Sialic acid-gold nanomaterial compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of novel glycoconjugates, and provides a preparation method of a sialic acid-gold nanomaterial compound. The preparation method of the sialic acid-gold nanomaterial compound comprises the following steps: S1, synthesizing sialic acid ligand functional molecules; s2, synthesizing PC molecules; s3, stable molecules on the surface of the gold nanomaterial are replaced with modified molecules under the action of an accelerant, and a surface modified material intermediate is obtained; and S4, activating surface carboxyl of the surface modified material intermediate, reacting with the PC molecules and the sialic acid ligand functional molecules, and connecting through amido bonds. The invention provides a method for preparing a sialic acid-gold nano composite material with a novel surface structure. The sialic acid molecule is obtained by modifying a natural sialic acid molecular structure and can specifically recognize and combine with the new coronavirus S protein. The non-specific interference of a complex environment on the material is reduced by utilizing the super-hydrophilic characteristic of a PC group, and meanwhile, the biological effect of the sialic acid-gold nano composite material is kept.

Description

Technical Field

[0001] The present invention belongs to the field of novel glycoconjugates, and particularly relates to a sialic acid-gold nanomaterial complex, a preparation method thereof, and an application thereof. Background Art

[0002] A sugar-gold nanomaterial complex refers to a complex formed by immobilizing functional sugar molecules on the surface of gold nanomaterials, which has important application potential in many fields. For example, by immobilizing the sugar ligand of the target protein on the surface of gold nanorods to construct a sugar-gold nanoprobe, it can be directly used for the detection of the target protein (Chem. Lett., 2015, 44, 132-134); by immobilizing sugar molecules on the surface of gold nanomaterials to form sugar clusters, and further improving the biological effects of sugar molecules through the sugar cluster effect (including enhancing the binding force with the target protein, enhancing the targeting effect, etc.) (Polym. Chem., 2015, 6, 5503-5514); preparing a sugar-gold nanocomposite material as a colloidal gold material for the preparation of test kits such as test strips (ACS Cent. Sci., 2020, 6, 2046-2052), etc.

[0003] Sialic acid is a class of naturally occurring nine-carbon monosaccharide compounds, also known as "N-acetylneuraminic acid", which often exists in the form of oligosaccharides, glycolipids or glycoproteins in organisms. It is an important component of various receptors on the cell membrane surface and mediates various physiological and pathological processes. The sialic acid molecules on the surface of human cells can be recognized and bound by the surface protein antigens of various pathogens, mediating or assisting in mediating the invasion process of various pathogens into the human body. Especially in recent years, the novel coronavirus that has caused a global pandemic, its surface S protein antigen can recognize and bind to the sialic acid ligand on the surface of human cells and assist in mediating the invasion of the novel coronavirus particles into the human body (Science, 2022, 377, eabm3125). Sialic acid molecules can be used as S protein ligand molecules of the novel coronavirus for the related detection of the S protein of the novel coronavirus, or for the chemical biology basic research on the recognition and binding of sialic acid-S protein, etc.

[0004] In a piece of literature, a sialic acid sugar-gold nanosphere probe material was disclosed. The surface of this sugar-gold nanosphere material has an SB molecular layer, and the superhydrophilic property of zwitterionic sulfobetaine is used to achieve anti-nonspecific interference. Its preparation process is as follows: First, replace the sodium citrate molecules on the surface of gold nanospheres with mercaptoundecanoic acid, and then introduce SB group stabilizing molecules and sialic acid sugar molecules on the surface of gold nanospheres simultaneously through amide condensation to construct a sugar-gold nanosphere probe material with an SB group layer on the surface. This sugar-gold nanosphere material uses the recognition and binding of sialic acid and virus protein antigens for the detection of virus protein antigens (Langmuir, 2019, 35, 1798-1806).

[0005] The current sugar-gold nanomaterial complex has insufficient specificity and binding strength in the recognition and binding during the detection of the novel coronavirus, which affects the accuracy of the virus detection results. Summary of the Invention

[0006] In order to improve the accuracy of the novel coronavirus detection results, the present invention provides a preparation method of a sialic acid-gold nanomaterial complex.

[0007] The preparation method of the sialic acid-gold nanomaterial complex comprises the following steps:

[0008] S1: Synthesize a sialic acid ligand functional molecule;

[0009] S2: Synthesize a PC molecule;

[0010] S3: The stabilizing molecule on the surface of the gold nanomaterial is replaced by a modified molecule under the action of a promoter to obtain an intermediate of the surface-modified material;

[0011] S4: Activate the surface carboxyl group of the surface-modified material intermediate, and react with the PC molecule and the sialic acid ligand functional molecule, and connect through an amide bond.

[0012] The structure of the sialic acid ligand functional molecule is selected from at least one of the following formulas:

[0013]

[0014] Wherein, X is a hydrogen atom or a halogen atom with indefinite quantity and position on the benzene ring, and the halogen atom is selected from a fluorine atom F, a chlorine atom Cl, and a bromine atom Br, and n 1 ranges from 0 to 10, and m 1 ranges from 1 to 5;

[0015] The structure of the PC molecule is selected from at least one of the following formulas:

[0016]

[0017] Wherein, n 2 ranges from 5 to 10, and m 2 ranges from 2 to 5;

[0018] The molecular structural formula of the modified molecule is HS-(CH 2 ) m -COOH, and the range of m is 10 - 25.

[0019] Further, the sialic acid ligand functional molecule in step S1 is prepared by the following method:

[0020] S11: The carboxyl group of sialic acid Neu5Ac undergoes methylation under acid catalysis and reacts with acyl chloride to obtain a chloro sugar intermediate;

[0021] S11: The chlorinated sugar intermediate undergoes glycosylation reaction to derivatize the side chain;

[0022] S13: Convert the acetamido group at the 5-position of the sugar ring into an azide group, and the 5-position azide intermediate of the sugar ring constructs a derivatized group at the 5-position of the sugar ring through Click reaction;

[0023] S14: After introducing an amino precursor group by converting the terminal group of the derivatized group at the 5-position of the sugar ring, deprotection is carried out to obtain the modified sialic acid molecule, or the modified sialic acid molecule is directly obtained through the deprotection process.

