A sialic acid-gold nanomaterial composite, its preparation method and application

Abstract

This invention belongs to the field of novel glycoconjugates and provides a method for preparing a sialic acid-gold nanomaterial composite. The method includes the following steps: S1: synthesizing a sialic acid ligand functional molecule; S2: synthesizing a PC molecule; S3: 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; S4: activating the surface carboxyl groups of the surface-modified material intermediate, reacting it with the PC molecule and the sialic acid ligand functional molecule, and connecting them through amide bonds. This invention provides a method for creating a sialic acid-gold nanocomposite material with a novel surface structure. The sialic acid molecule is obtained by modifying the structure of natural sialic acid molecules and can specifically recognize and bind to the SARS-CoV-2 S protein. The superhydrophilic properties of the PC group are utilized to reduce non-specific interference of complex environments on the material, while maintaining the biological effects of the sialic acid-gold nanocomposite material.

Description

Technical Field

[0001] This invention belongs to the field of novel glycoconjugates, specifically relating to a sialic acid-gold nanomaterial composite, its preparation method, and its application. Background Technology

[0002] Sugar-gold nanomaterial complexes refer to complexes formed by immobilizing functional sugar molecules on the surface of gold nanomaterials, and they have significant application potential in many fields. For example, sugar-gold nanoprobes can be constructed by immobilizing glycoligands of target proteins on the surface of gold nanorods, which can be directly used for the detection of target proteins (Chem. Lett., 2015, 44, 132-134); sugar clusters can be formed by immobilizing sugar molecules on the surface of gold nanomaterials, and the biological effects of sugar molecules can be further enhanced through the sugar cluster effect (including improving the binding force with target proteins and enhancing the targeting effect, etc.) (Polym. Chem., 2015, 6, 5503-5514); sugar-gold nanocomposite materials can be prepared as colloidal gold materials for the preparation of test strips and other reagent kits (ACS Cent. Sci., 2020, 6, 2046-2052), etc.

[0003] Sialic acid is a naturally occurring 9-carbon monosaccharide compound, also known as N-acetylneuraminic acid. In organisms, it often exists in the form of oligosaccharides, glycolipids, or glycoproteins. It is an important component of various receptors on the cell membrane surface, mediating a variety of physiological and pathological processes. Sialic acid molecules on the surface of human cells can be recognized and bound by surface protein antigens of various pathogens, mediating or assisting in the invasion of various pathogens into the human body. In particular, the SARS-CoV-2 virus, which has caused a global pandemic in recent years, can recognize and bind to sialic acid ligands on the surface of human cells, thus assisting in the invasion of SARS-CoV-2 particles into the human body (Science, 2022, 377, eabm3125). Sialic acid molecules can serve as ligand molecules for the SARS-CoV-2 S protein, used for related detection of the SARS-CoV-2 S protein, or for basic chemical and biological research on sialic acid-S protein recognition and binding.

[0004] A sialic acid-gold nanosphere probe material has been disclosed in a paper. This sugar-gold nanosphere material has an SB molecular layer on its surface, utilizing the superhydrophilic properties of zwitterionic sulfobetaine to resist nonspecific interference. The preparation process involves first replacing sodium citrate molecules on the surface of the gold nanospheres with mercaptoundecanoic acid, and then simultaneously introducing SB group stabilizing molecules and sialic acid sugar molecules onto the surface of the gold nanospheres via amide condensation to construct a sugar-gold nanosphere probe material with an SB group layer on its surface. This sugar-gold nanosphere material utilizes the recognition and binding of sialic acid with viral protein antigens for viral protein antigen detection (Langmuir, 2019, 35, 1798-1806).

[0005] Current sugar-gold nanomaterial complexes lack sufficient specificity and binding strength in the detection of the novel coronavirus, affecting the accuracy of virus detection results. Summary of the Invention

[0006] To improve the accuracy of COVID-19 test results, this invention provides a method for preparing a sialic acid-gold nanomaterial composite.

[0007] The preparation method of this sialic acid-gold nanomaterial composite includes the following steps:

[0008] S1: Synthesize functional molecules for sialic acid ligands;

[0009] S2: Synthetic PC molecules;

[0010] S3: The stable molecules on the surface of gold nanomaterials are replaced by modified molecules under the action of a promoter, thus obtaining a surface-modified material intermediate.

[0011] 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, linking them through amide bonds.

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

[0013]

[0014] Where X is a hydrogen atom or a halogen atom on a benzene ring with an indefinite number and position, the halogen atom is selected from fluorine atom F, chlorine atom Cl, bromine atom Br, n1 ranges from 0 to 10, and m1 ranges from 1 to 5;

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

[0016]

[0017] Where n2 ranges from 5 to 10, and m2 ranges from 2 to 5;

[0018] The molecular structure of the modified molecule is HS-(CH2). m -COOH, m ranges from 10 to 25.

