O-GlcNAc deglycosylation inducer as well as preparation method and application thereof

Abstract

The invention provides an O-GlcNAc deglycosylation inducer as well as a preparation method and application thereof. The O-GlcNAc deglycosylation inducer comprises a gold nanocluster and polypeptide modified on the surface of the gold nanocluster, the polypeptide modified on the surface of the gold nanocluster comprises a polypeptide targeting a target protein and a polypeptide targeting O-connected N-acetyl glucosamine hydrolase; the target protein comprises any one or a combination of at least two of P53, HER2 or XIAP. According to the O-GlcNAc deglycosylation inducer provided by the invention, polypeptide can be effectively delivered into cells, so that the off-target effect of a traditional small molecule drug is overcome; the O-GlcNAc deglycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells is realized, and the O-GlcNAc deglycosylation modification shows remarkable timeliness and controllability in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and particularly relates to an O-GlcNAc deglycosylation inducer, a preparation method thereof, and an application thereof. Background Art

[0002] Protein translational modifications (PTMs) introduce structural changes into proteins by performing certain chemical modifications on existing proteins, thereby regulating the functions of proteins. There are various types of PTMs, including glycosylation, ubiquitination, phosphorylation, acetylation, etc., and these modifications all play important roles in various physiological activities of living organisms. Among them, protein glycosylation modification is one of the most important PTMs. The O-GlcNAc glycosylation modification of proteins is jointly regulated by O-linked N-acetylglucosamine transferase (OGT) and O-linked N-acetylglucosaminidase (OGA), and is a reversible process. The disruption of O-GlcNAc homeostasis will trigger various human diseases. Controlling the O-linked N-acetylglucosaminidase to specifically regulate the function of target proteins provides a new idea for disease treatment.

[0003] Heterobifunctional small molecules use a linker as a connecting body, which can bind to endogenous effectors and target proteins simultaneously, enabling two proteins to approach each other and play a role. However, small molecule drugs have defects such as off-target effects, low permeability, and poor pharmacokinetics, which are the biggest obstacles to the clinical application of small molecule drugs. At present, the research on O-GlcNAc deglycosylation still remains at the level of in vitro verification of the effect of O-GlcNAc deglycosylation using tagged proteins, lacking in vivo verification of its specific targeting ability in complex biological environments.

[0004] Based on this, it is necessary to develop an O-GlcNAc deglycosylation inducer to achieve specific targeted therapy in vivo. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an O-GlcNAc deglycosylation inducer, a preparation method thereof, and an application thereof. The inducer provided by the present invention can effectively deliver polypeptides into cells, overcome the off-target effects of traditional small molecule drugs, and achieve rapid and efficient regulation of O-GlcNAc deglycosylation of target proteins.

[0006] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an O-GlcNAc deglycosylation inducer, which comprises gold nanoclusters and a polypeptide modified on the surface of the gold nanoclusters; the polypeptide modified on the surface of the gold nanoclusters comprises a polypeptide targeting a target protein and a polypeptide targeting O-linked N-acetylglucosaminidase; the target protein comprises any one or a combination of at least two of P53, HER2 or XIAP.

[0008] The O-GlcNAc deglycosylation inducer provided by the present invention is based on the concept of a heterobifunctional small molecule drug. Polypeptides with high affinity for the target protein and OGA respectively are connected through gold nanoclusters, which can effectively deliver the polypeptides into cells, overcoming the off-target effect of traditional small molecule drugs; realizing O-GlcNAc deglycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells, showing significant timeliness and controllability in vivo.

[0009] Preferably, the molar ratio of the gold nanoclusters to the polypeptide modified on the surface of the gold nanoclusters is (0.5 - 2):(1 - 4).

[0010] Specific point values in (0.5 - 2) can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.; specific point values in (1 - 4) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc.

[0011] Preferably, the molar ratio of the polypeptide targeting the target protein to the polypeptide targeting O-linked N-acetylglucosaminidase is (1 - 4):(1 - 4).

[0012] Specific point values in the first (1 - 4) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc.; specific point values in the second (1 - 4) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc.

[0013] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0014] Preferably, the amino acid sequence of the polypeptide targeting the target protein comprises any one or a combination of at least two of the sequences shown in SEQ ID No.1 - SEQ ID No.3.

