Preparation method and application of biomimetic phospholipid membrane monolithic column for screening and separation of antimicrobial peptides

Abstract

The present invention belongs to the technical field of phospholipid membrane monolithic columns, and specifically relates to a preparation method and application of a bionic phospholipid membrane monolithic column for screening and separation of antimicrobial peptides. The bionic phospholipid membrane monolithic column is prepared by using phospholipid monomers or mixed phospholipids, and is directly prepared in a quartz capillary through an in-situ polymerization reaction. The monolithic column has richer phospholipid species and more complex phospholipid membrane structures, can more accurately simulate the biomembrane environment, and improve separation efficiency and selectivity. The obtained mixed phospholipid membrane monolithic column has good polymerization, and is tightly combined with the capillary wall. The spherical pores are condensed into larger dispersed macroporous channels, and the structural performance is excellent. It can realize the separation of alkyl phenone compounds, and has the ability to resist nonspecific adsorption. It can also be used for the separation, purification and screening of antimicrobial peptides, which is of great significance and solves many deficiencies in the existing screening and purification methods of antimicrobial peptides, such as low efficiency, high cost, activity loss and other problems.

Description

Technical Field

[0001] The invention belongs to the technical field of phospholipid membrane monolithic columns, and in particular relates to a preparation method and application of a bionic phospholipid membrane monolithic column for screening and separating antimicrobial peptides. Background Art

[0002] In the immune defense mechanism of organisms, antimicrobial peptides (AMPs) are often called antimicrobial peptides. As a key class of endogenous small molecule polypeptides, their unique mechanism of action - directly targeting bacterial cell membranes rather than specific molecular recognition sites - is significantly different from traditional antibiotics. Since the structure and components of bacterial cell membranes are highly conserved during evolution, AMPs have the advantage of being difficult to induce widespread drug resistance. In addition to their broad-spectrum antibacterial properties, excellent thermal stability, good water solubility and low immunogenicity, AMPs are considered to be ideal substitutes for antibiotics. However, despite their huge potential, the types of AMPs found in nature are extremely limited to date, with an exploration rate of less than 0.1%, which is mainly restricted by two factors: first, the content of AMP in organisms is extremely low, making it difficult to meet the needs of large-scale screening; second, the existing screening methods mostly use the classic Waksman method, which is based on the activity-guided separation strategy, but the process is complex, inefficient, and costly, and it is easy to cause the loss of active ingredients and damage to peptide activity.

[0003] Given the core role of AMP binding to cell membranes in its antibacterial mechanism, membrane-targeted strategies provide an important idea for the affinity enrichment of AMP. Although studies have achieved AMP screening by immobilizing Escherichia coli cell membranes, the easy inactivation and short life of real cell membranes limit their widespread application. Further studies have shown that AMP exerts its effects by specifically recognizing specific phospholipid components (such as PE, PG, CL, etc.) in cell membranes, which provides a theoretical basis for the development of highly targeted biomimetic phospholipid membrane materials. By simulating the phospholipid bilayer structure of bacterial cell membranes, not only the material preparation process is simplified and the service life is extended, but also the selectivity and stability are enhanced, ensuring the specificity in the screening process.

[0004] However, the existing simulated cell membrane materials are still insufficient in bionics and specificity, and traditional affinity enrichment columns face many challenges when processing complex samples, such as high protein consumption, long analysis time, high back pressure, unstable materials and poor chromatographic performance, etc., which makes it difficult to effectively screen trace AMP components. With the rapid development of precision micro-machining technology and new materials, capillary affinity monolithic chromatography technology combining nanoliter liquid phase with monolithic column has emerged, providing new ideas for solving the above problems. Among them, organic polymer monolithic materials have become the key to improving AMP screening efficiency with their unique advantages, such as simple preparation, acid and alkali resistance, stable properties, high column efficiency, good permeability and good biocompatibility. The inventor team of this application has been committed to research in this field for a long time, and has successfully prepared a series of simulated cell membrane monolithic chromatographic columns of immobilized phospholipids, showing great potential in AMP screening, and laying a solid foundation for the development of efficient and specific AMP separation, purification and screening technology. Based on this, this patent aims to provide an innovative bionic phospholipid membrane monolithic material, in order to achieve a technological breakthrough in the field of separation, purification and screening of AMP. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a method for preparing and applying a bionic phospholipid membrane monolithic column for screening and separation of antimicrobial peptides.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a method for preparing a bionic phospholipid membrane monolithic column for screening and separating antimicrobial peptides, comprising the following steps:

[0008] 1) Preparation of phospholipid monomers:

[0009] Through chemical synthesis, PE / PC / PG / CL polymerizable methacrylate monomers MDPE, MDPC, MDPG, and MDCL containing different fatty chain lengths and saturation degrees are obtained, which are functional phospholipid monomers;

[0010] The preparation of MDPE comprises: using 1,12-dodecanediol as a raw material, performing an acylation reaction to obtain 2-methacryloyloxydodecyl alcohol, reacting with COP to obtain 12-(2-oxy-1,3,2-dioxaphospha-2-yl)methacrylate dodecyl, and then performing an amination reaction to obtain 2-methacryloyloxydodecylphosphoethanolamine MDPE;

[0011] The preparation of MDPC comprises: reacting MDPE with trimethylamine to obtain 2-methacryloyloxydodecylphosphocholine MDPC;