[0024] Further, the PC molecule in step S2 is prepared by the following method:

[0025] S21: Commercial or self-made raw materials include a terminal thiol protecting group and a terminal hydroxyl group, and the terminal hydroxyl group introduces a PC group to obtain an intermediate;

[0026] S22: After removing the terminal thiol protecting group from the intermediate, a small molecule containing a terminal thiol group and a terminal PC zwitterionic group is obtained.

[0027] Further, the commercial or self-made raw materials in S21 include a terminal thiol protecting group and a terminal hydroxyl group, and the intermediate obtained by introducing a PC group to the terminal hydroxyl group is prepared by the following method:

[0028] The commercial or self-made raw materials first form an ester with phosphorus oxychloride POCl 3 to introduce a phosphoryl chloride group, then the phosphoryl chloride group forms an ester with choline, and finally the remaining phosphoryl chloride group is hydrolyzed to introduce a PC group at the terminal hydroxyl group position to obtain the intermediate, wherein the molar ratio of the raw material: the phosphorus oxychloride: the choline is 1:10:5.

[0029] Further, the commercial or self-made raw materials in S21 include a terminal thiol protecting group and a terminal hydroxyl group, and the intermediate obtained by introducing a PC group to the terminal hydroxyl group is prepared by the following method:

[0030] The commercial or self-made raw materials first react with a cyclic phosphoryl halide reagent to introduce a cyclic phosphate group to obtain intermediate 1, and intermediate 1 undergoes a ring-opening substitution reaction with trimethylamine to construct a PC group to obtain the intermediate, wherein the molar ratio of the raw material: the cyclic phosphoryl chloride is 1:1, the molar ratio of intermediate 1: the trimethylamine is 1:1.05 - 1.1, and Y in the cyclic phosphoryl halide reagent is selected from chlorine and bromine atoms.

[0031] Further, the stable molecule on the surface of the gold nanomaterial in S3 is replaced by a modified molecule under the action of a promoter, and the steps for obtaining the surface-modified material intermediate include:

[0032] Under the action of a promoter, the surface stabilizing molecules of commercially purchased or self-made gold nanomaterials are replaced by the modified molecules to form S-Au bonds, obtaining an intermediate of the surface-modified material. Among them, the surface stabilizing molecules of the gold nanomaterials include one or more of cetyltrimethylammonium bromide CTAB, sodium citrate, and polyvinylpyrrolidone PVP. The promoter is selected from sodium hydroxide, potassium hydroxide, sodium borohydride, sodium cyanoborohydride, or potassium borohydride, with a dosage of 50-150 uL and a concentration of 10-100 mM. The dosage of the modified molecule is 50-100 ul, and the concentration is 10-20 mM.

[0033] Further, in S4, S4: Activate the surface carboxyl groups of the surface-modified material intermediate, and react with the PC molecule and the sialic acid ligand functional molecule, and connect through an amide bond, including the following steps:

[0034] Activate the surface carboxyl groups of the surface-modified material intermediate with an activator, and then simultaneously carry out an amide condensation reaction with the PC molecule and the sialic acid ligand functional molecule to obtain the sialic acid-gold nanomaterial complex. Among them, the activator is an aqueous solution of a combination of N-hydroxysulfosuccinimide Sulfo-NHS and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCl. The molar ratio of Sulfo-NHS:EDCl is 1:1.5-3, the molar ratio of Sulfo-NHS: the modified molecule is 0.8-1.5:1, the composition ratio of the PC molecule: the sialic acid ligand functional molecule is 99:1-50:50, and the molar ratio of (PC molecule + sialic acid ligand functional molecule): Sulfo-NHS is 1-1.2:1.

[0035] An object of the present invention is to provide a sialic acid-gold nanomaterial complex.

[0036] The sialic acid-gold nanomaterial complex is prepared by the preparation method of the sialic acid-gold nanomaterial complex described in any one of the above.

[0037] Further, the proportion of PC groups: sialic acid groups in the sialic acid-gold nanomaterial complex is 99%:1% - 50%:50%.

[0038] An object of the present invention is to provide an application of the sialic acid-gold nanomaterial complex as described above in a new coronavirus detection tool or treatment tool.

[0039] The present invention provides a sialic acid-gold nanocomposite with a novel surface structure and a preparation method thereof. The sialic acid molecules immobilized on the surface of the sialic acid-gold nanocomposite are obtained by modifying the structure of natural sialic acid molecules, and can specifically recognize and bind to the S protein of the novel coronavirus. The stable molecular layer on the surface of the composite is obtained by covalently immobilizing small molecules through S-Au bonds, while closing the gold atom sites on the surface of the gold nanomaterial. There are zwitterionic PC groups outside the stable molecular layer, and the superhydrophilic property of the PC groups is used to reduce the non-specific interference of the complex environment on the sialic acid-gold nanocomposite, while maintaining the biological effect of the sialic acid-gold nanocomposite. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the key surface structure of the sialic acid-gold nanomaterial composite provided by the present invention;

[0041] Figure 2 It is the functional molecular structure of the sialic acid ligand provided by the present invention;

[0042] Figure 3 It is the PC molecular structure provided by the present invention;

[0043] Figure 4 It is the preparation method of the sialic acid-gold nanomaterial composite provided by the present invention;

[0044] Figure 5 It is the method for replacing the stable molecules on the surface of the gold nanomaterial given by the present invention;

[0045] Figure 6 It is the method for constructing the sialic acid-gold nanocomposite in one step by surface carboxyl-modified gold nanomaterials given by the present invention;