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

[0020] S11: Sialic acid Neu5Ac undergoes methylation of its carboxyl group under acid catalysis, and reacts with acyl chloride to yield a chlorosaccharide intermediate;

[0021] S11: The chloroglycosylated intermediate undergoes a glycosylation reaction to generate side chains;

[0022] S13: The acetamino group at the 5-position of the sugar ring is converted into an azide group, and the sugar ring 5-position azide intermediate is used to construct the sugar ring 5-position derivative group via the Click reaction;

[0023] S14: The modified sialic acid molecule is obtained by deprotection after the amino precursor group is introduced by the group conversion of the terminal group of the 5-position derivative group of the sugar ring, or by directly obtaining the modified sialic acid molecule through the deprotection process.

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

[0025] S21: Commercial or homemade raw materials include a terminal thiol protecting group and a terminal hydroxyl group, wherein the terminal hydroxyl group is introduced into 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] Furthermore, the commercial or homemade raw material in S21 includes a terminal thiol protecting group and a terminal hydroxyl group, wherein the terminal hydroxyl group is introduced into a PC group to obtain an intermediate prepared by the following method:

[0028] Commercial or homemade raw materials are first esterified with phosphorus oxychloride (POCl3) to introduce phosphoryl chloride groups. Then, the phosphoryl chloride groups are esterified with choline. Finally, the remaining phosphoryl chloride groups are hydrolyzed to introduce PC groups at the terminal hydroxyl positions to obtain the intermediate. The molar ratio of the raw materials, phosphorus oxychloride, and choline is 1:10:5.

[0029] Furthermore, the commercial or homemade raw material in S21 includes a terminal thiol protecting group and a terminal hydroxyl group, wherein the terminal hydroxyl group is introduced into a PC group to obtain an intermediate prepared by the following method:

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

[0031] Furthermore, the stable molecules on the surface of the gold nanomaterial in S3 are replaced by modified molecules under the action of a promoter to obtain a surface-modified material intermediate, which includes the following steps:

[0032] Under the action of an accelerator, commercially purchased or self-made surface-stabilizing molecules of gold nanomaterials are replaced by the modified molecules to form S-Au bonds, resulting in a surface-modified material intermediate. The surface-stabilizing molecules of the gold nanomaterials include one or more of cetyltrimethylammonium bromide (CTAB), sodium citrate, and polyvinylpyrrolidone (PVP). The accelerator is selected from sodium hydroxide, potassium hydroxide, sodium borohydride, sodium cyanoborohydride, or potassium borohydride, and is used in an amount of 50-150 μL with a concentration of 10-100 mM. The modified molecules are used in an amount of 50-100 μL with a concentration of 10-20 mM.

[0033] Further, in S4: activating the surface carboxyl groups of the surface-modified material intermediate, reacting with the PC molecule and the sialic acid ligand functional molecule, and connecting them through amide bonds, includes the following steps:

[0034] The surface carboxyl groups of the surface-modified material intermediate are activated by an activator, and then undergo an amide condensation reaction with the PC molecule and the sialic acid ligand functional molecule to obtain the sialic acid-gold nanomaterial composite. The activator is an aqueous solution 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 PC molecule to 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.

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

[0036] The sialic acid-gold nanomaterial composite is prepared by any of the methods described above.

[0037] Furthermore, the ratio of PC groups to sialic acid groups in the sialic acid-gold nanomaterial composite is 99%:1% to 50%:50%.

[0038] One object of the present invention is to provide an application of the sialic acid-gold nanomaterial composite as described above in COVID-19 detection or treatment tools.

[0039] This invention provides a novel sialic acid-gold nanocomposite material with a novel surface structure and its preparation method. The sialic acid molecules immobilized on the surface of the sialic acid-gold nanocomposite material are obtained by modifying the structure of natural sialic acid molecules, which can specifically recognize and bind to the SARS-CoV-2 S protein. The stable molecular layer on the surface of the complex is obtained by covalently immobilizing small molecules through S-Au bonds, simultaneously sealing the gold atom sites on the surface of the gold nanomaterials. The outer side of the stable molecular layer has zwitterionic PC groups, utilizing the superhydrophilic properties of the PC groups to reduce non-specific interference from complex environments on the sialic acid-gold nanocomposite material while maintaining its biological effects. Attached Figure Description

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

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

[0042] Figure 3 The PC molecular structure provided by this invention;

[0043] Figure 4 The present invention provides a method for preparing sialic acid-gold nanomaterial composites;

[0044] Figure 5 This invention provides a method for replacing stable molecules on the surface of gold nanomaterials.

[0045] Figure 6 This invention provides a method for one-step construction of sialic acid-gold nanocomposites using surface carboxyl-modified gold nanomaterials.

[0046] Figure 7 This invention provides a functional sialic acid ligand molecule and its preparation method.

[0047] Figure 8 This invention provides a small PC molecule and its preparation method.

