Temperature-sensitive probes, methods of making and using the same to label core fucosylation and / or o-glcnaclylation

Abstract

The present application relates to a kind of temperature-sensitive probe, its preparation method and the method for marking core fucosylation and / or O-GlcNAc glycosylation, the present application provides a kind of chemical enzyme method marking method using new temperature-sensitive probe of formula 1, including: using sugar chain probe compound, and the ENGase mutant with the selectivity of core fucose and / or the ENGase mutant with the selectivity of O-GlcNAc are labeled, enrichment is carried out using temperature-sensitive probe shown in formula 1, and is released using the ENGase with the selectivity of core fucose and / or the ENGase with the selectivity of O-GlcNAc again.The method can simultaneously realize the labeling of two glycosylations, can be used in the research of core fucosylation and O-GlcNAc glycosylation at glycopeptide level, protein level, etc., provides effective means for the site research of core fucosylation and O-GlcNAc glycosylation.

Description

Technical Field

[0001] The present invention belongs to the technical field of glycosylation labeling, and in particular relates to a temperature-sensitive probe, a preparation method thereof, and a method for labeling core fucosylation and / or O-GlcNAc glycosylation using the same. Background Art

[0002] Glycosylation is a common post-translational modification of proteins. Common protein glycosylation modifications include N-glycosylation, O-glycosylation, oxygen-linked N-acetylglucosamine (O-GlcNAc) glycosylation, etc. Glycosylation, like other post-translational modifications of proteins, regulates protein stability and function, and participates in many physiological and pathological processes.

[0003] Core-fucosylation refers to a modification in which fucose is linked to the innermost GlcNAc of an N-glycan in the form of an α1,6 glycosidic bond. As one of the most important N-glycosylation modifications, core-fucosylation is gaining increasing attention, and many studies have shown its association with numerous physiological and pathological processes. Abnormal expression of core-fucosylation on the cell surface is common in liver cancer, colorectal cancer, ovarian cancer, prostate cancer, and breast cancer. Specific core-fucosylation of certain glycoproteins is a more reliable cancer biomarker than total protein expression levels. For example, alpha-fetoprotein (AFP) is an FDA-approved biomarker for hepatocellular carcinoma (HCC), but it is also associated with benign liver diseases. Elevated levels of core-fucosylated AFP are also a more specific marker for HCC. Studies have shown that core fucose promotes tumor growth, invasion and metastasis by regulating a variety of cell surface growth factors, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and transforming growth factor-β receptor (TGF-βR). In addition, core fucosylation plays an important role in regulating the immune system. Core fucosylation of the Fc segment of IgG inhibits antibody-dependent cellular cytotoxicity (ADCC). Compared with patients with mild symptoms, patients with severe COVID-19 have more core fucose-deficient IgG in their plasma, which amplifies the release of pro-inflammatory cytokines and acute phase responses. This shows that core fucosylation is a very important glycosylation modification.

[0004] O-GlcNAcylation is one of the most common glycosylation modifications. It involves the attachment of N-acetylglucose (GlcNAc) to serine or threonine residues on proteins via a glycosidic bond. O-GlcNAcylation consists of a monosaccharide structure and is not elongated or modified into more complex glycan structures. It is primarily found in the cytoplasm and nucleus, making it a highly dynamic protein modification. The intracellular enzymes O-GlcNAcylation transferase (OGT) and O-GlcNAcase (OGA) catalyze the addition or removal of O-GlcNAcylation from target proteins, regulating the level of O-GlcNAcylation. O-GlcNAcylation participates in numerous important biological processes. Numerous studies have shown that O-GlcNAcylation is associated with transcription and epigenetic inheritance. O-GlcNAcylation is a key regulator of gene expression by influencing higher-order chromatin structure, transcription, and the regulation of RNA polymerase II. O-GlcNAcylation modifies four histones, competing with or promoting phosphorylation or ubiquitination, thereby influencing transcription and epigenetic inheritance. Many other cellular processes are affected by O-GlcNAc, such as the cell cycle, apoptosis, and stress response. In summary, O-GlcNAc glycosylation is a very important glycosylation, and therefore its research is of great significance.