[0012] The preparation of MDPG comprises: reacting 2-methacryloyloxydodecanol, benzyl N,N-tetraisopropyl phosphoramidite and 1H-tetrazole to obtain 2-methacryloyloxydodecylphosphoglycerol benzyl ester, and then reducing the ester by palladium-carbon catalytic hydrogenation to remove the benzyl protecting group to obtain 2-methacryloyloxydodecylphosphoglycerol;

[0013] The preparation of MDCL comprises: preparing 1,2-diacyl-substituted glycerol with 3-p-toluenesulfonyl-1,2-propanediol and fatty acid chloride, reacting with benzyl N,N-tetraisopropylphosphoramidite to obtain an intermediate, condensing the intermediate with 2-benzyloxy-1,3-propanediol to obtain an intermediate 2-benzyl-1,3-di[(1,2-diacyl-substituted-glycerol-3)-phosphoryl]glycerol dibenzyl ester, performing palladium-carbon catalytic hydrogenation reduction to remove the benzyl protecting group to obtain 1,3-di[(1,2-dilauroyl-glycerol-3)-phosphoryl]glycerol, and condensing the intermediate with methacryloxydodecanoic acid to obtain 2-methacryloyloxydodecyl cardiolipin;

[0014] The fatty acid chloride includes lauroyl chloride;

[0015] 2) Preparation of biomimetic phospholipid membrane monolithic column materials:

[0016] One or more of the phospholipid monomers prepared above are mixed, a cross-linking agent, a pore-generating agent and an initiator are added to form a polymerization reaction solution, which is injected into a carrier matrix to obtain a phospholipid monolithic column through an in-situ polymerization reaction;

[0017] The cross-linking agent includes but is not limited to one or more of ethylene glycol dimethacrylate (EDMA), N,N'-methylenebisacrylamide (MBA), polyethylene glycol dimethacrylate (PEGDA), bis(2-methacryloyloxyethyl)phosphocholine (MMPC) glycidyl methacrylate (GMA), divinylbenzene (DVB);

[0018] The porogen is not limited to one or more of isopropanol, cyclohexanol, 1,4-butanediol, methanol, and tetrahydrofuran;

[0019] The initiator includes but is not limited to one or more of azobisisobutyronitrile (AIBN), dibenzoyl peroxide (BPO), potassium persulfate, and ammonium persulfate;

[0020] The carrier matrix includes modified quartz capillaries, modified silica gel, and pipette tips;

[0021] The modified quartz capillary is subjected to modification pretreatment, specifically, the capillary is rinsed with 1 mol / L NaOH solution, then filled with 1 mol / L NaOH and both ends of the capillary are sealed and placed in a high-temperature water bath, after the water bath is completed, the capillary is rinsed with deionized water until the pH is neutral, then the capillary is rinsed with methanol and dried in nitrogen, then a mixture of silane coupling agent and methanol is filled into the capillary and both ends are sealed and heated in a water bath, after the reaction, the capillary is rinsed with methanol and water respectively and then dried with nitrogen, that is, the modification pretreatment of the capillary is completed, and the modified capillary can remove impurities while allowing the inner wall of the capillary to undergo polymerization reaction;

[0022] The mixing volume ratio of the silane coupling agent and methanol is (1~2): (1~2);

[0023] The silane coupling agent includes one or more of γ-aminopropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltriethoxysilane, γ-(methacryloyloxy)propyltrimethoxysilane, and γ-glycidyloxypropyltrimethoxysilane;

[0024] The phospholipid monolithic column includes MDCL phospholipid membrane monolithic column, MDPE phospholipid monolithic column, MDPG, MDPC phospholipid membrane monolithic column, MDPE / MDPG phospholipid membrane monolithic column, MDPG / MDCL phospholipid membrane monolithic column, MDPC / MDCL phospholipid membrane monolithic column and the like.

[0025] The present invention also provides the application of the prepared bionic phospholipid membrane monolithic column in screening, separating and purifying antimicrobial peptides.

[0026] The beneficial effects of the present invention are as follows:

[0027] The method for preparing a bionic phospholipid membrane monolithic column for screening and separating antimicrobial peptides of the present invention adopts phospholipid monomers or mixed phospholipids to prepare a bionic phospholipid membrane monolithic column, and directly prepares the column in a quartz capillary through an in-situ polymerization reaction, thereby simplifying the preparation process and reducing the cost. In addition, due to the adoption of multiple phospholipid monomers, the monolithic column has more abundant phospholipid species and more complex phospholipid membrane structures, can more accurately simulate the biological membrane environment, and improve the separation efficiency and selectivity. Compared with the traditional silica gel matrix phospholipid membrane chromatographic column, the present invention develops a highly efficient and stable bionic phospholipid membrane monolithic material, the obtained phospholipid membrane monolithic column has better acid-base tolerance, the polymerization of the mixed phospholipid membrane monolithic column is good, can be closely combined with the capillary wall, and has excellent structural performance, the spherical pores of the monolithic column are condensed into larger dispersed macroporous channels, can realize the separation of alkyl phenone compounds, and has the ability to resist nonspecific adsorption, and can also be used for the separation, purification and screening of antimicrobial peptides, and solves many deficiencies of the existing screening and purification methods of antimicrobial peptides, such as low efficiency, high cost, activity loss and the like, and has important significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation of MDPE / MDPG phospholipid membrane monolithic column and scanning electron microscope image of the cross section of the capillary monolithic column;

[0029] Figure 2 The figure is a schematic diagram of the preparation of the MDPG / MDCL phospholipid membrane monolithic column and a scanning electron microscope image of the cross section of the capillary monolithic column;