[0048] Figure 9 The synthetic procedure for PC-1, a stable molecule with a PC group, is given in Example 1;

[0049] Figure 10 The synthesis procedure for PC-2, a stable molecule with a PC group, is given in Example 2;

[0050] Figure 11 The synthesis procedure for the sialic acid ligand functional molecule Sia-1 given in Example 3 is as follows;

[0051] Figure 12 The synthesis procedure for the sialic acid ligand functional molecule Sia-2 given in Example 4 is as follows;

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

[0053] Figure 14 The preparation process of the sialic acid-gold nanorod complex Sia-GNR2 given in Example 6 is as follows. Detailed Implementation

[0054] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but this should not be construed as limiting the scope of the present invention.

[0055] This invention provides a sialic acid-gold nanomaterial composite, the key surface structure of which is as follows: Figure 1 As shown, a modified molecular layer is introduced onto the surface of gold nanomaterials by forming S-Au bonds with surface-modifying molecules. Subsequently, the carboxyl groups on the outer side of the modified molecular layer form amide bonds and PC groups to stabilize the molecules, as well as sialic acid functional molecules, forming a novel sialic acid-gold nanomaterial composite. The morphologies of the gold nanomaterials include common forms such as gold nanorods, gold nanospheres, gold nanostars, gold nanooctahedrons, gold nanotriangular pieces, and gold nanocubes, as well as composite nanomaterials with a gold-plated surface. The sialic acid functional molecules are sialic acid derivatives with terminal amino functional side chains.

[0056] Sialic acid ligand functional molecular structure such as Figure 2 Where X is a hydrogen atom or a halogen atom on a benzene ring with an indefinite number and position, the halogen atom is selected from fluorine atom F, chlorine atom Cl, bromine atom Br, n1 ranges from 0 to 10, and m1 ranges from 1 to 5.

[0057] This invention provides a sialic acid-gold nanomaterial complex. By modifying the structure of the sialic acid ligand molecules on the surface of the complex, the complex can be further matched to occupy the CRD pocket space of the S protein, thereby improving the specificity and strength of recognition and binding, and enhancing the interaction between the complex and the SARS-CoV-2 S protein.

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

[0059] The sialic acid-gold nanocomposite provided by this invention uses a stable molecular layer structure with zwitterionic groups PC on the surface of the composite to seal the gold atom sites on the surface of the gold nanomaterial, and achieves resistance to non-specific interference through the superhydrophilic properties of the stable molecular layer, so as to maintain the normal biological effects of the sialic acid-gold nanocomposite in complex environments.

[0060] This invention provides a method for preparing sialic acid-gold nanomaterial composites, see reference. Figure 4 ,include:

[0061] a) The method of replacing the surface stabilizing molecules of gold nanomaterials with a surface carboxyl-stabilizing molecular layer, such as Figure 5 As 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 stable molecules are completely replaced by carboxyl-modified molecules. After purification to remove the replaced stable molecules and excess carboxyl-modified molecules, the modified gold nanomaterial intermediate is obtained.

[0063] b) A one-step method for constructing sialic acid-gold nanocomposites using surface carboxyl-modified gold nanomaterials, such as... Figure 6 As shown.

[0064] Modified gold nanomaterial intermediates (OD=1, 1 mL) were activated by an activator to activate the carboxyl groups on the surface of the material. Then, PC molecules and sialic acid ligand functional molecules were added and incubated. After purification to remove the activator reagents and the remaining PC group stabilizing molecules and sialic acid ligand functional molecules, a novel sialic acid-gold nanomaterial complex was 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) under the action of an accelerator, their surface stabilizing molecules are modified by molecules (carboxylic acid molecules containing terminal thiol groups HS-(CH2)). m By completely replacing -COOH groups and forming S-Au bonds, a surface-modified intermediate is obtained by introducing a monolayer molecule containing terminal carboxyl groups onto the surface of gold nanomaterials. The surface-stabilizing molecules for the gold nanomaterials include hexadecyltrimethylammonium bromide (CTAB), sodium citrate, and polyvinylpyrrolidone (PVP). The accelerators are selected from sodium hydroxide, potassium hydroxide, sodium borohydride, sodium cyanoborohydride, and potassium borohydride, used in amounts of 50-150 μL and at concentrations of 10-100 mM. The accelerators are carboxylic acid molecules containing terminal thiol groups (HS-(CH2)). m The range of m in (-COOH) is 10-25, the dosage is 50-100 μL, and the concentration is 10-20 mM. It is prepared by mixing terminal mercaptocarboxylic acid molecules in water and adding 0.1 M NaOH dropwise. a.q. Prepared by dissolving.