[0015] SEQ ID No.1: CREDEDEIEW。

[0016] SEQ ID No.2: LTVSPWYC。

[0017] SEQ ID No.3: AVPIAQKC。

[0018] In the present invention, the sequence shown in SEQ ID No.1 can specifically target the target protein P53, the sequence shown in SEQ ID No.2 can specifically target the target protein HER2, and the sequence shown in SEQ ID No.3 can specifically target the target protein XIAP.

[0019] The O-GlcNAc deglycosylation inducer provided by the present invention can simultaneously achieve O-GlcNAc deglycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells.

[0020] Among them, the O-GlcNAc deglycosylation modification of P53 will promote the proliferation of tumor cells, the O-GlcNAc deglycosylation modification of HER2 will inhibit the proliferation of tumor cells, and the O-GlcNAc deglycosylation modification of XIAP will promote the proliferation of tumor cells.

[0021] Preferably, the amino acid sequence of the polypeptide targeting O-linked N-acetylglucosaminidase includes the sequence shown in SEQ ID No.4.

[0022] SEQ ID No.4: VSSNISEDPVPVC。

[0023] The present invention uses peptide microarray technology to screen out the polypeptide sequence SEQ ID No.4 that can bind to OGA with high affinity and has O-GlcNAc deglycosylation function, which can stably bind to OGA in vivo and activate its hydrolase activity, thereby achieving O-GlcNAc deglycosylation of the target protein.

[0024] In a second aspect, the present invention provides a preparation method of the O-GlcNAc deglycosylation inducer as described in the first aspect, and the preparation method includes:

[0025] (1) Dispersing gold nanoclusters in deionized water to prepare an aqueous solution of gold nanoclusters;

[0026] (2) Mixing the aqueous solution of gold nanoclusters with the polypeptide to obtain the O-GlcNAc deglycosylation inducer.

[0027] The synthesis route of the O-GlcNAc deglycosylation inducer provided by the present invention is simple and the synthesis efficiency is high.

[0028] Preferably, the temperature of the mixing in step (2) is 34 - 40 °C, for example, it can be 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, 40 °C, etc.; the mixing time is 12 - 24 h, for example, it can be 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, etc.

[0029] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0030] Preferably, the preparation method of the gold nanoclusters includes:

[0031] (1) Mix chloroauric acid, glutathione and deionized water to obtain a reaction solution;

[0032] (2) Mix the reaction solution with isopropanol and centrifuge to obtain the gold nanoclusters.

[0033] Preferably, the molar ratio of chloroauric acid to glutathione is (2.5 - 3.5):(1.5 - 2.5).

[0034] The specific point values in (2.5 - 3.5) can be 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, etc.; the specific point values in (1.5 - 2.5) can be 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0035] Preferably, the temperature of the mixing in step (1) is 65 - 75 °C, for example, it can be 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, etc.; the mixing time is 16 - 26 h, for example, it can be 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, etc.

[0036] Preferably, the volume ratio of the reaction solution to isopropanol is (1 - 1.5):(0.8 - 1.2).

[0037] The specific point values in (1 - 1.5) can be 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, etc.; the specific point values in (0.8 - 1.2) can be 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, etc.

[0038] Preferably, the rotation speed of the centrifugation is 12,000 rpm - 14,000 rpm, and for example, it can be 12,000 rpm, 12,500 rpm, 13,000 rpm, 13,500 rpm, 14,000 rpm, etc.

[0039] Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0040] In a third aspect, the present invention provides the use of the O-GlcNAc deglycosylation inducer as described in the first aspect in the preparation of a tumor regulation reagent.

[0041] The use of the O-GlcNAc deglycosylation inducer of the present invention in the preparation of a tumor regulation reagent includes the preparation of a tumor treatment reagent or related reagents in the basic research of O-GlcNAc glycosylation modification, such as a tumor model for constructing O-GlcNAc deglycosylation of a target protein.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The O-GlcNAc deglycosylation inducer provided by the present invention is based on the concept of a heterobifunctional small molecule drug. Polypeptides with high affinity for a target protein and OGA respectively are connected through gold nanoclusters, and the polypeptide can be effectively delivered into cells, overcoming the off-target effect of traditional small molecule drugs; realizing O-GlcNAc glycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells, showing significant timeliness and controllability in vivo. Description of the Drawings