[0046] Figure 7 It is the sialic acid ligand functional molecule and its preparation method given by the present invention;

[0047] Figure 8 It is the PC small molecule and its preparation method given by the present invention;

[0048] Figure 9 It is the synthesis process of the stable molecule PC-1 with PC groups given in Example 1;

[0049] Figure 10 It is the synthesis process of the stable molecule PC-2 with PC groups given in Example 2;

[0050] Figure 11 It is the synthesis process of the sialic acid ligand functional molecule Sia-1 given in Example 3;

[0051] Figure 12 It is the synthesis process of the sialic acid ligand functional molecule Sia-2 given in Example 4;

[0052] Figure 13 The preparation process of the sialic acid-gold nanorod complex Sia-GNR1 given in Example 5;

[0053] Figure 14 This is the preparation process of the sialic acid-gold nanorod complex Sia-GNR2 given in Example 6. DETAILED DESCRIPTION

[0054] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0055] The present invention provides a sialic acid-gold nanomaterial complex, the key surface structure of the sialic acid-gold nanomaterial complex is as follows Figure 1 As shown. The gold nanomaterial introduces a modified molecular layer on the surface of the gold nanomaterial by forming an S-Au bond with the surface modified molecule, and then forms an amide bond with the PC group stable molecule and the sialic acid functional molecule through the carboxyl group on the outside of the modified molecular layer to form a new sialic acid-gold nanomaterial complex. Among them, the gold nanomaterial morphology includes common morphologies such as gold nanorods, gold nanospheres, gold nanostars, gold nanooctahedrons, gold nanotriangular sheets, gold nanocubes, and composite nanomaterials with a gold-plated layer on the surface, and the sialic acid functional molecule is a sialic acid derivative containing a terminal amino functional side chain.

[0056] Functional molecular structure of sialic acid ligands Figure 2 Wherein, X is a hydrogen atom or a halogen atom of an indefinite number and position on a benzene ring, the halogen atom is selected from a fluorine atom F, a chlorine atom Cl, and a bromine atom Br, n1 ranges from 0 to 10, and m1 ranges from 1 to 5.

[0057] The present invention provides a sialic acid-gold nanomaterial complex, which further matches the CRD pocket space occupied by the S protein by structurally modifying the sialic acid ligand molecules on the surface of the complex, thereby improving the specificity and strength of recognition and binding, and enhancing the interaction between the complex and the S protein of the new coronavirus.

[0058] The PC group stabilizes the molecule (PC molecule) structure as Figure 3 As shown in the figure, n2 ranges from 5 to 10, and m2 ranges from 2 to 5.

[0059] The sialic acid-gold nanocomposite provided by the present invention has a zwitterionic group PC on the surface of the complex to stabilize the molecular layer structure, thereby sealing the gold atomic sites on the surface of the gold nanomaterial, and realizing resistance to nonspecific interference through the super-hydrophilic property of the stable molecular layer, thereby maintaining the sialic acid-gold nanocomposite to normally exert the corresponding biological effect in a complex environment.

[0060] The present invention provides a method for preparing a sialic acid-gold nanomaterial composite. Refer to Figure 4 , including:

[0061] a) A method for replacing the surface stabilizing molecules of gold nanomaterials with a surface carboxyl stabilizing molecular layer, as Figure 5 shown.

[0062] Commercially purchased or self-made gold nanomaterials (OD = 1, 1 mL) are incubated with carboxyl-modified molecules under the action of a promoter until the surface stabilizing molecules are completely replaced by the carboxyl-modified molecules. After purification to remove the replaced stabilizing molecules and excess carboxyl-modified molecules, an intermediate of modified gold nanomaterials is obtained.

[0063] b) A method for constructing a sialic acid-gold nanocomposite in one step from the surface carboxyl-modified gold nanomaterials, as Figure 6 shown.

[0064] The intermediate of modified gold nanomaterials (OD = 1, 1 mL) is activated to activate the carboxyl groups on the material surface under the action of an activator, and then PC molecules and sialic acid ligand functional molecules are added for incubation reaction. After purification to remove the activator reagent and the remaining PC group stabilizing molecules and sialic acid ligand functional molecules, a novel sialic acid-gold nanomaterial composite is obtained.

[0065] Specifically, a method for preparing a sialic acid-gold nanomaterial composite includes:

[0066] a) Commercially purchased or self-made gold nanomaterials (OD = 1, 1 mL, aqueous solution) are incubated under the action of a promoter, and the surface stabilizing molecules are completely replaced by modified molecules (carboxylic acid molecules containing terminal mercapto HS-(CH 2 ) m -COOH). By forming S-Au bonds, a monolayer molecule containing a terminal carboxyl group is introduced onto the surface of the gold nanomaterials to obtain an intermediate of surface-modified materials. Among them, the surface stabilizing molecules of the gold nanomaterials include cetyltrimethylammonium bromide CTAB, sodium citrate, and polyvinylpyrrolidone PVP. The promoter is selected from sodium hydroxide, potassium hydroxide, sodium borohydride, sodium cyanoborohydride, and potassium borohydride, with a dosage of 50 - 150 uL and a concentration of 10 - 100 mM. The range of m in the carboxylic acid molecule containing a terminal mercapto (HS-(CH 2 ) m -COOH) is 10 - 25, the dosage is 50 - 100 ul, and the concentration is 10 - 20 mM. The terminal mercapto carboxylic acid molecules are mixed in water and dissolved by dropping 0.1 M NaOH a.q. .