[0067] b) The surface-modified intermediate activates the surface carboxyl groups under the action of an activator, and then undergoes an amide condensation reaction with PC molecules containing terminal amino groups and sialic acid ligand functional molecules. This constructs a PC-stabilized layer on the gold nanoparticle surface while simultaneously immobilizing the sialic acid functional molecules to build a functional surface, thus constructing a sugar-gold nanocomposite with a novel surface structure in one step. The activator is an aqueous solution of N-hydroxysulfosuccinimide (Sulfo-NHS) and the condensing agent 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 PC molecules to sialic acid ligand functional molecules is 99:1–50:50, and the molar ratio of (PC molecules + sialic acid ligand functional molecules) to Sulfo-NHS is 1–1.2:1.

[0068] Sialic acid ligand functional molecules and their preparation methods, such as Figure 7 As shown.

[0069] Commercial raw material sialic acid Neu5Ac undergoes carboxyl methylation protection under acid catalysis, followed by a one-step reaction that simultaneously achieves hydroxy acetylation protection and terminal chlorination to obtain a chlorosaccharide intermediate. The chlorosaccharide intermediate undergoes a glycosylation reaction to derivatize side chains, and then undergoes a series of group conversion reactions to convert the acetamino group at the 5-position of the sugar ring into an azide group. The 5-azide intermediate is used to construct a 5-position derivatized group through a Click reaction, and group conversion is carried out according to the type of terminal group of the side chain to introduce an amino precursor group. After deprotection, the sialic acid ligand functional molecule given in this invention is obtained, or the sialic acid ligand functional molecule given in this invention is obtained directly through a deprotection process.

[0070] PC small molecules and their preparation methods, such as Figure 8 As shown.

[0071] Commercial or self-made raw materials undergo a three-step reaction (first, esterification with phosphorus oxychloride POCl3 to introduce phosphoryl chloride, then esterification of phosphoryl chloride with choline, and finally hydrolysis of the remaining phosphoryl chloride) to introduce a PC group at the terminal hydroxyl position to obtain a key intermediate. Alternatively, a cyclic phosphate ester group is introduced by first reacting with a cyclic phosphoryl halide reagent, followed by a ring-opening substitution reaction with trimethylamine to construct a PC group, yielding a key intermediate. After removing the terminal thiol protecting group from the key intermediate, a small molecule containing a terminal thiol group and a terminal PC zwitterionic group is obtained. The molar ratio of starting material: phosphorus oxychloride: choline is 1:10:5; the molar ratio of 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 or bromine atoms.

[0072] Based on the preparation method of the sialic acid-gold nanomaterial complex provided above, Examples 1-6 are provided to prepare sialic acid-gold nanorod complexes, and the anti-nonspecific interference properties, binding force to the SARS-CoV-2 S protein antigen, and ability to detect the SARS-CoV-2 S protein antigen in complex physiological environments of the sialic acid-gold nanorod complexes are tested.

[0073] Example 1

[0074] For the synthetic procedure of PC-1, a stable molecule with a PC group, please refer to [link / reference]. Figure 9 .

[0075] Commercially available 11-mercapto-undecanol (5 g, 24.47 mmol) was dissolved in dry acetonitrile (100 mL), and triphenylmethyl chloride (10.23 g, 36.7 mmol) and potassium carbonate (6.76 g, 48.93 mmol) were added. The mixture was refluxed under an Ar atmosphere for 12 hours. The reaction was confirmed to be complete by TLC. The reaction solvent was evaporated, and the residue was dissolved in dichloromethane and 5% HCl. a.q. Washed, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain intermediate 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 added dropwise to a dichloromethane solution (40 mL) containing phosphorus oxychloride (3.43 g, 22.39 mmol). The mixture was stirred at room temperature for 3 hours, the solvent was evaporated, and the residue was dissolved in pyridine (20 mL). Choline chloride (1.56 g, 11.19 mmol) was added at an ice bath temperature, and the mixture was stirred vigorously. The reaction solution was slowly heated to room temperature and stirred 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 5% HCl. a.q. Washed, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain compound P-2 (0.42 g, 30.7% yield). ESI-MS m / z calcd for [C 35 H 51 NO4PS]+(M+H)+:612.32,found:612.33.

[0077] Compound P-2 (0.4 g, 0.65 mmol) was added to 10 mL of 50% TFA / DCM solution containing triethylsilane (0.23 g, 1.98 mmol). The mixture was stirred at room temperature for 1 hour. TLC analysis showed that the reaction was complete. The solvent was removed by evaporation, and compound PC-1 (0.21 g, 87% yield) was purified by column chromatography. ESI-MS m / z calcd for [C 16 H 37 NO4PS]+(M+H)+:370.21,found:370.20.

[0078] Example 2

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

[0080] The preparation process of compound P-3 is the same as that of compound P-1 in Example 1. [C 27 H 33 O4S]+(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-dioxophosphazenecyclopentane (1.57 g, 11.05 mmol) was slowly added dropwise at -10 °C. The mixture was slowly heated to room temperature and stirred for 3 hours. The solid was filtered off, and the filtrate was added to diethyl ether. The insoluble precipitate was filtered off, and the solvent was evaporated to obtain the crude compound P-4, which was used directly in the next reaction.