[0005] To further understand glycosylation modifications, researchers are continuously developing methods for labeling and enriching glycosylated proteins. Currently, the most commonly used methods include antibodies, lectins, chemoenzymatic labeling, and metabolic labeling. Antibodies have low affinity. Lectins are glycoproteins or sugar-binding proteins extracted from plants, invertebrates, and higher animals. They are named lectins because they can bind to red blood cells, but they have poor specificity. Metabolic labeling involves chemically labeled non-natural monosaccharide precursors that are metabolized in cells. Through normal cellular metabolism, the non-natural monosaccharides replace normal monosaccharides in sugar chain synthesis, thereby labeling glycosylation sites. Once entering the cell, non-natural monosaccharides enter different metabolic pathways, resulting in low selectivity. Chemoenzymatic labeling involves the use of glycosyltransferases or other enzymes with glycosyltransferase properties to modify target oligosaccharide determinants with non-natural sugar molecules containing bioorthogonal reactive groups, natural sugar molecules, or molecules directly carrying detectable or enrichable molecules, in an in vitro reaction catalyzed by glycosyltransferases, enabling qualitative and quantitative analysis of target oligosaccharides. Methods for studying O-GlcNAc glycosylation include antibodies (RL21, CTD110.6, etc.), lectins (wheat germ agglutinin WAG, castor bean agglutinin RCA-I, Agrocybe aegerita agglutinin AANLs, etc.), metabolic labeling (sugar analogs used: tetraacetylated N-azidoacetylglucosamine, tetraacetylated N-azidoacetylgalactosamine, etc.), and chemoenzymatic labeling (using a mutant of the classic bovine milk galactosyltransferase GalT1 (GalT1-Y289L) and UDP-GalNAz, combined with a cleavable tag). Core fucosylation research methods primarily involve lectin binding (lentil lectin LCA, pea lectin PSA, coleopteran lectin PhoSL, Aspergillus oryzae lectin AOL, Dictyosporum aurantium aurantium agglutinin AAL, etc.) and chemoenzymatic labeling (endoglycosidase EndoF3 and its mutant EndoF3-D165A). Summary of the Invention

[0006] This study establishes a strategy for reversibly labeling two types of glycosylation (core fucosylation and O-GlcNAc glycosylation) using a chemoenzymatic method and a temperature-sensitive probe in the same system. This method, followed by reversible release of core fucosylated and O-GlcNAc glycosylated peptides or proteins, can be directly used for mass spectrometry and proteomic analysis, providing an effective means for discovering new targets and sites for core fucosylation and O-GlcNAc glycosylation.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] In a first aspect, the present invention provides a temperature-sensitive probe as shown in Formula 1,

[0009]

[0010] Wherein, n is an integer from 17 to 90, for example, n is 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 87, 90, but is not limited thereto. Preferably, n is 87.

[0011] In a second aspect, the present invention provides a method for preparing a temperature-sensitive probe represented by Formula 1, comprising the following steps:

[0012]

[0013] ⅰ Compound a and tert-butyl (3-aminopropyl) carbamate are condensed in the presence of a condensing agent and a solvent, diluted, washed, and rotary evaporated to obtain a crude product;

[0014] ii. The crude product was redissolved, purified by silica gel column, and dried to obtain compound b;

[0015] iii. Compound b and the carboxylic acid-terminated poly (N-isopropylacrylamide) of Formula 3 are subjected to a condensation reaction in the presence of a condensing agent and a solvent, diluted, purified by dialysis, concentrated to obtain a crude product, and then purified by gel column and dried to obtain a temperature-sensitive probe shown in Formula 1.

[0016]

[0017] In Formula 3, n is an integer from 17 to 90, for example, n is 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 87, or 90, but is not limited thereto. Preferably, n is 87.

[0018] In some embodiments, in step i, the condensing agent is O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA); and / or

[0019] The solvent is selected from one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and acetonitrile (MeCN).

[0020] In some embodiments, in step i, the reaction molar ratio of compound a to tert-butyl (3-aminopropyl) carbamate is 1:1-1:5; and / or

[0021] The reaction temperature is room temperature and the reaction time is 4-8h.

[0022] In some embodiments, in step iii, the condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA); and / or

[0023] The solvent is selected from one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and acetonitrile (MeCN).

[0024] In some embodiments, in step iii, the molar ratio of carboxylic acid-terminated poly(N-isopropylacrylamide) and compound b is 1:1-1:5; and / or

[0025] The reaction temperature is room temperature, and the reaction time is 4-8 h.

[0026] In some specific embodiments, the method for preparing the temperature-sensitive probe represented by formula 1 comprises the following steps:

[0027] i. Compound a (DBCO-acid), tert-butyl (3-aminopropyl)carbamic acid tert-butyl ester, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) are dissolved in N,N-dimethylformamide (DMF) and stirred at room temperature under nitrogen for 4 h. Dichloromethane is added to dilute the reaction, and the mixture is washed twice with saturated brine. Rotary evaporation is performed to obtain a crude product;

[0028] ii. The obtained crude product is dissolved in 50% trifluoroacetic acid in dichloromethane, and stirred at room temperature for 1 h. Silica gel column purification is performed, and the product is evaporated to obtain compound b as a black oil;

[0029] iii. Compound b and carboxylic acid-terminated poly(N-isopropylacrylamide) (PNIPAM) dendrimer (n is an integer from 17 to 90) are dissolved in N,N-dimethylformamide (DMF), and HATU and DIPEA are added. Stirring is performed at room temperature under nitrogen for 8 h. The reaction solution is diluted with 1 volume of water, centrifuged to obtain the supernatant, and then purified by dialysis (MWCO 5000). After dialysis, the solution is precipitated, the supernatant is concentrated, and freeze-drying is performed. The obtained crude product is purified by Sephadex G50 column (water), and freeze-drying is performed to obtain the temperature-sensitive probe 1.