[0030] Figure 3 The figure is a schematic diagram of the preparation of MDPC / MDCL phospholipid membrane monolithic column and a scanning electron microscope image of the cross section of the capillary monolithic column;

[0031] Figure 4 The Fourier transform infrared spectroscopy analysis results of MDPC column and MDPC / MDCL phospholipid membrane monolithic column are shown;

[0032] Figure 5 This is a graph showing the separation test results of MDPC / MDCL phospholipid membrane monolithic column on alkyl phenone compounds;

[0033] Figure 6 This is the result diagram of the anti-nonspecific adsorption ability of the MDPC / MDCL phospholipid membrane monolithic column;

[0034] Figure 7 This is the mass spectrum of bee venom peptide adsorbed on MDPC / MDCL phospholipid membrane monolithic column. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0036] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0037] Example 1

[0038] Synthesis of Tetralauroyl Cardiolipin MDCL

[0039] 1) Synthesis of 1,2-dilauroylglycerol

[0040]

[0041] 3-p-Toluenesulfonyl-1,2-propanediol (2.46 g, 10 mmol) was dissolved in 30 mL of dichloromethane, cooled in an ice-water bath, and triethylamine (6.9 mL, 50 mmol) was added. Lauroyl chloride (24 mmol) was slowly added dropwise in an ice-water bath, followed by 4-dimethylaminopyridine (2 mmol). After stirring at room temperature for 12 hours, 100 mL of water was added, dichloromethane was separated, and the aqueous phase was extracted with dichloromethane (50 mL × 3). The dichloromethane was combined, washed with saturated brine, and dried over sodium sulfate. After concentration, it was separated and purified on a silica gel column (petroleum ether: ethyl acetate = 10:1) to obtain 4.5 g (74%) of 1,2-dilauroyl-3-p-toluenesulfonyl glycerol. The product was added to 50 mL of water, the pH was adjusted to about 3, and the reaction was heated overnight. After removing the p-toluenesulfonyl group, it was extracted with ethyl acetate three times. The ethyl acetate layers were combined and dried over anhydrous sodium sulfate overnight. The product was obtained after concentration. 1 H NMR (DMSO-d6,400 MHz) δ 0.86 (t, J = 7.0 Hz, 6H), 1.17-1.34 (m, 32H), 1.49-1.62 (tt, J =7.7, 7.4 Hz, 4H), 2.20-2.32 (m, 4H), 3.47 (d, J = 7.2 Hz, 2H), 4.53 (d, J =7.4 Hz, 2H), 5.07 (m, 1H). 13 C NMR (DMSO-d6, 100 MHz) δ 14.0, 22.6, 24.8, 29.3, 31.7, 33.8, 62.2, 62.4, 71.3, 173.0.

[0042] 3) 2-Benzyl-1,3-di[(1,2-dilauroyl-glycerol-3-yl)-phosphoryl]glycerol dibenzyl ester

[0043]

[0044] 1,2-Dilauroylglycerol (4.6 g, 10 mmol), benzyl N,N-tetraisopropylphosphoramidite (3.70 g, 11 mmol) and 1H-tetrazole (11 mmol) were added to 30 mL of dichloromethane and stirred at room temperature for 3 hours. 2-Benzyloxy-1,3-propanediol (820 mg, 4.5 mmol) was dissolved in 10 mL of dichloromethane and slowly added dropwise to the reaction solution and stirred for 3 hours. After the reaction system was cooled to -40°C, m-CPBA (5.75 g, 20 mmol) was slowly added dropwise and maintained at -40°C for 30 minutes. After the reaction solution was warmed to room temperature, 30 mL of water was added to quench the reaction. Dichloromethane was separated and the aqueous phase was extracted with dichloromethane (30 mL × 3). The dichloromethane was combined, washed with saturated brine, and dried over sodium sulfate. The residue was filtered, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 3:2 as eluent to obtain 4.2 g (60%) of the product. 1 H NMR (DMSO-d6, 400 MHz) δ 0.80 (s, 3H), 0.84-0.88 (m, 9H), 1.23-1.34 (m, 64H), 1.52-1.60 (m, 8H), 2.24-2.29 (m, 8H), 4.04(m, 1H), 4.32-4.34 (m, 8H), 4.46-4.48 (d, J = 7.0 Hz, 4H), 4.64 (s, 2H), 4.98(m, 4H), 5.03 (m, 2H), 7.29 (s, 4H), 7.33 (s, 2H), 7.41 (m, 3H), 7.49 (m, 6H). 13 C NMR (DMSO-d6, 100 MHz) δ 14.0, 22.6, 24.8, 29.3, 31.7, 33.8, 62.4,67.7, 68.5, 71.3, 72.2, 127.7, 128.4, 136.5, 173.0.

[0045] 4) 1,3-bis[(1,2-dilauroyl-3-propanediyl)-phosphoryl]glycerol diammonium salt

[0046]

[0047] Dissolve 2-benzyl-1,3-di[(1,2-dilauroyl-glycerol-3-yl)-phosphoryl] dibenzyl glycerol ester (1.4 g, 1 mmol) in 20 mL tetrahydrofuran and perform reduction hydrogenation with 10% Pd / C and hydrogen for 10 hours. After filtering through diatomaceous earth to remove palladium carbon, concentrate to obtain the target product. Add 2 mL of 30% ammonia water, stir and concentrate to obtain the product. 1 H NMR (DMSO-d6,400 MHz) δ 0.81-0.92 (m, 12H), 1.23-1.34 (m, 64H), 1.51-1.59 (m, 8H), 2.24-2.29 (m, 8H), 4.01 (m, 1H), 4.34-4.38 (m, 8H), 4.47 (d, J = 7.0 Hz, 4H), 4.98-5.06 (m, 2H). 13 C NMR (DMSO-d6, 100 MHz) δ 14.0, 22.6, 24.8, 29.3, 31.7,33.8, 62.4, 68.5, 70.6, 71.3, 173.0.