[0082] The crude product of formula P-4 obtained in the previous step was mixed with trimethylamine (0.69 g, 11.67 mmol) in dry dichloromethane (50 mL), and the mixture was refluxed under sealed conditions for 2 days. The reaction solution was cooled to room temperature to give a white solid, which was purified by column chromatography to give compound P-5 (5.7 g, 83.5% yield in 2 steps). ESI-MS m / z calcd for [C 32 H 45 NO7PS]+(M+H)+:618.26,found:618.26.

[0083] The process for preparing compound PC-2 from compound P-5 is the same as the process for preparing compound PC-1 from compound P-2. ESI-MS m / z calcd for [C 13 H 33 NO7PS]+(M+H)+:376.15,found:376.15.

[0084] Example 3

[0085] The synthetic procedure for the sialic acid ligand functional molecule Sia-1 is as follows: 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 portions. The mixture was stirred until the solution became clear, and then saturated NaHCO3 was added. a.q. Neutralize the reaction solution, remove the solvent by evaporation, add toluene to the residue and azeotropically evaporate three times, then dry to obtain crude intermediate S-1 (10.1 g, ~96% yield), which can be used directly in the next reaction.

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

[0088] The crude product S-2 obtained in the previous step and HOC6H 12 Cl (8.45 g, 61.86 mmol) was mixed with dry dichloromethane (300 mL), and silver carbonate (25.6 g, 92.8 mmol) was added under ice bath conditions. The reaction was carried out at room temperature in the dark for 24 h. The reaction was confirmed to be complete by TLC (n-hex:Acetone = 2:1). The silver carbonate solid was filtered off, and the residue was concentrated 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 Boc₂O (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 confirmed to be complete by TLC (n-hex:Acetone = 2:1). The reaction was cooled to room temperature, the solvent was evaporated, and the residue was dissolved in dichloromethane and saturated with NaHCO₃. a.qWash, dry with anhydrous Na₂SO₄, concentrate, dissolve the residue in methanol (50 mL), add MeONa dropwise to adjust the pH of the solution to 8-10, stir at room temperature for 2 hours, and TLC shows complete reaction. Neutralize the reaction solution to neutral, evaporate the methanol solvent, dissolve the residue in pyridine (30 mL), add acetic anhydride (15 mL) in an ice bath, stir at room temperature overnight, and TLC shows complete reaction. Quench the reaction with excess methanol dropwise in an ice bath, evaporate the solvent, dissolve the residue in dichloromethane, and then irradiate with 1N HCl. a.q. ,sat.NaHCO 3a.q. Washed with brine, dried over anhydrous Na2SO4, concentrated, and then subjected to 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] Compound S-4 (4.5 g, 6.74 mmol) was suspended in 30% trifluoroacetic acid / dichloromethane solution (30 mL) and stirred at room temperature for 30 minutes. TLC analysis showed that the reaction was complete. The reaction solution was diluted with dichloromethane and then dissolved in sodium hydroxide solution. a.q. Wash, dry with anhydrous Na₂SO₄, concentrate, dissolve the residue in toluene, add trifluoromethanesulfonate azidocyanate (2.36 g, 13.47 mmol) and copper sulfate (0.32 g, 2.02 mmol), stir overnight at room temperature, evaporate the solvent, and after mixing the residue, column chromatography yielded compound S-5 (3.2 g, 80%), a pale yellow foam. ESI-MS m / z calcd for [C 24 H 37 ClN3O 12 ]+(M+H)+:594.20,found:594.21.

[0091] Compound S-5 (3 g, 5.05 mmol) was dissolved in tetrahydrofuran (50 mL), and phenylacetylene (0.77 g, 7.58 mmol), cuprous iodide (192 mg, 1.01 mmol), and N,N-diisopropylethylamine (1.16 g, 10.1 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction was confirmed to be complete by TLC. The insoluble solids were filtered off, the solvent was evaporated, and the residue was purified by column chromatography to obtain compound S-6 (3.26 g, 92.7%). ESI-MS m / z calcd for [C 32 H 43 ClN3O 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 (NaN3) (0.84 g, 12.93 mmol) was added. The reaction solution was placed in an oil bath at 80 °C for 24 hours. The solvent was evaporated, and 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 N6O 12 ]+(M+H)+:703.29,found:703.28.

[0093] Compound S-7 (3 g, 4.27 mmol) was dissolved in methanol (30 mL). Sodium methoxide was added dropwise to adjust the pH of the reaction solution to 8–10. The mixture was stirred at room temperature for 2 hours. TLC confirmed the reaction was complete. The reaction solution was neutralized, and the solvent was evaporated. The residue was dissolved in a MeOH / H₂O mixed solvent (v / v = 1:2, 30 mL). LiOH (0.3 g, 12.8 mmol) was added, and the mixture was stirred at room temperature for 3 hours. TLC confirmed the reaction was complete. H⁺ cation exchange resin was added to neutralize the reaction solution. 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 N6O8]+(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 and reacted under a hydrogen atmosphere at 4 atm for 4 hours. TLC analysis showed the reaction was 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 N4O8]+(M+H)+:495.24,found:495.25.