[0030] In a third aspect, the present application provides a use of the temperature-sensitive probe represented by formula 1 described above in marking and detecting core fucosylation and / or O-GlcNAc glycosylation in vitro.

[0031] In a fourth aspect, the present application provides a method for marking core fucosylation and / or O-GlcNAc glycosylation, which comprises the following steps: Figure 2As shown, the following steps are included:

[0032] S01, Marking:

[0033] Adding a sugar chain probe compound represented by the following formula 2 and an ENGase mutant selective for core fucose and / or an ENGase mutant selective for O-GlcNAc to a buffer containing a glycosylated peptide segment with core fucosylation and / or O-GlcNAc glycosylation to carry out a reaction;

[0034]

[0035] Wherein, the ENGase mutant selective for core fucose is selected from one or more of EndoF3-D165A and EndoF2-D124A;

[0036] The ENGase mutant selective for O-GlcNAc is selected from one or more of EndoCC-N180H and EndoM-N75Q;

[0037] Preferably, the reaction temperature is 25-37°C and the reaction time is 1-3h;

[0038] S02, Enrichment:

[0039] Adding the temperature-sensitive probe shown in Formula 1 to the labeled system to react, removing the unlabeled miscellaneous peptide segments, and obtaining a solid;

[0040] Preferably, the reaction temperature is 20-30°C and the reaction time is 1-3h;

[0041] Preferably, removing the unlabeled miscellaneous peptide segments comprises: adding sodium chloride, heating for a period of time to produce a precipitate, discarding the supernatant, adding water to redissolve the precipitate on ice, continuing to add sodium chloride, heating for a period of time to produce a precipitate, discarding the supernatant, and repeating the process;

[0042] Preferably, the heating temperature for removing unlabeled miscellaneous peptide segments is 40-60° C., and the heating time is 5-10 min;

[0043] S03, Release:

[0044] The washed solid is dissolved in a buffer solution, and an ENGase selective for core fucose and / or an ENGase selective for O-GlcNAc is added to react. After the reaction is completed, sodium chloride is added, and the mixture is heated for a period of time and then separated to obtain the supernatant, thereby obtaining the labeled peptide fragments.

[0045] Wherein, the ENGase selective for core fucose is selected from one or more of EndoF3 and EndoF2;

[0046] The ENGase selective for O-GlcNAc is selected from one or more of EndoCC and EndoM;

[0047] Preferably, the reaction temperature is 4-30°C and the reaction time is 3-12h;

[0048] Preferably, the heating temperature is 40-60° C., and the heating time is 5-10 min.

[0049] In some embodiments, the method for labeling core fucosylation and / or O-GlcNAc glycosylation comprises the following steps:

[0050] Labeling: To a PBS (200 mM, pH 6.5-8.0, preferably pH 7.5) buffer solution containing two glycosylated peptides, core fucosylation and / or O-GlcNAc glycosylation, add sugar chain probe compound 2 (in portions or all at once), an ENGase mutant selective for core fucose (EndoF3-D165A or EndoF2-D124A, preferably EndoF3-D165A, with a final concentration of 0.05 mg / mL to 1 mg / mL, preferably 0.2 mg / mL) and / or an ENGase mutant selective for O-GlcNAc (EndoCC-N180H or EndoM-N75Q, preferably EndoCC-N180H, with a final concentration of 0.05 mg / mL to 1 mg / mL, preferably 0.2 mg / mL), and react at a suitable temperature (25°C-37°C, preferably 30°C) for 1 to 3 h (preferably 3 h).

[0051] Among them, EndoF3-D165A, EndoF2-D124A, EndoCC-N180H, and EndoM-N75Q are all β-N-acetylglucosaminylase (ENGase) mutants. EndoF3-D165A and EndoF2-D124A are derived from Elizabethkingiameningoseptica and are selective for core fucose, EndoCC-N180H is derived from Coprinopsis cinerea, and EndoM-N75Q is derived from Mucor hiemalis and is selective for O-GlcNAc.