[0048] 5) Synthesis of Tetralauroyl Cardiolipin (MDCL)

[0049]

[0050] Under argon protection, add 0.2 g 12-(methacryloyloxy) dodecanoic acid (0.63 mmol), 0.1 g 1-ethyl-(3-dimethylaminopropyl) diimide carbonate hydrochloride (EDCI, 0.6836 mmol), 0.2 g DMAP (1.58 mmol), 0.1 g HOBt (0.68 mmol) and 10 mL DCM, and replace the air three times. Dissolve 1,3-di[(1,2-dilauroyl-glycerol-3)-phosphoryl] diammonium glycerol (565 mg, 0.5 mmol) in 5 mL of DCM, wait until the temperature drops to 0°C, and slowly add it dropwise to the reaction solution. After stirring for half an hour, transfer to room temperature and react for 48 hours. After the reaction is completed, filter out the white precipitate and concentrate the filtrate. Ethyl acetate was added and extracted three times. The organic layer was washed once with saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM: MeOH = 20: 1) to obtain MDCL (353 mg, 50%). 1H NMR(DMSO-d6, 400 MHz) δ 0.80 (s, 3H), 0.84-0.88 (m, 9H), 1.10 (s, 2H), 1.11 (s,2H), 1.13 (m, 6H), 1.18 (m, 4H), 1.21-1.34 (m, 64H), 1.52-1.59 (m, 10H), 1.69(s, 2H), 1.86 (s, 3H), 2.24-2.28 (m, 10H), 4.29-4.33 (m, 10H), 4.46 (s, 2H), 4.49 (s, 2H), 4.95-5.05 (m, 3H), 5.53 (d, J = 3.9 Hz, 1H), 6.05 (d, J = 3.9Hz, 1H). 13 C NMR (DMSO-d6, 100 MHz) δ 14.0, 18.3, 22.6, 24.8, 25.8, 29.3, 29.3,31.7, 33.8, 62.4, 65.2, 68.5, 71.3, 126.1, 135.8, 167.3, 173.0.

[0051] Example 2

[0052] Synthesis of 2-methacryloyloxydodecylglycerol phosphate (MDPG)

[0053] 1) Synthesis of 2-methacryloyloxydodecylglycerol benzyl phosphate

[0054]

[0055] Dissolve glycerol (920 mg, 10 mmol) in 10 mL of dichloromethane and cool in an ice-water bath. Add 2-methacryloyloxydodecanol (2.7 g, 10 mmol), benzyl N,N-tetraisopropylphosphoramidite (3.70 g, 11 mmol) and 1H-tetrazole (11 mmol) to 30 mL of dichloromethane. Stir at room temperature for 3 hours, then slowly dropwise add to the glycerol solution and stir for 3 hours. After cooling the reaction system to -40°C, slowly dropwise add m-CPBA (5.75 g, 20 mmol) and maintain -40°C for 30 minutes. After the reaction solution is warmed to room temperature, add 30 mL of water to quench the reaction. Separate the dichloromethane and extract the aqueous phase with dichloromethane (30 mL×3). Combine the dichloromethane, wash with saturated brine, and dry with sodium sulfate. The residue was filtered, concentrated, and separated by column chromatography with petroleum ether:ethyl acetate = 3:2 as eluent to obtain 3.3 g (65%) of the product. 1H NMR (DMSO-d6, 400 MHz) δ 1.16-1.45(16H, m), 1.65-1.79 (4H, m), 1.86 (3H, s), 2.56 (2H, t, J = 7.4 Hz), 3.50(2H, d, J = 5.5 Hz), 3.97 (1H,m), 4.24-4.38 (4H, m), 4.97 (2H, s), 5.55 (1H,d, J = 3.9 Hz), 6.05 (1H, d, J = 3.9 Hz), 7.27-7.45 (5H, m).

[0056] 2) Synthesis of 2-methacryloyloxydodecylglycerol phosphate

[0057]

[0058] Dissolve 2-methacryloyloxydodecylglycerol benzyl phosphate (510 mg, 1 mmol) in 20 mL tetrahydrofuran and reduce and hydrogenate with 10% Pd / C and hydrogen for 10 hours. After filtering through diatomaceous earth to remove palladium carbon, concentrate to obtain the target product. Add 2 mL of 30% ammonia water, stir and concentrate to obtain the product. 1 H NMR (DMSO-d6, 100 MHz): 1.16-1.45 (16Hm), 1.66-1.80 (4H, m), 1.86 (3H, s), 2.47 (2H, t, J = 7.4 Hz), 3.50 (2H, d, J= 5.5 Hz), 3.96 (1H, m), 4.24-4.36 (4H, m), 5.55 (1H, d, J = 3.9 Hz), 6.05(1H, d, J = 3.9 Hz).