[0095] Example 4

[0096] The synthetic procedure for the sialic acid ligand functional molecule Sia-2 is as follows: Figure 12 .

[0097] The process of preparing sialic acid ligand functional molecule Sia-2 from compound S-2 is the same as the process of preparing sialic acid ligand functional molecule Sia-1 from compound S-2. The only difference in the process of preparing compound S-9 from compound S-2 is the addition of the reagent HO-C6H. 12 -Cl is replaced with HO-C2H4OC2H4-NHCbz. In the process of preparing compound S-13 from compound S-11, the phenylacetylene reagent is replaced with 3,4,5-trifluorophenylacetylene reagent, and the sodium azide substitution reaction step for the terminal chlorine atom of the side chain in the preparation of compound S-7 from compound S-6 is omitted. ESI-MS m / z calcd for [C 21 H 28 F3N4O9]+(M+H)+:537.17,found:537.16.

[0098] Example 5

[0099] For the preparation process of the sialic acid-gold nanorod complex Sia-GNR1, please refer to [link / reference]. Figure 13 .

[0100] Commercially purchased CTAB-stabilized gold nanorods (aspect ratio approximately 3, maximum absorption wavelength approximately 800 nm) were diluted with deionized water to OD = 1. 1 mL of this solution was taken and 50 μL of sodium borohydride (10 mM) was added as an accelerator. The mixture was incubated at 37 °C for 30 minutes. Then, 100 μL of a modified 11-mercapto-undecanoic acid aqueous solution (10 mM, 100 μL, with 11-mercapto-undecanoic acid suspended in deionized water) was added, followed by the addition of 0.1 M NaOH. a.q. Dissolve the GNR solution and incubate it at 37°C overnight. Centrifuge (8000 rpm, 10 min), discard the supernatant, and disperse the residue in deionized water to obtain the modified GNR solution.

[0101] The modified GNR solution obtained in the previous step was adjusted to OD=1. 1 mL of the solution was taken and a freshly prepared Sulfo-NHS / EDCl mixed aqueous solution (Sulfo-NHS:EDCl=1:2, total concentration 30mM, 100uL) was added. After incubation at room temperature for 30 minutes, a mixed aqueous solution of PC-1 / Sia-1 (PC-1:Sia-1=3:1, total concentration 10mM, 100uL) was added. The reaction was maintained for 2 hours and then centrifuged (8000rpm, 10min) to obtain the sialic acid-gold nanorod complex Sia-GNR1.

[0102] Example 6

[0103] For the preparation process of the sialic acid-gold nanosphere complex Sia2-GNP1, please refer to [link / reference]. Figure 14 .

[0104] Commercially purchased sodium citrate-stabilized gold nanospheres (approximately 20 nm in diameter) were diluted with deionized water to OD = 1. 1 mL of this solution was then added to 0.1 M NaOH. a.q. Adjust the solution pH to 10, add modified 11-mercapto-11-undecanoic acid (10 mM, 100 μL, suspended in deionized water), and add 0.1 M NaOH dropwise. a.q. Dissolve the sample (as prepared by dissolving), incubate at room temperature for 24 hours, centrifuge (7000 rpm, 10 min), discard the supernatant, disperse the residue in deionized water, and add 0.1 M NaOH dropwise. a.q. Maintain the pH of the solution at 10 to obtain the modified GNP solution.

[0105] The process of preparing the complex Sia-GNP1 from modified GNP is the same as that of preparing Sia-GNR1 from 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 and 10 min.

[0106] Example 7

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

[0108] The non-specific interference resistance properties of the sialic acid-gold nanorod complex Sia-GNR1 were verified by using a bovine serum albumin (BSA) adsorption assay on gold nanorods. BSA is a commonly used reagent in biomedical research and is frequently used as a non-specific protein in various interference experiments. Gold nanorods exhibit optical absorption signals that are highly sensitive to changes in the surface environment due to the localized surface plasmon resonance (LSPR) effect. By measuring the changes in the optical absorption signal of the Sia-GNR1 solution before and after the addition of BSA, the non-specific interaction between the Sia-GNR1 complex and BSA was explored, thereby characterizing the non-specific interference resistance properties of the sialic acid-gold nanorod complex Sia-GNR1.

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

[0110] The test results showed that when a concentration of 10 was added... -4 ~10 -5When BSA was added to the control gold nanorod solution, a significant interaction occurred between the control solution and BSA, resulting in a marked change in its optical absorption signal. However, when different concentrations of BSA were added to the sialic acid-gold nanorod complex Sia-GNR1, no significant change in its optical absorption signal was observed, indicating that the sialic acid-gold nanorod complex Sia-GNR1 has excellent anti-nonspecific interaction properties.