[0067] b) The surface carboxyl groups of the surface-modified material intermediate are activated by an activator, and then an amide condensation reaction occurs simultaneously with the PC molecule containing a terminal amino group and the sialic acid ligand functional molecule. While constructing a stable PC layer on the gold nanoparticle surface, the sialic acid functional molecule is immobilized to construct a functional surface, and a sugar-gold nanoparticle composite with a novel surface structure is constructed in one step. Among them, the activator refers to an aqueous solution composed of N-hydroxysulfosuccinimide (Sulfo-NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl). The molar ratio of Sulfo-NHS to EDCl is 1:1.5 - 3, the molar ratio of Sulfo-NHS to the modified molecule is 0.8 - 1.5:1, the composition ratio of the PC molecule to the sialic acid ligand functional molecule is 99:1 - 50:50, and the molar ratio of (PC molecule + sialic acid ligand functional molecule) to Sulfo-NHS is 1 - 1.2:1.

[0068] The sialic acid ligand functional molecule and its preparation method are as Figure 7 shown.

[0069] The commercial raw material sialic acid Neu5Ac undergoes carboxymethylation protection under acid catalysis, and then hydroxyl acetylation protection and terminal chlorination are achieved simultaneously in one step to obtain a chlorinated sugar intermediate. The chlorinated sugar intermediate undergoes glycosylation reaction to derivatize the side chain, and then through a series of group conversion reactions, the acetamido group at the 5-position of the sugar ring is converted into an azide group. The 5-position azide intermediate constructs a 5-position derivative group through a Click reaction, and according to the type of the terminal group of the side chain, a group conversion is carried out to introduce an amino precursor group and a deprotection process to obtain the sialic acid ligand functional molecule given in the present invention, or directly obtain the sialic acid ligand functional molecule given in the present invention through a deprotection process.

[0070] The PC small molecule and its preparation method are as Figure 8 shown.

[0071] Commercial or self-made raw materials undergo three consecutive reactions (first forming an ester with phosphorus oxychloride POCl 3 to introduce phosphoryl chloride, then phosphoryl chloride forming an ester with choline, and finally hydrolyzing the remaining phosphoryl chloride) to introduce a PC group at the terminal hydroxyl position to obtain a key intermediate, or first reacting with a cyclic phosphoryl halide reagent to introduce a cyclic phosphate group, and then undergoing a ring-opening substitution reaction with trimethylamine to construct a PC group to obtain a key intermediate; after the key intermediate removes the terminal mercapto protecting group, a small molecule containing a terminal mercapto group and a terminal PC zwitterionic group is obtained; among them, the molar ratio of the starting material: phosphorus oxychloride: choline is 1:10:5; the molar ratio of the raw material: cyclic phosphoryl chloride is 1:1; the molar ratio of intermediate 1: trimethylamine is 1:1.05 - 1.1, and Y in the cyclic phosphoryl halide reagent is selected from chlorine and bromine atoms.

[0072] According to the preparation method of the sialic acid-gold nanomaterial composite provided above, Examples 1-6 were provided to prepare the sialic acid-gold nanorod composite, and the anti-nonspecific interference characteristics, the binding force to the SARS-CoV-2 S protein antigen, and the ability to detect the SARS-CoV-2 S protein antigen in a complex physiological environment of the sialic acid-gold nanorod composite were tested.

[0073] Example 1

[0074] For the synthesis process of the PC group-stabilized molecule PC-1, refer to Figure 9 。

[0075] The commercial raw material 11-mercapto-undecanol (5 g, 24.47 mmol) was dissolved in dry acetonitrile (100 mL), triphenylmethyl chloride (10.23 g, 36.7 mmol) and potassium carbonate (6.76 g, 48.93 mmol) were added, and the mixture was refluxed in an Ar atmosphere for 12 hours. The reaction was monitored by TLC until completion. The reaction solvent was evaporated, and the residue was dissolved in dichloromethane and washed with 5% HCl a.q. , dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the intermediate of formula P-1 (9.4 g, 86% yield). ESI-MS m / z calcd for [C 30 H 39 OS]+(M+H)+: 447.26, found: 447.25.

[0076] The intermediate of formula P-1 (1 g, 2.24 mmol) and triethylamine (2.27 g, 22.39 mmol) were dissolved in dichloromethane (10 mL), and the solution was added dropwise to a dichloro solution (40 mL) containing phosphorus oxychloride (3.43 g, 22.39 mmol). The mixture was stirred at room temperature for 3 hours, and the solvent was evaporated. The resulting residue was dissolved in pyridine (20 mL), and choline chloride (1.56 g, 11.19 mmol) was added under ice bath temperature. The mixture was stirred vigorously, and the reaction solution was slowly warmed to room temperature. After stirring for 24 hours, water (5 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 6 hours. The solvent was evaporated, and the residue was dissolved in dichloromethane and washed with 5% HCl a.q. , dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the compound of formula P-2 (0.42 g, 30.7% yield). ESI-MS m / z calcd for [C 35 H 51 NO 4 PS]+(M+H)+: 612.32, found: 612.33.

[0077] Compound P-2 (0.4 g, 0.65 mmol) was added to a 50% TFA / DCM solution (10 mL) of triethylsilane (0.23 g, 1.98 mmol). The reaction mixture was stirred at room temperature for 1 hour. TLC detection showed that the reaction was complete. The solvent was evaporated, and the residue was purified by column chromatography to obtain compound PC-1 (0.21 g, 87% yield). ESI-MS m / z calcd for [C 16 H 37 NO 4 PS]+(M+H)+: 370.21, found: 370.20.

[0078] Example 2

[0079] For the synthesis of the molecule PC-2 with a PC group, refer to Figure 10 .

[0080] The preparation process of compound P-3 was the same as that of compound P-1 in Example 1. [C 27 H 33 O 4 S]+(M+H)+: 453.20, found: 453.19.

[0081] Compound P-3 (5 g, 11.05 mmol) and triethylamine (2.24 g, 22.09 mmol) were dissolved in anhydrous tetrahydrofuran (50 mL). 2-Chloro-2-oxo-1,3,2-dioxaphospholane (1.57 g, 11.05 mmol) was slowly added dropwise at -10°C. The reaction mixture was slowly warmed to room temperature and stirred for 3 hours. The solid was filtered off. Ether was added to the filtrate, and the insoluble precipitate was filtered off. The solvent was evaporated to obtain the crude product of compound P-4, which was directly used in the next step.