[0059] Example 3

[0060] Synthesis of 2-methacryloyloxydodecylphosphoethanolamine (MDPE)

[0061] 1) Synthesis of MDPA

[0062] ①Synthesis of 2-methacryloyloxydodecyl alcohol

[0063]

[0064] Take a three-necked round-bottom flask dried in an oven, add 10.0 g 1,12-dodecanediol (49.45 mmol, 1.0 eq), 2.3 g anhydrous Py (29.67 mmol, 0.6 eq) and 200 mL anhydrous THF under argon protection, and replace the air three times. A clear solution is obtained under magnetic stirring, 3.1 g methacryloyl chloride (29.67 mmol, 0.6 eq) is dissolved in 50 mL THF, and methacryloyl chloride is slowly added dropwise at 0°C using a constant pressure dropping funnel for 0.5 hours, and the addition is completed in two times, and the reaction is carried out at -5°C for 6 hours. After the reaction is completed, the white precipitate is filtered out, and then the filtrate is concentrated under reduced pressure to obtain a white solid crude product.

[0065] Purification by silica gel column chromatography (PE: EA = 30: 1) and spin-drying to obtain a colorless oily liquid (compound A) (TLC: R f =0.2, 40.6%). 1 H NMR (400 MHz, CDCl3) δ 6.09 (m, 1H), 5.53 (m, 1H), 4.13 (m, 2H), 3.62 (m, 2H), 1.93 (m, 3H), 1.66 (m, 2H), 1.54 (m, 2H), 1.41-1.19 (m,16H); 13 CNMR (101 MHz, CDCl3) δ 166.70, 137.79, 125.16, 64.85, 63.05, 32.79, 29.96, 29.58, 29.52, 29.48, 29.42, 29.23, 28.60, 25.96, 24.54, 18.32.

[0066] ②Synthesis of MDPA

[0067]

[0068] Take a three-necked flask dried in an oven, add 0.9 g (5.77 mmol, 1.3 eq) of anhydrous phosphorus oxychloride and 30 mL of anhydrous THF under argon protection, and replace the air three times. When the temperature drops to 0°C, take 1.2 g of 2-methacryloyloxydodecyl alcohol (4.44 mmol, 1.0 eq) and 0.5 g of triethylamine (TEA) (4.88 mmol, 1.1 eq) and dissolve them in 40 mL of THF, and slowly drop them in. After stirring at low temperature for a period of time, move to room temperature and react for 4 hours. After the reaction is completed, the next step can be carried out without post-treatment. Slowly add 5 mL of ice-water mixture to remove unreacted phosphorus oxychloride, extract with ethyl acetate three times, combine the organic phases, wash the organic layer once with saturated brine, add anhydrous sodium sulfate to dry, filter, and concentrate the filtrate under reduced pressure to obtain a yellow oily liquid. The compound MDPA was purified by silica gel column chromatography (DCM: MeOH = 10: 1) and dried to obtain a yellow oily liquid (TLC: R f =0.2, 81.1%). HRMS (ESI): m / z calcd for C 16 H 32 O6P[M+H] + :351.1858, found 351.1938; 1 H NMR (400 MHz, CDCl3) δ 6.11 (s, 1H), 5.56 (s,1H), 4.22-4.09 (m, 3H), 1.96 (s, 3H), 1.75- 1.57 (m, 3H), 1.30 (m, 23H), 0.90(m, 2H); 13 C NMR (101 MHz, CDCl3) δ 167.90, 138.09, 125.89, 66.33, 64.59,44.64, 30.17, 29.37, 29.32, 29.03, 28.69, 28.37, 25.77, 25.33, 17.27.

[0069] 2) Synthesis of 2-methacryloyloxydodecylphosphoethanolamine (MDPE)

[0070] ①Synthesis of 12-(2-oxy-1,3,2-dioxaphospha-2-yl) dodecyl methacrylate (Compound B)

[0071]

[0072] Take a three-necked flask dried in an oven, add 0.6 g of compound A (2.22 mmol, 1.0 eq), 0.3 g of anhydrous TEA (2.66 mmol, 1.2 eq) and 25 mL of anhydrous THF under argon protection, and replace the air three times. Dissolve 0.6 g of COP (4.44 mmol, 2.0 eq) in THF, then lower the temperature to -20°C, slowly add COP with a constant pressure dropping funnel, stir for 0.5 hours, and place it at room temperature to react for 4 hours until the reaction is complete. After the reaction is complete, add 5 mL of anhydrous n-hexane to the mixture, filter out the trimethylammonium chloride precipitate, and concentrate the filtrate under reduced pressure to obtain a colorless oily liquid compound B (TLC: R f =0.2, 90.1%). 1 H NMR (400 MHz, CDCl3) δ 6.11 (s, 1H), 5.56 (m, 1H), 4.33-4.21 (m, 1H), 4.15 (m, 3H), 4.10-4.00 (m, 1H), 3.93-3.57 (m, 3H), 1.95 (s,3H), 1.68 (m, 4H), 1.45-1.20 (m, 19H). 13 C NMR (101 MHz, CDCl3) δ 166.85,137.17, 125.16, 68.07, 64.84, 63.01, 46.04, 32.57, 29.58, 29.41, 29.14,27.55, 26.88, 25.53, 25.41, 17.76, 9.33.