[0111] Example 8

[0112] The sialic acid-gold nanosphere complex Sia-GNP1 enhances the binding affinity of sialic acid to the SARS-CoV-2 spike protein antigen.

[0113] The sialic acid-gold nanomaterial complex provided by this invention has a multivalent cluster structure on its surface, which can enhance the binding force between individual sialic acid ligand molecules on the complex surface and target proteins through the cluster effect. The binding force of the Sia-2 ligand molecule obtained in Example 4 and the complex Sia-GNP1 prepared from Sia-2 in Example 6 to the SARS-CoV-2 S protein was verified by SPR (surface plasmon resonance) method.

[0114] Testing instrument: Biacore 3000.

[0115] Detection method: After activation by EDC / NHS, the carboxylic acid-functionalized CM-6 chip was bubbled with PBS solution containing the SARS-CoV-2 spike protein to immobilize the spike protein on the chip surface. PBS was then continuously bubbled in to wash away any unimmobilized spike protein, resulting in an SPR chip with immobilized spike protein. This chip was placed in a Biacore 3000 instrument, and PBS buffer was bubbled in until the signal baseline stabilized. Different concentrations of Sia-2 ligand and the complex Sia-GNP1 were then bubbled in, and the SPR signal response value was measured. The binding force was calculated and expressed as the Kd value (Kd is the protein-ligand equilibrium dissociation constant, commonly used to characterize the binding force of small molecule ligands to large target proteins; it represents the concentration of ligand when half of the protein is bound, with half expressed as a molar concentration; the smaller the value, the higher the binding force of the ligand to the protein).

[0116] Test results: The binding force of sialic acid ligand Sia-2 to the SARS-CoV-2 S protein was measured to be Kd ~ 16 μM, and the binding force of monovalent sialic acid ligand on the complex Sia-GNP1 to the SARS-CoV-2 S protein was Kd ~ 150 nM, which is about 107 times higher than that of SARS-CoV-2. This shows that by forming multivalent sialic acid molecular clusters on the surface of gold nanomaterials, the recognition and binding of sialic acid ligands to the SARS-CoV-2 S protein is greatly improved through the cluster effect.

[0117] Example 9

[0118] Detection of SARS-CoV-2 S protein antigen in complex physiological environments using the sialic acid-gold nanorod complex Sia-GNR1

[0119] The sialic acid-gold nanomaterial complex provided by this invention can be used to detect the SARS-CoV-2 S protein by recognizing and binding to the SARS-CoV-2 S protein using the sialic acid ligands on the surface of the complex. This was verified by the detection of SARS-CoV-2 S protein using the complex Sia-GNR1. The SARS-CoV-2 S protein is a trimer, and the surface of the complex Sia-GNR1 has a multivalent sialic acid ligand structure, which can bind to the SARS-CoV-2 S protein in a "many-to-many" multivalent manner, thereby inducing probe aggregation. The optical absorption signal exhibited by its LSPR effect changes significantly.

[0120] Test method: Take 1 mL of Sia-GNR1 solution (OD=1), add 100 uL of a certain concentration of SARS-CoV-2 S protein sample (positive group), saliva sample from healthy individuals (negative group), and a mixture of saliva from healthy individuals and SARS-CoV-2 S protein (mixed group), and deionized water (blank control group). Use saliva sample from healthy individuals and the added sample to simulate a complex physiological testing environment. Let stand at room temperature for 15 minutes, measure the absorbance spectrum of each mixed solution, and record the maximum absorption wavelength and absorbance value.

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

[0122]

[0123]

[0124] The results showed that the sialic acid-gold nanorod complex Sia-GNR1 provided by this invention was almost unaffected by non-specific interference from physiological saliva samples. It detects the S protein antigen by recognizing and binding to the SARS-CoV-2 S protein antigen and inducing probe aggregation. At the same time, the potential of Sia-GNR1 to detect the SARS-CoV-2 S protein antigen in complex physiological environments was verified by mixed sample simulation of complex physiological sample detection experiments, thus proving that Sia-GNR1 has application prospects in the detection of SARS-CoV-2 S protein antigen.

Claims

1. A method for preparing a sialic acid-gold nanomaterial complex, characterized in that, The method comprises the following steps: S1: synthesizing a sialic acid ligand functional molecule; S2: synthesizing a PC molecule; S3: replacing the stabilizing molecules on the surface of the gold nanomaterial with modified molecules under the action of a promoter to obtain a surface modified material intermediate; S4: activating the surface carboxyl groups of the surface modified material intermediate, and reacting with the PC molecule and the sialic acid ligand functional molecule to connect through an amide bond, The structure of the sialic acid ligand functional molecule is selected from at least one of the following structures: Wherein, X is a hydrogen atom or a number of halogen atoms on the benzene ring, the halogen atoms are selected from fluorine atoms F, chlorine atoms Cl, bromine atoms Br, n1 is 0 to 10, m1 is 1~5; The structure of the PC molecule is selected from at least one of the following structures: Wherein, n2 is 5~10, m2 is 2~5; The molecular structural formula of the modified molecule is HS-(CH2) m -COOH, and m ranges from 10 to 25.

2. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 1, characterized in that, The sialic acid ligand functional molecule in step S1 is prepared by the following method: S11: Under the action of acid catalysis, the carboxyl group of sialic acid Neu5Ac is methylated, and reacted with acyl chloride to obtain a chloro sugar intermediate; S11: The chloro sugar intermediate undergoes glycosylation reaction to derive a side chain; S13: The acetylamino group at the 5-position of the sugar ring is converted into an azido group, and the 5-position azido intermediate of the sugar ring is constructed by Click reaction to construct a 5-position derivative group of the sugar ring; S14: The terminal group of the 5-position derivative group of the sugar ring is converted to introduce an amino precursor group, and then deprotected to obtain the modified sialic acid molecule, or directly deprotected to obtain the modified sialic acid molecule.

3. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 1, characterized in that, The PC molecule in step S2 is prepared by the following method: S21: Commercial or self-made raw materials including terminal mercapto protecting group and terminal hydroxyl group, the terminal hydroxyl group introduces PC group to obtain an intermediate; S22: After removing the terminal mercapto protecting group of the intermediate, a small molecule containing terminal mercapto and terminal PC zwitterion group is obtained.

4. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 3, characterized in that, The commercial or self-made raw materials including terminal mercapto protecting group and terminal hydroxyl group in S21, which introduces PC group to obtain an intermediate, are prepared by the following method: The commercial or self-made raw materials are first esterified with phosphorus oxychloride POCl3 to introduce a phosphorus oxychloride group, then the phosphorus oxychloride group is esterified with choline, and finally the remaining phosphorus oxychloride group is hydrolyzed to introduce a PC group at the terminal hydroxyl position to obtain the intermediate, wherein the material molar ratio of the raw material: the phosphorus oxychloride: the choline is 1:10:

5.

5. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 3, characterized in that, The commercial or self-made raw materials including terminal mercapto protecting group and terminal hydroxyl group in S21, which introduces PC group to obtain an intermediate, are prepared by the following method: The commercial or self-made raw materials are first reacted with a cyclic phosphorus oxyhalogenated reagent to introduce a cyclic phosphate group to obtain an intermediate 1, and then the intermediate 1 is subjected to ring-opening substitution reaction with trimethylamine to construct a PC group to obtain the intermediate, wherein the material molar ratio of the raw material: the cyclic phosphorus oxychloride is 1:1, the material molar ratio of the intermediate 1: the trimethylamine is 1:1.05~1.1, and Y in the cyclic phosphorus oxyhalogenated reagent is selected from chlorine and bromine atoms.

6. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 1, characterized in that, The replacement of the stabilizing molecules on the surface of the gold nanomaterial with modified molecules under the action of a promoter in S3 to obtain a surface modified material intermediate comprises the following steps: The surface stabilizing molecules of the commercially purchased or self-made gold nanomaterials are replaced by the modification molecules under the action of the promoter to form S-Au bonds, and a surface modification material intermediate is obtained, wherein 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, the amount is 50-150 uL, and the concentration is 10-100 mM, the amount of the modification molecules is 50-100 ul, and the concentration is 10-20 mM.

7. The method for preparing the sialic acid-gold nanomaterial composite as described in claim 1, characterized in that, In S4, S4: the surface carboxyl groups of the surface modification material intermediate are activated, and the PC molecules and the sialic acid ligand functional molecules are reacted to be connected by amide bonds, including the following steps: The surface carboxyl groups of the surface modification material intermediate are activated by an activator, and then amide condensation reactions of the PC molecules and the sialic acid ligand functional molecules occur simultaneously to obtain the sialic acid-gold nanomaterial complex, wherein the activator is a combination of N-hydroxysulfosuccinimide (Sulfo-NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCl) aqueous solution, the material molar ratio of Sulfo-NHS:EDCl is 1:1.5-3, the material molar ratio of Sulfo-NHS:the modification molecules is 0.8-1.5:1, the composition ratio of the PC molecules:the sialic acid ligand functional molecules is 99:1-50:50, and the material molar ratio of (PC molecules+ sialic acid ligand functional molecules):Sulfo-NHS is 1-1.2:

1.

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

9. The sialic acid-gold nanomaterial complex of claim 8, wherein, The proportion of PC groups:sialic acid groups is 99%:1%-50%:50%.

10. Use of the sialic acid-gold nanomaterial complex in claim 8 in the preparation of a new coronavirus detection tool.

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