[0052]

[0053] Enrichment: Add a thermosensitive probe (compound of formula 1, wherein n is 87) to the completed labeled system at a final concentration of 75 mg / mL. React at room temperature for 1 to 3 hours (preferably 1 hour). After the reaction is completed, add sodium chloride with a final concentration of 1M. Heat at 40 to 60°C (preferably 40°C) for 5 to 10 minutes (preferably 5 minutes). A clear white precipitate is visible. Centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and dissolve the precipitate in water on ice. Add sodium chloride with a final concentration of 1M, heat at 40 to 60°C (preferably 40°C) for 5 to 10 minutes (preferably 5 minutes), centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant. This process is called washing, the purpose of which is to remove unlabeled miscellaneous peptide segments. Repeat the washing step 3 to 10 times.

[0054] Release: After washing, the solid was dissolved in PBS (100 mM, pH 6.5-8.0, preferably pH 7.0) buffer solution. An ENGase selective for core fucose (EndoF3 or EndoF2, preferably EndoF3, at a final concentration of 0.05 mg / mL to 1 mg / mL, preferably 0.1 mg / mL) and / or an ENGase selective for O-GlcNAc (EndoCC or EndoM, preferably EndoCC, at a final concentration of 0.05 mg / mL to 1 mg / mL, preferably 0.1 mg / mL) were added. The reaction was allowed to proceed at a suitable temperature (4°C-30°C, preferably 25°C) for 3 to 12 hours (preferably 12 hours). After the reaction, sodium chloride was added to a final concentration of 1 M. The mixture was heated at 40 to 60°C (preferably 40°C) for 5 to 10 minutes (preferably 5 minutes). Centrifuged at 10,000 rpm for 5 minutes to obtain the supernatant.

[0055] Among them, EndoF3, EndoF2, EndoCC, and EndoM are all β-N-acetylglucosaminylases (ENGases). EndoF3 and EndoF2 are derived from Elizabethkingia meningoseptica and are selective for core fucose, EndoCC is derived from Coprinopsis cinerea, and EndoM is derived from Mucor hiemalis and is selective for O-GlcNAc.

[0056] The above method can perform reversible chemoenzymatic labeling of two glycosylations (core fucosylation and O-GlcNAc glycosylation) in the same system.

[0057] The above method can also be used for the separate study of core fucose and O-GlcNAc glycosylation, that is, when studying core fucosylation alone, use an enzyme that can specifically recognize core fucosylation (EndoF3-D165A, EndoF2-D124A, EndoF3, EndoF2); when studying O-GlcNAc glycosylation alone, use an enzyme that can specifically recognize O-GlcNAc glycosylation (EndoCC-N180H, EndoM-N75Q, EndoCC, EndoM).

[0058] In a fifth aspect, the present invention provides a method for detecting core fucosylation and / or O-GlcNAc glycosylation in vitro, comprising: labeling according to the above method, and then detecting and analyzing the labeled peptides.

[0059] Analytical methods include but are not limited to liquid chromatography, mass spectrometry, liquid chromatography / mass spectrometry, etc.

[0060] For example, the supernatant obtained in the fourth aspect is subjected to omics analysis using mass spectrometry to identify the target glycosylation modification sites.

[0061] In a sixth aspect, the present invention provides an in vitro detection kit for core fucosylation and / or O-GlcNAc glycosylation, comprising:

[0062] The temperature-sensitive probe represented by formula 1 described above;

[0063] The sugar chain probe compound represented by Formula 2 described above;

[0064] ENGase mutants selective for core fucose and / or ENGase mutants selective for O-GlcNAc;

[0065] ENGase selective for core fucose and / or ENGase selective for O-GlcNAc;

[0066] and buffer,

[0067] Wherein, the ENGase mutant selective for core fucose is selected from one or more of EndoF3-D165A and EndoF2-D124A;

[0068] The ENGase mutant selective for O-GlcNAc is selected from one or more of EndoCC-N180H and EndoM-N75Q;

[0069] The ENGase selective for core fucose is selected from one or more of EndoF3 and EndoF2;

[0070] The ENGase selective for O-GlcNAc is selected from one or more of EndoCC and EndoM;

[0071] The buffer solution includes PBS buffer solution.

[0072] Beneficial effects:

[0073] The present invention provides a novel chemical enzymatic labeling method using temperature-sensitive materials, which can simultaneously achieve labeling of two glycosylation groups and has good selectivity and high labeling efficiency.

[0074] The method of the present invention can be used for the study of core fucosylation and O-GlcNAc glycosylation at the glycopeptide level, protein level, etc., and provides an effective means for the site study of core fucosylation and O-GlcNAc glycosylation.

[0075] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The H NMR of the thermosensitive probe 1 (n=87, Mn 10000) is shown. 1 H NMR) spectrum.

[0077] Figure 2 Schematic diagram of the labeling, enrichment, release and proteomics research strategies of core fucosylated peptides and O-GlcNAc glycosylated peptides. Among them, ■ in the glycosylated peptide represents N-acetylglucosamine, represents fucose.