[0082] The crude product of compound P-4 obtained in the previous step and trimethylamine (0.69 g, 11.67 mmol) were mixed in dry dichloromethane (50 mL). The reaction mixture was refluxed with sealing for 2 days. The reaction solution was cooled to room temperature to obtain a white solid, which was purified by column chromatography to obtain compound P-5 (5.7 g, 83.5% yield in 2 steps). ESI-MS m / z calcd for [C 32 H 45 NO 7 PS]+(M+H)+: 618.26, found: 618.26.

[0083] The process for preparing compound PC-2 from compound P-5 was the same as that for preparing compound PC-1 from compound P-2. ESI-MS m / z calcd for [C 13 H 33 NO 7PS]+(M+H)+: 376.15, found: 376.15。

[0084] Example 3

[0085] The synthesis process of the sialic acid ligand functional molecule Sia-1 is shown in Figure 11 。

[0086] Concentrated hydrochloric acid (1 mL) was added dropwise to methanol (100 mL), and natural sialic acid Neu5Ac (10 g, 32.3 mmol) was added in batches. The mixture was stirred until the solution became clear. Then saturated NaHCO 3 a.q. was used to neutralize the reaction solution. The solvent was evaporated, and the residue was co-evaporated with toluene three times by azeotropic distillation and then dried under reduced pressure to obtain the crude product of intermediate S-1 (10.1 g, ~96% yield), which was directly used in the next step of the reaction.

[0087] The crude product of S-1 obtained in the previous step (10 g, ~30.9 mmol) was dissolved in chloroacetyl chloride (100 mL), and the mixture was stirred at room temperature for 24 hours until the reaction was complete. The solvent was evaporated to obtain the crude product of S-2, which was directly used in the next step of the reaction.

[0088] The crude product of S-2 obtained in the previous step was mixed with HOC 6 H 12 Cl (8.45 g, 61.86 mmol) in dry dichloromethane (300 mL). Silver carbonate (25.6 g, 92.8 mmol) was added under an ice bath, and the mixture was reacted at room temperature in the dark for 24 h. The reaction was monitored by TLC (n-hex:Acetone = 2:1). After the reaction was complete, the silver carbonate solid was filtered off, and the residue was concentrated, mixed with silica gel, and separated by column chromatography to obtain compound S-3 (14.1 g, 74.2% in 2 steps), a foamy solid. ESI-MS m / z calcd for [C 26 H 41 ClNO 13 +(M+H)+: 610.22, found: 610.21。

[0089] Compound S-3 (5 g, 8.2 mmol) was dissolved in tetrahydrofuran THF (100 mL), and Boc 2 O (2.68 g, 12.3 mmol) and N,N-dimethylaminopyridine (0.6 g, 4.92 mmol) were added. The mixture was refluxed in an oil bath at 70 °C for 2 hours. The reaction was monitored by TLC (n-hex:Acetone = 2:1). After the reaction was complete, the reaction mixture was cooled to room temperature, and the solvent was evaporated. The residue was dissolved in dichloromethane and washed with saturated NaHCO 3 a.q and dried over anhydrous Na 2 SO 4Dry, concentrate, and dissolve the resulting residue in methanol (50 mL). Dropwise add MeONa to adjust the pH of the solution to 8 - 10, and stir the reaction at room temperature for 2 hours. Monitor the reaction by TLC until completion. Neutralize the reaction mixture to neutrality, evaporate the methanol solvent, dissolve the residue in pyridine (30 mL), add acetic anhydride (15 mL) in an ice bath, and stir the reaction at room temperature overnight. Monitor the reaction by TLC until completion. Quench the reaction by dropwise adding excess methanol in an ice bath, evaporate the solvent, dissolve the residue in dichloromethane, and wash successively with 1N HCl a.q. 、sat. NaHCO 3a.q. and brine, and dry over anhydrous Na 2 SO 4 Dry, concentrate, and then perform column chromatography to obtain Compound S-4 (4.7 g, 85.8%), a foamy solid. ESI-MS m / z calcd for [C 29 H 47 ClNO 14 +(M + H)+: 668.26, found: 668.27.

[0090] Suspend Compound S-4 (4.5 g, 6.74 mmol) in 30% trifluoroacetic acid / dichloromethane solution (30 mL), and stir the reaction at room temperature for 30 minutes. Monitor the reaction by TLC until completion. Dilute the reaction mixture with dichloromethane, and wash with sat. NaHCO 3 a.q. and dry over anhydrous Na 2 SO 4 Dry, concentrate, dissolve the residue in toluene, add azidotrifluoromethanesulfonate (2.36 g, 13.47 mmol) and copper sulfate (0.32 g, 2.02 mmol), and stir the reaction at room temperature overnight. Evaporate the solvent, load the residue onto a column for column chromatography to obtain Compound S-5 (3.2 g, 80%), a pale yellow foam. ESI-MS m / z calcd for [C 24 H 37 ClN 3 O 12 +(M + H)+: 594.20, found: 594.21.