[0073] ②Synthesis of MDPE

[0074]

[0075] Take a three-necked flask dried in an oven, add 0.5 g of compound B (1.33 mmol, 1.0 eq) and 20 mL of anhydrous ACN under argon protection, and replace the air three times. Dissolve 0.1 g of anhydrous ammonia (3.32 mmol, 2.5 eq) in ACN, lower the temperature to -20°C, quickly add anhydrous ammonia with a syringe, stir for a period of time, raise the temperature to 70°C, and react for 48 hours. After the reaction is completed, the reaction mixture is redissolved in water, precipitated, and filtered. Then, after adding 10 mL of ether, a sticky precipitate is formed, which is washed 3 times with THF and acetone respectively, and finally recrystallized in acetonitrile to obtain yellow solid MDPE (298 mg, 58.4%). HRMS(ESI): m / z calcd for C 18 H37 NO6P[M+H] + : 393.2280, found394.2362; 1 H NMR (400 MHz, CDCl3) δ 6.02 (m, 1H), 5.49 (m, 1H), 4.16-4.03 (m,2H), 4.02-3.61 (m, 4H), 3.53 (m, 1H), 2.81 (s, 2H), 1.87 (m, 3H), 1.64-1.42(m, 4H), 1.23 (m, 16H); 13 C NMR (101 MHz, CDCl3) δ 167.50, 138.09, 125.74,67.94, 66.33, 64.76, 62.98, 44.99, 29.52, 29.47, 29.43, 29.29, 29.17, 28.53,25.90, 25.77, 25.65, 18.25.

[0076] Example 4

[0077] Synthesis of 2-methacryloyloxydodecylphosphocholine (MDPC)

[0078] The MDPE (393 mg, 1 mmol) prepared in Example 3 was dissolved in 20 mL of acetonitrile / isopropanol, and a 45% trimethylamine solution (0.4 g, 3 mmol) was added, and the mixture was reacted at room temperature overnight. After the solvent was removed by rotary evaporation under reduced pressure, the obtained solid was redissolved in water, precipitated, and filtered. Then, 10 mL of ether was added to form a viscous precipitate, which was washed three times with THF and acetone, respectively, and finally recrystallized in acetonitrile to obtain 2-methacryloyloxydodecylphosphocholine MDPC. 1 H NMR (400MHz, CDCl3-d) δ 6.06-6.08 (m, 1H), 5.59-5.92 (m, 1H), 4.22-4.29 (m, 2H), 4.14(m, 2H), 3.87 (m, 2H), 3.64 (m, 2H), 3.23 (s, 9H), 1.92 (m, 3H), 1.63-1.71 (m, 4H), 1.33-1.44 (m, 8H), 1.25-1.35 (m, 8H).

[0079] Example 5

[0080] Preparation of MDPE / MDPG phospholipid membrane monolithic column

[0081] Functional monomers MDPE (Example 3) and MDPG (Example 2), crosslinking agent EDMA, pore-generating agent (isopropanol and tetrahydrofuran in a mass ratio of 65:35) and initiator AIBN were placed in a 1 mL glass vial at a mass ratio of 30:40:400:1 to prepare a polymerization reaction solution. After ultrasonic degassing, the polymerization reaction solution was injected into a pre-olefinized quartz capillary with an inner diameter of 100 μm (100 μm ID capillary) with nitrogen. Both ends were sealed and placed in a constant temperature water bath at 60°C for 12 hours to perform in-situ polymerization. Methanol was pumped into the capillary with a high-pressure pump to flush the unreacted reagents, and finally an MDPE / MDPG phospholipid membrane monolithic column was prepared. The preparation schematic diagram and the scanning electron microscope image of the cross section of the capillary monolithic column are shown in FIG. Figure 1 As shown, it can be seen that the polymer polymerization is good, with a uniform distribution of granules in the shape of rice, and is tightly integrated with the capillary wall without collapse; the spherical pores are condensed into larger dispersed macroporous channels, which contributes to the excellent performance of the material.

[0082] Example 6

[0083] Preparation of MDPG / MDCL phospholipid membrane monolithic column

[0084] Functional monomers MDPG (Example 2) and MDCL (Example 1), crosslinking agent EDMA, pore-generating agent (methanol and tetrahydrofuran in a mass ratio of 65:35) and initiator AIBN were placed in a 1 mL glass vial at a mass ratio of 30:35:400:1 to prepare a polymerization reaction solution. After ultrasonic degassing, the polymerization reaction solution was injected into a pre-olefinized quartz capillary with an inner diameter of 100 μm (100 μm ID capillary) with nitrogen. Both ends were sealed and placed in a constant temperature water bath at 60°C for 12 hours to perform in-situ polymerization. Methanol was pumped into the capillary with a high-pressure pump to flush the unreacted reagents, and finally an MDPG / MDCL phospholipid membrane monolithic column was prepared. The preparation schematic diagram and the scanning electron microscope image of the cross section of the capillary monolithic column are shown in FIG. Figure 2 As shown, it can be seen that the polymer polymerization is good, with a uniform distribution of granules in the shape of rice, and is tightly integrated with the capillary wall without collapse; the spherical pores are condensed into larger dispersed macroporous channels, which contributes to the excellent performance of the material.

[0085] Example 7

[0086] Preparation of MDPC / MDCL phospholipid membrane monolithic column

[0087] Functional monomers MDPC (Example 4) and MDCL (Example 1), crosslinking agent EDMA, pore-forming agent (1,4-butanediol and tetrahydrofuran in a mass ratio of 65:35) and initiator AIBN were placed in a 1 mL glass vial at a mass ratio of 35:35:400:1 to prepare a polymerization reaction solution. After ultrasonic degassing, the polymerization reaction solution was injected into a pre-olefinized capillary with an inner diameter of 100 μm (100 μm ID capillary) with nitrogen. Both ends were sealed and placed in a constant temperature water bath at 60°C for 12 hours to perform in-situ polymerization. Methanol was pumped into the capillary with a high-pressure pump to flush the unreacted reagents, and finally an MDPC / MDCL phospholipid membrane monolithic column was prepared. The preparation schematic diagram and the scanning electron microscope image of the cross section of the capillary monolithic column are shown in FIG. Figure 3 As shown, it can be seen that the polymer polymerization is good, with a uniform distribution of granules in the shape of rice, and is tightly integrated with the capillary wall without collapse; the spherical pores are condensed into larger dispersed macroporous channels, which contributes to the excellent performance of the material.