[0078] Figure 3 The following figure shows the results of labeling, enrichment, and release HPLC detection of core fucosylated standard glycopeptide and O-GlcNAc glycosylated standard glycopeptide, wherein P1 is the core fucosylated standard glycopeptide, P2 is the O-GlcNAc standard peptide, and P3 is the internal reference peptide (SEQ ID NO: 3).

[0079] Figure 4 The chromatogram (A) and mass spectrum (B) of a mixed complex system of core fucosylated standard glycopeptides, O-GlcNAc glycosylated standard glycopeptides and BSA peptides before and after labeling and enrichment were shown. DETAILED DESCRIPTION

[0080] The present invention will be further described below with reference to the examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed in the present invention.

[0081] Unless otherwise indicated, the starting materials, reagents, methods, etc. employed in the examples are of conventional character and are available from commercial sources.

[0082] Materials

[0083] Egg yolk powder was purchased from Anhui Rongda Food Company.

[0084] Thiamet-G (TMG, CAS No. 1009816-48-1), carboxy-terminated poly(N- isopropylacrylamide) (Mn 10000, Cat. No. 724459) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0085] Compound a (DBCO-acid, CAS No. 1353016-70-2) was purchased from Shanghai Biotechmed Co., Ltd.

[0086] tert-Butyl (3-aminopropyl)carbamate (CAS No. 75178-96-0) was purchased from Shaoyuan Technology (Shanghai) Co., Ltd.

[0087] O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS No. 148893-10-1) was purchased from Accela.

[0088] N,N-Diisopropylethylamine (DIPEA; CAS No. 7087-68-5) was purchased from Macklin.

[0089] Methanol (CAS No. 67-56-1), dichloromethane (CAS No. 75-09-2) were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0090] Triethylamine (Et3N, CAS No. 121-44-8), N,N-dimethylformamide (CAS No. 68-12-2), trifluoroacetic acid (CAS No. 76-05-1) were purchased from Biotang.

[0091] Core fucose standard glycopeptide P1 and O-GlcNAc standard glycopeptide P2 were synthesized by GenScript (Shanghai) Co., Ltd.

[0092] RIPA lysis buffer was purchased from Biyun Tian Biological Technology Co., Ltd.

[0093] Sugar chain probe (compound 2) was synthesized in the laboratory. 9-azido sialic acid was synthesized according to the method reported in the known literature (ACS Central Science 2020, 6(3), 382-389.).

[0094] Examples

[0095] Example 1: Enzyme expression and purification

[0096] Enzymes NanA (sialidase from Streptococcus pneumoniae, Uniprot ID: P62576), Pd2,6ST (sialyltransferase from Photobacterium damselae, Uniprot ID: O66375), NmCSS (N-acylneuraminic acid cytidyltransferase from Neisseria meningitidis, Uniprot ID: P0A0Z8), EndoF3 (Uniprot ID: P36913), EndoF3-D165A (Uniprot ID: P36913, amino acid 165 is mutated from aspartic acid to alanine), EndoF2 (Uniprot ID: P36912), EndoF2-D124A (Uniprot ID: P36912, amino acid 124 is mutated from aspartic acid to alanine after removal of the signal peptide (1-45 amino acids), EndoCC ... Expression and purification of EndoCC-N180H (Uniprot ID: A8P7P2, amino acid 180 was mutated from asparagine to histidine), EndoM (Uniprot ID: Q9C1S6), and EndoM-N175Q (Uniprot ID: Q9C1S6, amino acid 175 was mutated from asparagine to glutamine).

[0097] The NanA, Pd2,6ST, NmCSS, EndoF3, EndoF3-D165A, EndoF2, EndoF2-D124A, EndoCC, EndoCC-N180H, EndoM, and EndoM-N175Q genes were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. and ligated into the pET28a vector (kanamycin resistance). Competent cells were transformed, positive clones were selected, and cultured. Escherichia coli expression strain BL21(DE3) (Thermo Fisher Scientific, EC0114) containing the above-mentioned vectors was cultured at 37°C. When the OD600 value was approximately 0.7, 0.1 mM isopropyl-β-D-thiogalactose (IPTG) and 10% glycerol were added and protein expression was induced at 16°C for 30 h. The cells were harvested by centrifugation at 4°C. Bacteria were resuspended in PBS and disrupted by sonication. The supernatant was centrifuged and incubated with gel affinity resin (Ni-NTA agarose, Thermo Fisher Scientific, R90101) at 4°C for 1 hour. The supernatant was washed with 10 mM imidazole in PBS and eluted with 300 mM imidazole in PBS. The imidazole was removed using ultrafiltration tubes (Millipore, UFC9010). 20% glycerol was added to the protein solution in PBS, and the aliquots were frozen. Protein purity and concentration were determined by SDS-PAGE.