[0091] Dissolve Compound S-5 (3 g, 5.05 mmol) in tetrahydrofuran (50 mL), add phenylacetylene (0.77 g, 7.58 mmol), copper(I) iodide (192 mg, 1.01 mmol), and N,N-diisopropylethylamine (1.16 g, 10.1 mmol), and stir the reaction at room temperature for 2 hours. Monitor the reaction by TLC until completion. Filter off the insoluble solids, evaporate the solvent, and purify the residue by column chromatography to obtain Compound S-6 (3.26 g, 92.7%). ESI-MS m / z calcd for [C 32 H43 ClN 3 O 12 +(M + H)+: 696.25, found: 696.26。

[0092] Compound S - 6 (3 g, 4.31 mmol) was dissolved in DMF (30 mL), and sodium azide NaN 3 (0.84 g, 12.93 mmol) was added. The reaction mixture was placed in an oil bath at 80 °C and reacted for 24 hours. The solvent was evaporated. The residue was dissolved in dichloromethane, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound S - 7 (2.6 g, 85.8%), a white foam. ESI - MS m / z calcd for [C 32 H 43 N 6 O 12 +(M + H)+: 703.29, found: 703.28。

[0093] Compound S - 7 (3 g, 4.27 mmol) was dissolved in methanol (30 mL), and sodium methoxide was added dropwise to adjust the pH of the reaction mixture to 8 - 10. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC and found to be complete. The reaction mixture was neutralized to neutrality, and the solvent was evaporated. The residue was dissolved in a MeOH / H 2 O mixed solvent (v / v = 1:2, 30 mL), and LiOH (0.3 g, 12.8 mmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC and found to be complete. H+ cation exchange resin was added to neutralize the reaction mixture to neutrality. The resin was filtered off, and the solvent was evaporated to obtain compound S - 8 (2.1 g, 94.5%), a white solid. ESI - MS m / z calcd for [C 23 H 33 N 6 O 8 +(M + H)+: 521.23, found: 521.23。

[0094] Compound S - 8 (2 g, 3.84 mmol) was dissolved in methanol (20 mL), and 5% Pd / C powder (0.2 g) was added. The mixture was stirred evenly and placed in a hydrogen atmosphere at 4 atm and stirred for 4 hours. The reaction was monitored by TLC and found to be complete. The Pd / C powder was filtered off, and the solvent was evaporated to obtain the sialic acid ligand functional molecule Sia - 1 (1.85 g, 97.4%), a white solid. ESI - MS m / z calcd for [C 23 H 35 N 4 O 8 +(M + H)+: 495.24, found: 495.25。

[0095] Example 4

[0096] The synthesis process of the sialic acid ligand functional molecule Sia-2 is shown in Figure 12 。

[0097] The process of preparing the sialic acid ligand functional molecule Sia-2 from compound S-2 is the same as that of preparing the sialic acid ligand functional molecule Sia-1 from compound S-2. In the process of preparing compound S-9 from compound S-2, only the reagent HO-C 6 H 12 -Cl is replaced by HO-C 2 H 4 OC 2 H 4 -NHCbz. In the process of preparing compound S-13 from compound S-11, the phenylacetylene reagent is replaced by 3,4,5-trifluorophenylacetylene reagent, and the reaction step of replacing the chlorine atom at the end of the side chain with sodium azide in the process of preparing compound S-7 from compound S-6 is omitted. ESI-MS m / z calcd for [C 21 H 28 F 3 N 4 O 9 +(M + H)+: 537.17, found: 537.16.

[0098] Example 5

[0099] For the preparation process of the sialic acid-gold nanorod complex Sia-GNR1, refer to Figure 13 。

[0100] Commercially purchased CTAB-stabilized gold nanorods (aspect ratio approximately 3, maximum absorption wavelength approximately 800 nm) are diluted with deionized water to OD = 1. Take 1 mL and add the promoter sodium borohydride (10 mM, 50 μL), and incubate at 37 °C for 30 minutes. Then add an aqueous solution of the modified molecule 11-mercapto-undecanoic acid (10 mM, 100 μL, prepared by suspending 11-mercapto-undecanoic acid in deionized water and dropping in 0.1 M NaOH a.q. for dissolution), and keep incubating overnight at 37 °C. Centrifuge (8000 rpm, 10 min), pour off the supernatant, and disperse the residue in deionized water to obtain the modified GNR solution.

[0101] Adjust the OD of the modified GNR solution obtained in the previous step to 1. Take 1 mL and add it to a freshly prepared mixed aqueous solution of Sulfo-NHS / EDCl (Sulfo-NHS:EDCl = 1:2, total concentration 30 mM, 100 μL). Incubate at room temperature for 30 minutes, then add a mixed aqueous solution of PC-1 / Sia-1 (PC-1:Sia-1 = 3:1, total concentration 10 mM, 100 μL). Maintain the reaction for 2 hours, centrifuge (8000 rpm, 10 min) to obtain the sialic acid-gold nanorod complex Sia-GNR1.

[0102] Example 6

[0103] The preparation process of the sialic acid-gold nanosphere complex Sia2-GNP1 is referred to Figure 14 。

[0104] Commercially purchased citrate-stabilized gold nanospheres (particle size about 20 nm) are diluted with deionized water to OD = 1. Take 1 mL and add 0.1 M NaOH a.q. Adjust the solution pH to 10, add the modifying molecule 11-mercapto-11-undecanoic acid (10 mM, 100 μL, prepared by suspending 11-mercapto-undecanoic acid in deionized water and dropping 0.1 M NaOH a.q. to dissolve), maintain the incubation reaction at room temperature for 24 hours, centrifuge (7000 rpm, 10 min), pour off the supernatant, and disperse the residue in deionized water, then drop 0.1 M NaOH a.q. to maintain the solution pH at 10 to obtain the modified GNP solution.

[0105] The process of preparing the complex Sia-GNP1 from the modified GNP is the same as the process of preparing Sia-GNR1 from the modified GNR, except that the ratio of PC-2:Sia-2 is adjusted to 3:2, and the centrifugation conditions in the purification step are replaced with 7000 rpm, 10 min.

