use of the natural product-based membrane-embedded conjugated oligoelectrolyte according to claim 1 or 2 in whole-cell catalysis.

8. The use according to claim 7, characterized in that The natural product-based membrane-embedded conjugated oligoelectrolyte increases the permeability of the Escherichia coli membrane and promotes the transport of membrane contents through the cell membrane.

9. The use according to claim 7, characterized in that The effective concentration of the natural product-based membrane-embedded conjugated oligoelectrolyte for Escherichia coli is 2-64 μM.

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