[0098] Example 2: Synthesis of Sugar Chain Probe Molecules

[0099] (1) Synthesis of octasaccharide (Compound d)

[0100]

[0101] According to the method reported in the known literature (Nat. Protoc. 2017, 12, 1702–1721.), sialylglycopeptide (SGP, compound c) was extracted from egg yolk powder. SGP was dissolved in PBS solution (pH 6.0), enzymes EndoM and NanA were added, and the reaction was carried out at 30°C for 4 h. The reaction was monitored by thin layer chromatography (TLC, isopropanol:ammonia:water = 7:3:2, v:v:v), and the octasaccharide (compound d) molecule was separated and purified by polyacrylamide gel P-2 (Bio-Rad, #1504118).

[0102] (2) Synthesis of sugar chain probe precursor (compound e)

[0103]

[0104] NmCSS (0.2 mg / mL) and Pd2,6ST (0.1 mg / mL) purified in Example 1 were added to a PBS solution (100 mM, pH 7.5, 10 mM MgCl2, 10 mL) containing compound d (200 mg, 0.14 mmol), cytidine triphosphate disodium (142 mg, 0.3 mmol) and 9-azidosialic acid (67 mg, 0.2 mmol). The reaction was incubated at 37°C and the reaction progress was monitored by TLC (developing solvent: isopropanol:ammonia:water = 7:3:2, v:v:v). After 4 h, the reaction was essentially complete. The supernatant was collected by centrifugation and separated and purified on polyacrylamide gel P-2 to obtain the target sugar chain probe precursor (compound e).

[0105] (3) Synthesis of sugar chain probe (Compound 2)

[0106]

[0107] DMC (245 mg, 1.5 mmol) and Et3N (628 μL, 4.5 mmol) were added to an aqueous solution of compound e (200 mg, 0.01 mmol) and reacted on ice for 0.5 h. The supernatant was centrifuged and purified on polyacrylamide gel P-2 (0.2% Et3N in water). The product fractions were collected and lyophilized with 3 equivalents of NaOH to obtain compound 2.

[0108] Example 3: Synthesis of temperature-sensitive probes

[0109] (1) Synthesis of thermosensitive probe intermediate b

[0110]

[0111] Compound a (305 mg, 1 mmol), tert-butyl(3-aminopropyl)carbamate (348 mg, 2 mmol), HATU (520 mg, 4 mmol), and DIPEA (1 mL, 6 mmol) were added to DMF (5 mL) and stirred under nitrogen for 4 h. 200 mL of dichloromethane was added to the reaction, and the resulting mixture was washed twice with saturated brine. The organic solvent was removed by rotary evaporation. The residue was dissolved in 50% trifluoroacetic acid in dichloromethane, stirred under nitrogen for 1 h, and monitored by TLC (developing system: dichloromethane:methanol = 30:1, v:v). The solvent was removed by rotary evaporation, and the product was purified by silica gel column (dichloromethane:methanol = 10:1) to obtain compound DBCO amine (compound b) as a black oil (280 mg, 76%). 1H NMR (600MHz, CDCl3) δ7.87 (s, 1H), 7.52 (d, J = 7.4Hz, 1H), 7.43-7.38 (m, 1H) ,7.34-7.28(m,3H),7.26-7.10(m,3H),5.00(d,J=14.0Hz,1H),3.90(s,2H), 3.57(d,J=14.0Hz,1H),3.14-2.94(m,2H),2.73(s,2H),2.68-2.52(m,1H), 2.22-2.14(m,1H),2.11-2.04(m,1H),1.89-1.80(m,1H),1.68-1.44(m,2H). 13 C NMR (200MHz, CDCl3) δ173.8,172.8,151.0,148.1,132.1,129.3,128.8,128.5,128.2, 127.9,127.2,125.6,123.1,122.4,114.6,107.8,55.7,37.2,35.9,30.7,30.2,26.9.

[0112] (2) Synthesis of temperature-sensitive probe (compound 1)

[0113] Compound b (36 mg, 0.1 mmol) and poly (N-isopropylacrylamide) (PNIPAM) dendrimer (n = 87, Mn 10000, 500 mg, 0.05 mmol) were dissolved in DMF (10 mL), HATU (76 mg, 0.2 mmol) and DIPEA (50 μL, 0.3 mmol) were added, and stirred under nitrogen for 8 h. The reaction solution was diluted with 1 volume of water, centrifuged and the supernatant was taken, and then dialyzed (MWCO5000) for purification. The precipitate was removed from the dialyzed solution, the supernatant was concentrated and lyophilized, and the crude product was purified with Sephadex G50 (water) and lyophilized to obtain the temperature-sensitive probe (Compound 1). According to the nuclear magnetic resonance spectrum ( Figure 1 ) calculation, the loading efficiency is about 75%.