[0106] Example 7

[0107] Anti-nonspecific interference property of the sialic acid-gold nanorod complex Sia-GNR1

[0108] The anti-nonspecific interference property of the sialic acid-gold nanorod complex Sia-GNR1 was verified by the experiment of adsorbing bovine serum albumin (BSA) with gold nanorods. BSA is a commonly used reagent in biomedical research and is often used as a nonspecific protein in various interference experiments. The optical absorption signal of gold nanorods due to the Localized Surface Plasmon Resonance effect (LSPR effect) is very sensitive to changes in the surface environment. By measuring the change in the optical absorption signal of the complex Sia-GNR1 solution before and after adding BSA protein, the nonspecific interaction between the complex Sia-GNR1 and BSA was explored, so as to characterize the anti-nonspecific interference property of the sialic acid-gold nanorod complex Sia-GNR1.

[0109] Test method: Take 1 mL of the sialic acid-gold nanorod complex Sia-GNR1 solution with OD = 1. Using the surface CTAB-GNR in Example 6 as a control and deionized water as a blank, add 100 μL of BSA aqueous solutions with different concentrations respectively, incubate at 37 °C for 15 minutes, and record the optical absorption signal of the mixed solution.

[0110] The measurement results show that: when adding BSA with a concentration of 10 -4 ~10 -5 M, there is an obvious interaction between the control gold nanorod solution and BSA, and its optical absorption signal changes significantly. After adding different concentrations of BSA to the sialic acid-gold nanorod complex Sia-GNR1, its optical absorption signal has no obvious change, indicating that the sialic acid-gold nanorod complex Sia-GNR1 has excellent anti-nonspecific interaction performance.

[0111] Example 8

[0112] The sialic acid-gold nanosphere complex Sia-GNP1 improves the binding ability of sialic acid to the S protein antigen of the new coronavirus

[0113] The sialic acid-gold nanomaterial complex provided by the present invention has a multivalent cluster structure on its surface, and can improve the binding ability of a single sialic acid ligand molecule on the surface of the complex to the target protein through the Cluster effect. The binding ability of the Sia-2 ligand molecule obtained in Example 4 and the complex Sia-GNP1 prepared from Sia-2 in Example 6 to the S protein of the new coronavirus was verified by the SPR (Surface Plasmon Resonance) method.

[0114] Detection instrument: Biacore 3000.

[0115] Detection method: After the carboxylic acid-functionalized CM-6 chip is activated by EDC / NHS, a PBS solution of the S protein of the novel coronavirus is introduced to immobilize the S protein on the chip surface. Then, PBS is continuously introduced to wash away the unimmobilized S protein, obtaining an SPR chip immobilized with the S protein. The chip is placed in a Biacore 3000 instrument. After introducing PBS buffer until the signal baseline is stable, different concentrations of Sia-2 ligand and complex Sia-GNP1 are introduced, and the SPR signal response value is measured, and the binding force is calculated, which is represented by the Kd value (the Kd value is the equilibrium dissociation constant of the protein-ligand, which is often used to characterize the binding force of a small molecule ligand to a large molecule target protein, that is: the concentration of the ligand when half of the protein is bound, and half is expressed as a molar concentration value. The smaller the value, the higher the recognition and binding force of the ligand to the protein).

[0116] Test results: The binding force Kd of the sialic acid ligand Sia-2 to the S protein of the novel coronavirus was measured to be ~16 μM, and the binding force Kd of the monovalent sialic acid ligand on the complex Sia-GNP1 to the S protein of the novel coronavirus was ~150 nM. The binding force increased by about 107 times, indicating that by forming multivalent sialic acid molecular clusters on the surface of gold nanomaterials, the recognition and binding of sialic acid ligands to the S protein of the novel coronavirus were significantly improved through the cluster effect.

[0117] Example 9

[0118] Detection of the S protein antigen of the novel coronavirus by the sialic acid-gold nanorod complex Sia-GNR1 in a complex physiological environment

[0119] The sialic acid-gold nanomaterial complex provided by the present invention can be used to detect the S protein of the novel coronavirus by using the sialic acid ligand on the surface of the complex to recognize and bind to the S protein of the novel coronavirus. This was verified by the experiment of detecting the S protein of the novel coronavirus with the complex Sia-GNR1. The S protein of the novel coronavirus is a trimer, and the surface of the complex Sia-GNR1 has a multivalent sialic acid ligand structure, which can undergo "many-to-many" multivalent binding with the S protein of the novel coronavirus, thereby causing probe aggregation, and the optical absorption signal presented by its LSPR effect changes significantly.

[0120] Test method: Take 1 mL of Sia-GNR1 solution (OD = 1), and add 100 μL of a certain concentration of the S protein sample of the novel coronavirus (positive group), healthy human saliva sample (negative group), and the mixed solution of healthy human saliva and the S protein of the novel coronavirus (mixed group), and deionized water (blank control group) respectively. Use healthy human saliva samples and added samples to simulate a complex physiological detection environment, and let it stand at room temperature for 15 minutes. Measure the absorption spectra of each mixed solution, and record the maximum absorption wavelength and absorption value.

[0121] The test results are summarized in the following table:

[0122]

[0123]

[0124] The results showed that the sialic acid-gold nanorod complex Sia-GNR1 provided by the present invention was hardly interfered by non-specificity of physiological saliva samples. It detected the S protein antigen by recognizing and binding to the S protein antigen of the novel coronavirus, triggering probe aggregation. At the same time, the potential of Sia-GNR1 to detect the S protein antigen of the novel coronavirus in a complex physiological environment was verified by a detection experiment of simulating complex physiological samples with mixed samples, thus proving that Sia-GNR1 has application prospects in the detection of the S protein antigen of the novel coronavirus.