[0088] The MDPC / MDCL phospholipid membrane monolithic column was subjected to the following structural and performance tests:

[0089] 1. Fourier transform infrared spectroscopy analysis

[0090] The single phospholipid monolithic column MDPC column and the mixed phospholipid monolithic column MDPC / MDCL phospholipid membrane monolithic column were analyzed by FT-IR spectroscopy. Figure 4 As shown, the two spectra are almost identical in the position and intensity of the main absorption, especially -P=O, -PO-alkyl and -N + (CH3) three extensions (1258, 1153 and 969 cm -1 ) of the phosphorylcholine head group. The difference worth noting is that for the MDPC / MDCL phospholipid membrane monolithic column, 1728 and 2928 cm -1 The relative intensity of the strong absorption peak at 240 nm is greater, which is the result of the significant contribution of the -C=O stretching of the carboxyl group in cardiolipin serine. These spectroscopically specific observations prove that both phosphorylcholine PC and cardiolipin CL serine groups are grafted onto the MDPC / MDCL phospholipid membrane monolithic column matrix.

[0091] 2. Elemental analysis

[0092] In order to measure the exact percentage of the final polymer composition, the carbon, nitrogen, and hydrogen contents of the MDPC / MDCL phospholipid membrane monolith were determined by elemental analysis. The average weight percentages of carbon, nitrogen, and hydrogen were 58.2%, 4.64%, and 0.91%, respectively. Since the MDPC molecule contains one nitrogen, 21 carbon, and 42 hydrogen atoms, while the MDCL molecule contains no nitrogen but 81 carbon and 14 hydrogen atoms, the molar ratio of MDCL to MDPC in the final polymer can be calculated to be 1:3.85, which is close to the desired molar ratio of the two monomers in the initial polymerization mixture (1:4). It can be seen that the percentage of cardiolipin in the mixed phospholipid functionalized monomers can be quantitatively controlled.

[0093] 3. Chromatographic separation

[0094] The MDPC / MDCL phospholipid membrane monolithic column was used to separate alkyl phenone compounds. Figure 5 As shown in the figure, due to the long alkyl chains of MDPC and MDCL monomers and the negatively charged polymer surface, both hydrophobic and electrostatic interactions can significantly promote the overall retention of analytes on mixed phospholipid functionalized monomers. In order to better understand the retention mechanism of mixed phospholipid functionalized monomers, a series of alkyl phenone compounds were separated on these stationary phases. Experimental conditions: 150 mm × 100 μm inner diameter column; UV detection wavelength was 214 nm; mobile phase was water (A), acetonitrile (B); gradient was 0 min / 10%B, 15.0 min / 100%B, 18.0 min / 100%B, 18.5 min / 10%B, 26.0 min / 10%B; flow rate, 600 nL / min; The figure shows the separation of eight alkyl phenones (from left to right: acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanone, octanophenone, decanophenone) on the MDPC / MDCL phospholipid membrane monolithic column. By using gradient elution, good separation of all analytes could be achieved on both columns within 17 min.

[0095] 5. Nonspecific adsorption

[0096] In order to investigate the anti-nonspecific adsorption ability of the monolithic column, the MDPC / MDCL phospholipid membrane monolithic column was treated with Tris-HCl buffer (50 mmol / L, pH 8.0) to quench the epoxy groups, then injected with fluorescently labeled BSA protein (FITC-BSA), and then rinsed with Tris-HCl buffer for 1 hour. The fluorescence images of the monolithic column before and after rinsing were recorded by fluorescence microscopy. The results are shown in Figure 2. Figure 6 As shown, before washing, both the monolithic column prepared with pure matrix EDMA and the monolithic column with MDPC / MDCL phospholipid membrane had strong fluorescence signals ( Figure 6A and C in the figure); after washing with Tris-HCl buffer, the fluorescence signal intensity of the monolithic column with strong non-specific adsorption is still strong ( Figure 6 B); and the MDPC / MDCL phospholipid membrane monolithic column of the present invention ( Figure 6 The fluorescence signal of (D) disappears, which indicates that the nonspecific adsorption effect of the material prepared by the present invention is weak.

[0097] 6. Enrichment of Antimicrobial Peptides

[0098] When performing the antimicrobial peptide enrichment operation, the pipette tip with the functionalized monolithic material prepared in Examples 6 to 8 was embedded into the top cover of the centrifuge tube with a hole in the top cover to complete the centrifugation operation during the enrichment process. Before enrichment, the biomimetic phospholipid membrane monolithic column was first activated and equilibrated using acetonitrile and the same loading solution used for bee venom.

[0099] The complex bee venom sample was diluted to a specific concentration using the loading solution, and 200 μL was added to the pipette tip. Under the action of centrifugation, it passed through the entire material at the tip of the pipette tip at a flow rate of about 30 μL / min, thereby completing the adsorption of antimicrobial peptides in the sample solution. The material was then washed twice with the loading solution of the same composition to remove nonspecific adsorption on the surface of the material.