[0114] Example 4: Reversible labeling of standard peptides

[0115] A glycan probe (Compound 2 shown below) was added to a PBS buffer (200 mM, pH 7.5) containing 0.5 mM standard peptides (core fucose standard glycopeptide P1 and O-GlcNAc standard glycopeptide P2, 0.5 mM each) at a final concentration of 2.5 mM. The reaction was carried out at 30°C for 3 h under the catalysis of EndoCC-N180H (0.2 mg / mL). EndoF3-D165A (0.2 mg / mL) and the glycan probe (2.5 mM) were then added to the reaction, and the reaction was continued at 30°C for 1 h. The reaction was terminated by heating, and HPLC analysis showed that the core fucose standard glycopeptide P1 was completely converted to the azido-core fucosyl peptide P1N, and that over 50% of the O-GlcNAc standard glycopeptide P2 was converted to the azido-O-GlcNAc peptide P2N.

[0116] The azido peptide solution was mixed with 45 mg of the temperature-sensitive probe (compound 1) in PBS buffer (100 mM, pH = 7.5, total volume of 600 μL) and reacted at room temperature for 1 hour. After the reaction, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 40°C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, and the supernatant was analyzed by HPLC to obtain the reduced amount after enrichment of the azido peptide. The precipitate was dissolved in water (600 μL) in an ice bath. Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. Heat at 40°C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes and the precipitate was collected. Repeat the dissolution of the precipitate three times to wash away the unlabeled peptides. The supernatant of the third time was retained for HPLC analysis to ensure that the impurities were washed away.

[0117] The third precipitate was dissolved in PBS buffer (100 mM, pH = 7.0, 600 μL) in an ice bath. Endoglycosidase (EndoF3 and EndoCC, 0.1 mg / mL each) was then added. The reaction was allowed to proceed at room temperature for 12 h. After the reaction was completed, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 40 ° C for 5 min. Centrifuged at 10,000 × g for 5 min, and the supernatant was analyzed by HPLC to obtain the amount of released standard glycopeptides. The enrichment and release process was monitored by reverse phase HPLC at 214 nm, using a linear gradient elution (elution conditions are as follows: A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile) by reverse phase HPLC (Shimadzu, C18 column: Spherisorb, 5 μm, 250 × 4.6 mm).

[0118]

[0119]

[0120] The experimental results are as follows Figure 3As shown in Figure 3, this method can efficiently label, enrich, and release core fucose standard peptides and O-GlcNAc standard peptides. After the complete process, both glycosylated peptides can be effectively enriched, which is beneficial for subsequent research.

[0121] Example 5: Reversible labeling of standard peptides in a temperature-sensitive probe complex system

[0122] (1) BSA peptide acquisition

[0123] Weigh 100 mg of BSA solid and dissolve it in 10 mL of 100 mM NH₄HCO₃ buffer. Add trypsin. Incubate at 37°C for 24 hours, adding trypsin once. After digestion, desalt the BSA peptide using C18 (Sep-Pak tC18) solid-phase extraction and lyophilize. Dissolve the lyophilized solid in ddH₂O, determine the concentration using a BCA protein assay kit, and store at -80°C until use.

[0124] For the C18 solid-phase extraction process described above, add 1 mL of methanol to a Sep-Pak tC18 cartridge for activation. Once no liquid remains above the cartridge packing, add 1 mL of 70% acetonitrile / 0.1% TFA solution for further activation. After activation, add 1 mL of 0.1% TFA for equilibration. Before loading the peptide, acidify with 0.1% TFA and centrifuge to remove precipitates. Keep the sample volume approximately 1 mL. Wash twice with 1 mL of 0.1% TFA. Then, elute with 70% acetonitrile / 0.1% TFA solution. The resulting eluate is lyophilized.

[0125] (2) Reversible labeling of standard glycopeptides in complex systems

[0126] Standard peptides (core fucose standard glycopeptide P1 (SEQ ID NO: 1 (EEQYN (GlcNAc-Fucose) STYR)) and O-GlcNAc standard glycopeptide P2 (SEQ ID NO: 2 (TAPT (GlcNAc) STIAPG)), 20 μg each) were mixed with digested BSA peptide (10 mg) at a ratio of 1:500 (w / w) in PBS buffer (200 mM, pH = 7.5). 2.5 mM of the sugar chain probe (Compound 2) was added to the resulting peptide mixture, and the reaction was carried out at 30°C for 3 hours under the catalysis of EndoCC-N180H (0.2 mg / mL). Subsequently, EndoF3-D165A (0.2 mg / mL) and the sugar chain probe (2.5 mM) were added to the reaction, and the reaction was continued at 30°C for 1 hour. The obtained azidated peptide fragments were desalted by C18 solid phase extraction (Sep-Pak tC18 cartridge) and lyophilized.