Claims

1. A preparation method of a sialic acid-gold nanomaterial composite, characterized in that, it comprises the following steps: S1: Synthesize a sialic acid ligand functional molecule; S2: Synthesize a PC molecule; S3: Under the action of a promoter, the stabilizing molecule on the surface of the gold nanomaterial is replaced with a modified molecule to obtain a surface-modified material intermediate; S4: Activate the surface carboxyl group of the surface-modified material intermediate, and react with the PC molecule and the sialic acid ligand functional molecule, and connect through an amide bond, the structure of the sialic acid ligand functional molecule is selected from at least one of the following figures: Wherein, X is a hydrogen atom or halogen atoms with indefinite quantity and position on the benzene ring, and the halogen atom is selected from a fluorine atom F, a chlorine atom Cl, and a bromine atom Br, and n 1 ranges from 0 to 10, and m 1 ranges from 1 to 5; the structure of the PC molecule is selected from at least one of the following figures: where n 2 ranges from 5 to 10, and m 2 ranges from 2 to 5; The molecular structural formula of the modified molecule is HS-(CH 2 ) m -COOH, and the range of m is 10 - 25.

2. The preparation method of the sialic acid-gold nanomaterial composite according to claim 1, characterized in that, in step S1, the sialic acid ligand functional molecule is prepared by the following method: S11: The carboxyl group of sialic acid Neu5Ac is methylated under acid catalysis and reacts with an acyl chloride to obtain a chloro sugar intermediate; S11: The chloro sugar intermediate undergoes a glycosylation reaction to derivatize the side chain; S13: Convert the acetamido group at the 5-position of the sugar ring into an azide group, and the 5-position azide intermediate of the sugar ring constructs a 5-position derivative group of the sugar ring through a Click reaction; S14: After the terminal group of the 5-position derivative group of the sugar ring is converted to introduce an amino precursor group and then deprotected, the modified sialic acid molecule is obtained, or the modified sialic acid molecule is directly obtained through a deprotection process.

3. The preparation method of the sialic acid-gold nanomaterial composite according to claim 1, characterized in that, in step S2, the PC molecule is prepared by the following method: S21: Commercial or self-made raw materials include a terminal thiol protecting group and a terminal hydroxyl group, and the terminal hydroxyl group introduces a PC group to obtain an intermediate; S22: After the intermediate removes the terminal thiol protecting group, a small molecule containing a terminal thiol and a terminal PC zwitterionic group is obtained.

4. The preparation method of the sialic acid-gold nanomaterial composite according to claim 3, characterized in that, in S21, the commercial or self-made raw materials include a terminal thiol protecting group and a terminal hydroxyl group, and the intermediate obtained by introducing a PC group to the terminal hydroxyl group is prepared by the following method: Commercial or self-made raw materials are first esterified with phosphorus oxychloride POCl 3 to introduce a phosphoryl chloride group, and then the phosphoryl chloride group is esterified with choline. Finally, the remaining phosphoryl chloride group is hydrolyzed to introduce a PC group at the terminal hydroxyl position to obtain the intermediate. Among them, the molar ratio of the raw material: phosphorus oxychloride: choline is 1:10:

5.

5. The preparation method of the sialic acid-gold nanomaterial composite according to claim 3, characterized in that, in S21, the commercial or self-made raw materials include a terminal thiol protecting group and a terminal hydroxyl group, and the intermediate obtained by introducing a PC group to the terminal hydroxyl group is prepared by the following method: The commercial or self-made raw materials first react with a cyclic phosphoryl halide reagent to introduce a cyclic phosphate group to obtain intermediate 1, and intermediate 1 undergoes a ring-opening substitution reaction with trimethylamine to construct a PC group to obtain the intermediate, wherein the molar ratio of the raw material to the cyclic phosphoryl chloride is 1:1, the molar ratio of intermediate 1 to trimethylamine is 1:1.05 - 1.1, and Y in the cyclic phosphoryl halide reagent is selected from chlorine and bromine atoms.

6. The preparation method of the sialic acid-gold nanomaterial composite according to claim 1, characterized in that, in S3, the step of replacing the stabilizing molecule on the surface of the gold nanomaterial with a modified molecule under the action of a promoter to obtain a surface-modified material intermediate includes the following steps: Under the action of a promoter, the surface stabilizing molecules of commercially purchased or self-made gold nanomaterials are replaced by the modified molecules to form S-Au bonds, obtaining an intermediate of the surface-modified material. Among them, the surface stabilizing molecules of the gold nanomaterials include one or more of cetyltrimethylammonium bromide CTAB, sodium citrate, and polyvinylpyrrolidone PVP. The promoter is selected from sodium hydroxide, potassium hydroxide, sodium borohydride, sodium cyanoborohydride, or potassium borohydride, with a dosage of 50-150 uL and a concentration of 10-100 mM. The dosage of the modified molecule is 50-100 ul, and the concentration is 10-20 mM.

7. The preparation method of the sialic acid-gold nanomaterial complex according to claim 1, characterized in that, In S4, S4: Activate the surface carboxyl groups of the surface-modified material intermediate, and react with the PC molecule and the sialic acid ligand functional molecule, and connect through an amide bond, including the following steps: Activate the surface carboxyl groups of the surface-modified material intermediate with an activator, and then simultaneously carry out an amide condensation reaction with the PC molecule and the sialic acid ligand functional molecule to obtain the sialic acid-gold nanomaterial complex. Among them, the activator is an aqueous solution of a combination of N-hydroxysulfosuccinimide Sulfo-NHS and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCl. The molar ratio of Sulfo-NHS:EDCl is 1:1.5-3, the molar ratio of Sulfo-NHS: the modified molecule is 0.8-1.5:1, the composition ratio of the PC molecule: the sialic acid ligand functional molecule is 99:1-50:50, and the molar ratio of (PC molecule + sialic acid ligand functional molecule):Sulfo-NHS is 1-1.2:

1.

8. A sialic acid-gold nanomaterial complex, characterized in that, It is obtained by the preparation method of the sialic acid-gold nanomaterial complex according to any one of claims 1-7.

9. The sialic acid-gold nanomaterial complex according to claim 8, characterized in that, The ratio of the PC group to the sialic acid group is 99%:1% - 50%:50%.

10. The application of the sialic acid-gold nanomaterial complex according to claim 8 in a new coronavirus detection tool or treatment tool.

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