[0100] Finally, 5 μL of MALDI mass spectrometry matrix solution composed of 20 mg / mL 2,5-dihydroxybenzoic acid, 50% acetonitrile and 1% phosphoric acid was used to elute the antimicrobial peptides adsorbed on the surface of the material. Take 1 μL of the collected eluate and drop it on the MALDI-MS target plate. After it is dried, it is analyzed using MALDI-ToF MS, such as Figure 7 As shown, it was confirmed to be melittin. After the elution, the material can be regenerated using acetonitrile / water 1:1, 5% trifluoroacetic acid solution for repeated use.

Claims

1. An application of a biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides, characterized in that: The steps include: The antimicrobial peptides include melittin; The preparation method of the bionic phospholipid membrane monolithic column comprises the following steps: 1) Preparation of phospholipid monomers: After the reaction, 2-methacryloyloxydodecylphosphoethanolamine MDPE, 2-methacryloyloxydodecylphosphocholine MDPC, 2-methacryloyloxydodecylphosphoglycerol MDPG and 2-methacryloyloxydodecylcardiolipin MDCL are obtained, which are functional phospholipid monomers; The preparation of MDPE comprises: using 1,12-dodecanediol as a raw material, performing an acylation reaction to obtain 2-methacryloyloxy dodecyl alcohol, reacting with 2-chloro-2-oxo-1,3,2-dioxaphosphane COP to obtain 12-(2-oxy-1,3,2-dioxaphosphane-2-yl) methacrylate dodecyl, and then performing an amination reaction to obtain 2-methacryloyloxy dodecyl phosphoethanolamine MDPE; The preparation of MDPC comprises: reacting MDPE with trimethylamine to obtain 2-methacryloyloxydodecylphosphocholine MDPC; The preparation of MDPG comprises: reacting 2-methacryloyloxydodecanol, benzyl N,N-tetraisopropyl phosphoramidite and 1H-tetrazole to obtain 2-methacryloyloxydodecylphosphoglycerol benzyl ester, and then reducing the ester by palladium-carbon catalytic hydrogenation to remove the benzyl protecting group to obtain 2-methacryloyloxydodecylphosphoglycerol MDPG; The preparation of MDCL comprises: preparing 1,2-diacyl-substituted glycerol with 3-p-toluenesulfonyl-1,2-propanediol and fatty acid chloride, reacting with benzyl N,N-tetraisopropylphosphoramidite to obtain an intermediate, condensing the intermediate with 2-benzyloxy-1,3-propanediol to obtain an intermediate 2-benzyl-1,3-di[(1,2-diacyl-substituted-glycerol-3)-phosphoryl]glycerol dibenzyl ester, performing palladium-carbon catalytic hydrogenation reduction to remove the benzyl protecting group to obtain 1,3-di[(1,2-dilauroyl-glycerol-3)-phosphoryl]glycerol, and condensing the intermediate with methacryloxydodecanoic acid to obtain 2-methacryloyloxydodecyl cardiolipin MDCL; 2) Preparation of biomimetic phospholipid membrane monolithic column materials: Two of the above-prepared phospholipid monomers are mixed, a cross-linking agent, a pore-generating agent and an initiator are added to form a polymerization reaction solution, which is injected into a carrier matrix to obtain a phospholipid monolithic column through an in situ polymerization reaction, including an MDPE / MDPG phospholipid membrane monolithic column, an MDPG / MDCL phospholipid membrane monolithic column, and an MDPC / MDCL phospholipid membrane monolithic column.

2. The application of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 1, characterized in that: The crosslinking agent includes one or more of ethylene glycol dimethacrylate, N,N'-methylenebisacrylamide, polyethylene glycol dimethacrylate, bis(2-methacryloyloxyethyl)phosphocholine, glycidyl methacrylate, and styrene.

3. The application of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 1, characterized in that: The porogen includes at least one of isopropanol, cyclohexanol, 1,4-butanediol, methanol and tetrahydrofuran.

4. The application of the biomimetic phospholipid membrane monolithic column according to claim 1 in screening and separating and purifying antimicrobial peptides, characterized in that: The initiator includes one or more of azobisisobutyronitrile, dibenzoyl peroxide, potassium persulfate, and ammonium persulfate.

5. The application of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 1, characterized in that: The carrier matrix includes modified quartz capillary, modified silica gel and pipette tip.

6. The use of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 5, characterized in that: The modified quartz capillary is subjected to modification pretreatment, specifically, the capillary is rinsed with 1 mol / L NaOH solution and then filled, both ends of the capillary are sealed and placed in a high-temperature water bath, after the water bath is completed, the capillary is rinsed with deionized water until the pH is neutral, then the capillary is rinsed with methanol and dried in nitrogen, and then a mixture of silane coupling agent and methanol is filled into the capillary and both ends are sealed and heated in a water bath, and after the reaction, it is rinsed with methanol and water respectively and then dried with nitrogen, thus completing the modification pretreatment of the capillary.

7. The use of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 6, characterized in that: The volume ratio of the silane coupling agent to methanol is (1~2):(1~2).

8. The use of the biomimetic phospholipid membrane monolithic column in screening and separating and purifying antimicrobial peptides according to claim 7, characterized in that: The silane coupling agent includes one or more of γ-aminopropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, γ-(methacryloyloxy)propyltrimethoxysilane, and γ-glycidyloxypropyltrimethoxysilane.

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