[0044] Figure 1 TEM morphology diagram of the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0045] Figure 2 Particle size distribution diagram of the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0046] Figure 3 Zeta potential diagram of the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0047] Figure 4 Immunoblotting diagram of the O-GlcNAc deglycosylation of the target protein P53 by the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 in tumor cells HCT116;

[0048] Figure 5Immunoblotting image of the O-GlcNAc deglycosylation inducer H-OAnGNPs prepared in Example 2 for deglycosylation of the target protein HER2 in tumor cells SKOV3;

[0049] Figure 6 Immunoblotting image of the O-GlcNAc deglycosylation inducer X-OAnGNPs prepared in Example 3 for deglycosylation of the target protein XIAP in tumor cells HCT116;

[0050] Figure 7 Immunoblotting image of the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 for deglycosylation of the target protein P53 in tumor cells SKBR3;

[0051] Figure 8 Cell viability curve of tumor cells HCT116 after treatment with the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0052] Figure 9 Cell viability curve of tumor cells SKOV3 after treatment with the O-GlcNAc deglycosylation inducer H-OAnGNPs prepared in Example 2;

[0053] Figure 10 Curve graph of tumor volume change in HCT116 tumor-bearing mice after injection with the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0054] Figure 11 Tumor image of HCT116 tumor-bearing mice on the 28th day after injection with the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1;

[0055] Figure 12 HE staining images of major organs and tumors of HCT116 tumor-bearing mice on the 28th day after injection with the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1. Detailed implementation manners

[0056] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solutions of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0057] For those not specifying specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0058] In the following examples, the polypeptide targeting O-linked N-acetylglucosamine hydrolase (OGA) was screened by peptide microarray technology to identify polypeptides with strong affinity for OGA. Briefly, first, a peptide library consisting of 6912 peptides was immobilized on a biochip by amino coupling to prepare a peptide microarray chip for subsequent experiments; the peptide microarray was printed, with each glass slide containing two identical array replicates, and each spot in the microarray represented an individual peptide; after the chip was fabricated, it was blocked with 10% BSA solution at 30 °C for 30 minutes and then washed three times; the Cy5-labeled OGA protein solution (1 μg / mL) was evenly distributed on the chip surface and incubated in the dark at 30 °C for 1 hour, and then the chip was washed three times with PBS buffer containing 0.05% Tween-20 to remove unbound OGA protein; after air drying, the glass slide was scanned using an ArrayWoRx biochip reader (Applied Precision), and the binding ability of the peptide to the OGA protein was determined by measuring the fluorescence intensity of the peptide spots, thereby identifying the targeting peptide VSSNISEDPVPV. To link the gold nanoclusters, cysteine (C) was introduced at the valine (V) terminus, and the sulfhydryl group of cysteine could form an Au-S chemical bond with the gold nanoclusters.

[0059] Example 1

[0060] This example provides an O-GlcNAc deglycosylation inducer, and its preparation method is as follows:

[0061] (1) Preparation of gold nanoclusters: 300 μL of aqueous chloroauric acid solution (50 mM) and 200 μL of aqueous glutathione solution (50 mM) were added to 3 mL of deionized water, and the mixture was stirred and reacted at 70 °C for 24 h; 3 mL of isopropanol was added, mixed evenly, and centrifuged at 13000 rpm for 10 min to obtain the gold nanoclusters;

[0062] (2) The obtained gold nanoclusters were dispersed in 3 mL of deionized water, and the polypeptide CREDEDEIEW targeting P53 (1.5 μmol) and the polypeptide VSSNISEDPVPVC targeting OGA (1.5 μmol) were added, and the mixture was stirred thoroughly at 37 °C for 18 h to obtain the O-GlcNAc deglycosylation inducer P-OAnGNPs.

[0063] Example 2

[0064] This example provides an O-GlcNAc deglycosylation inducer, and its preparation method is as follows:

[0065] (1) Preparation of gold nanoclusters: 250 μL of aqueous chloroauric acid solution (50 mM) and 150 μL of aqueous glutathione solution (50 mM) were added to 3 mL of deionized water, and the mixture was stirred at 65 °C for 26 h; 2.4 mL of isopropanol was added, mixed well, and centrifuged at 12000 rpm for 10 min to obtain the gold nanoclusters;

[0066] (2) The obtained gold nanoclusters were dispersed in 3 mL of deionized water, and the polypeptide LTVSPWYC (2 μmol) targeting HER2 and the polypeptide VSSNISEDPVPVC (1 μmol) targeting OGA were added, and the mixture was stirred thoroughly at 40 °C for 12 h to obtain the O-GlcNAc deglycosylation inducer H-OAnGNPs.

[0067] Example 3

[0068] This example provides an O-GlcNAc deglycosylation inducer, and its preparation method is as follows:

[0069] (1) Preparation of gold nanoclusters: 350 μL of aqueous chloroauric acid solution (50 mM) and 250 μL of aqueous glutathione solution (50 mM) were added to 3 mL of deionized water, and the mixture was stirred at 75 °C for 16 h; 3.6 mL of isopropanol was added, mixed well, and centrifuged at 14000 rpm for 10 min to obtain the gold nanoclusters;

[0070] (2) The obtained gold nanoclusters were dispersed in 3 mL of deionized water, and the polypeptide AVPIAQKC (1 μmol) targeting XIAP and the polypeptide VSSNISEDPVPVC (2 μmol) targeting OGA were added, and the mixture was stirred thoroughly at 34 °C for 24 h to obtain the O-GlcNAc deglycosylation inducer X-OAnGNPs.

[0071] Test Example 1

[0072] The O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 was characterized for its physicochemical properties: (1) Morphology observation: Transmission electron microscopy (TEM) was used to observe its morphology and record the size. The test results are as Figure 1 and Figure 2 shown. The particle size of the O-GlcNAc deglycosylation inducer provided by the present invention is 1-3 nm; (2) Zeta potential: The Zeta potential was detected using a dynamic light scattering instrument (DLS). The test results are as Figure 3 shown.

[0073] Test Example 2

[0074] The immunoblotting experiment was carried out on the O-GlcNAc deglycosylation inducers prepared in Examples 1-3 to detect the O-GlcNAc deglycosylation modification effect of the target protein:

[0075] ① The tumor cells HCT116 were cultured in a medium containing the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 for 24 h; ② The tumor cells SKOV3 were cultured in a medium containing the O-GlcNAc deglycosylation inducer H-OAnGNPs prepared in Example 2 for 24 h; ③ The tumor cells HCT116 were cultured in a medium containing the O-GlcNAc deglycosylation inducer X-OAnGNPs prepared in Example 3 for 24 h; ④ The tumor cells SKBR3 were cultured in a medium containing the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 for 24 h;

[0076] Collect the above-mentioned tumor cells, wash them three times with PBS buffer, lyse the cells with RIPA lysis buffer supplemented with 1% protease inhibitor (phenylmethylsulfonyl fluoride PMSF), measure the total protein concentration using a BCA kit, enrich the target protein with magnetic beads, perform SDS-PAGE electrophoresis, transfer to a PVDF membrane, incubate with the corresponding antibodies respectively, and finally develop color by chemiluminescence method to detect the O-GlcNAc deglycosylation effect of the target protein;

[0077] The test results are as Figures 4 - 7 shown. The O-GlcNAc deglycosylation inducer provided by the present invention can effectively deliver polypeptides into cells, realizing the O-GlcNAc deglycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells.

[0078] Test Example 3

[0079] The cytotoxicity test of the O-GlcNAc deglycosylation inducers prepared in Examples 1-2 on in vitro tumor cells was carried out: The tumor cells HCT116 and SKOV3 were respectively inoculated in 96-well plates. After culturing for 24 hours, the cells were treated with different concentrations of the O-GlcNAc deglycosylation inducer and cultured for another 24 hours; Remove the original medium and wash it once with PBS buffer. Add 100 μL / well of CCK-8 working solution (the volume ratio of CCK-8 to the medium is 1:10) to the 96-well plates, and incubate the cells in an incubator for 2 hours. After the incubation, measure the absorbance at 450 nm using a microplate reader;

[0080] The test results are as Figure 8 and Figure 9As shown, the O-GlcNAc deglycosylation inducer provided by the present invention can effectively deliver polypeptides into cells to achieve O-GlcNAc deglycosylation modification of target proteins. O-GlcNAc deglycosylation modification of P53 promotes the proliferation of tumor cells, while O-GlcNAc deglycosylation modification of HER2 inhibits the proliferation of tumor cells.

[0081] Test Example 4

[0082] The effect of the O-GlcNAc deglycosylation inducer P-OAnGNPs prepared in Example 1 in animals was tested: Female BALB / c nude mice were subcutaneously injected with HCT116 tumor cells (5×10 6 cells per mouse) to establish an HCT116 tumor-bearing mouse model; P-OAnGNPs were injected into the tail vein three times (injection dose: 30 mg / kg), with a three-day interval between each injection. During this period, the body weight and tumor volume of the mice were recorded every other day. After 28 days, all the mice were sacrificed, and the tumors and organs were collected for further analysis;

[0083] The changes in the tumor volume of HCT116 tumor-bearing mice treated with P-OAnGNPs are as Figure 10 shown, and the tumor images collected on the 28th day are as Figure 11 shown. O-GlcNAc deglycosylation modification of P53 promotes the proliferation of tumor cells; the HE staining of the main organs and tumors collected on the 28th day is as Figure 12 shown, indicating that the O-GlcNAc deglycosylation inducer provided by the present invention has excellent biosafety.

[0084] In summary, the O-GlcNAc deglycosylation inducer provided by the present invention can effectively regulate the O-GlcNAc deglycosylation modification of target proteins P53, HER2, and XIAP, achieving O-GlcNAc deglycosylation modification of different target proteins in the same type of cells or the same target protein in different types of cells, and showing significant timeliness and controllability in vivo.

[0085] The applicant declares that the present invention uses the above embodiments to illustrate an O-GlcNAc deglycosylation inducer and its preparation method and application, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0086] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0087] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. An O-GlcNAc deglycosylation inducer, characterized in that The O-GlcNAc deglycosylation inducer includes gold nanoclusters and polypeptides modified on the surface of the gold nanoclusters; the polypeptides modified on the surface of the gold nanoclusters include polypeptides targeting target proteins and polypeptides targeting O-linked N-acetylglucosamine hydrolases; the target proteins include any one of P53, HER2 or XIAP or a combination of at least two of them.

2. The O-GlcNAc deglycosylation inducer according to claim 1, characterized in that The molar ratio of the gold nanoclusters to the polypeptide modified on the surface of the gold nanoclusters is (0.5-2):(1-4); Preferably, the molar ratio of the polypeptide targeting the target protein to the polypeptide targeting the O-linked N-acetylglucosamine hydrolase is (1-4):(1-4).

3. The O-GlcNAc deglycosylation inducer according to claim 1 or 2, characterized in that The amino acid sequence of the polypeptide targeting the target protein includes any one or a combination of at least two of the sequences shown in SEQ ID No.1 to SEQ ID No.3; Preferably, the amino acid sequence of the polypeptide targeting O-linked N-acetylglucosamine hydrolase comprises the sequence shown in SEQ ID No.

4.

4. The method for preparing the O-GlcNAc deglycosylation inducer according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) dispersing gold nanoclusters in deionized water to prepare a gold nanocluster aqueous solution; (2) Mixing the gold nanocluster aqueous solution with the polypeptide to obtain the O-GlcNAc deglycosylation inducer.

5. The preparation method according to claim 4, characterized in that: The mixing temperature in step (2) is 34-40° C., and the mixing time is 12-24 h.

6. The preparation method according to claim 4, characterized in that: The preparation method of the gold nanoclusters comprises: (1) mixing tetrachloroauric acid, glutathione and deionized water to obtain a reaction solution; (2) Mixing the reaction solution with isopropanol and centrifuging to obtain the gold nanoclusters.

7. The preparation method according to claim 6, characterized in that: The molar ratio of tetrachloroauric acid to glutathione is (2.5-3.5):(1.5-2.5).

8. The preparation method according to claim 6, characterized in that: The mixing temperature in step (1) is 65-75° C., and the mixing time is 16-26 h.

9. The preparation method according to claim 6, characterized in that: The volume ratio of the reaction solution to isopropanol is (1-1.5):(0.8-1.2); Preferably, the centrifugal rotation speed is 12000rpm-14000rpm.

10. Use of the O-GlcNAc deglycosylation inducer according to any one of claims 1 to 3 in the preparation of a tumor regulating agent.

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