[0127] The azidated peptide solid was dissolved in PBS buffer (100 mM, pH = 7.5, 600 μL) and reacted with 45 mg of the thermosensitive probe (Compound 1) at room temperature for 1 hour. After the reaction, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 40 ° C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, the solid was collected and redissolved in water (600 μL). Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. Heat at 40 ° C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, and the precipitate was collected. Repeat the dissolution of the precipitate ten times to wash away the unlabeled peptide segment.

[0128] The washed solid was dissolved in PBS buffer (100 mM, pH = 7.0, 600 μL). Then, endoglycosidase (EndoF3 and EndoCC, 0.1 mg / mL) was added. The mixture was reacted at room temperature for 12 h. Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. The temperature of the mixture was raised to 40°C to precipitate the PNIPAM compound. The supernatant was collected by centrifugation (10,000 × g). The release of the standard peptide in the supernatant was subjected to C18 solid phase extraction desalting (Sep-Pak tC18 cartridge) and analyzed by LC-MS / MS. The analysis results are shown in FIG. Figure 4 .

[0129] Example 6: Reversible labeling of target peptides in cell peptides using temperature-sensitive probes

[0130] (1) Acquisition of tumor cell peptides

[0131] Human hepatoma cell lines HepG2 were cultured in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 U / ml streptomycin at 37°C and 5% CO2. 10 mM TMG (a small molecule compound that inhibits O-GlcNAc glycosylation hydrolysis) was added 24 hours before cell harvest.

[0132] Cells were harvested and lysed in RIPA lysis buffer (50 mM Tris, 150 mM sodium chloride, 0.1% (w / v) SDS, pH 7.4, EDTA-free protease inhibitor cocktail) for 20 min at 4°C. The mixture was centrifuged at 4°C, and the supernatant was collected. 10 mM dithiothreitol (DTT) was added to the supernatant and the reaction was incubated at 50°C for 30 min. The supernatant was then cooled to room temperature, and 30 mM iodoacetamide (IAA) was added to the reaction mixture. The reaction mixture was incubated in the dark for 60 min at room temperature. The reaction mixture was then subjected to protein precipitation (methanol / chloroform / water = 4:1:4). 8 M urea was added to the protein precipitate to dissolve the protein, and the protein solution was diluted to a final urea concentration of 1 M with 100 mM NH4HCO3 buffer. Trypsin (Promega, enzyme:substrate ratio 1:100) was added and digested at 37°C for 34 h. The resulting peptide solution was desalted using C18 solid-phase extraction (Sep-Pak tC18 cartridge) and lyophilized. PBS buffer (100 mM, pH 7.5) was added to dissolve the lyophilized peptides, and the peptide concentration was determined by BCA assay. The peptides were diluted with PBS to a final concentration of 5 mg / mL. EndoF3 (0.1 mg / mL) was then added and reacted at 37°C for 2 h to expose the core fucose sites. PNGase F (0.05 mg / mL) was then added and reacted at 30°C for 12 h to remove N-glycan residues other than the core fucose, minimizing interference with subsequent mass spectrometry site analysis. The deglycosylated peptides were then desalted using C18 solid-phase extraction (Sep-Pak tC18 cartridge) and lyophilized. The purified deglycosylated peptide solids were dissolved in water, aliquoted, and stored at -80°C. The concentration of the deglycosylated peptides was determined using a BCA protein assay kit.

[0133] (2) Reversible labeling of tumor cell peptides with double glycosylation

[0134] EndoCC-N180H (0.2 mg / mL) and 2.5 mM sugar chain probe (Compound 2) were added to 300 μL of PBS buffer (200 mM, pH = 7.5) containing 3 mg of the deglycosylated peptide obtained in (1) above (1) (10 mg / mL), and the mixture was reacted at 30°C for 3 h. EndoF3-D165A (0.2 mg / mL) and Compound 2 (2.5 mM) were then added to the reaction. The resulting mixture was reacted for an additional 1 h. The azido peptide was desalted by C18 solid-phase extraction (Sep-Pak tC18 cartridge) and lyophilized.

[0135] The azidated peptide solid was dissolved in PBS buffer (100 mM, pH = 7.5, 600 μL) and reacted with 45 mg of the thermosensitive probe (Compound 1) at room temperature for 1 hour. After the reaction, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 40 ° C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, the solid was collected and redissolved in water (600 μL). Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. Heat at 40 ° C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, and the precipitate was collected. Repeat the dissolution of the precipitate ten times to wash away the unlabeled peptide segment.

[0136] The washed solid was dissolved in PBS buffer (100 mM, pH = 7.0, 600 μL). Then, endoglycosidase (EndoF3 and EndoCC, 0.1 mg / mL) was added. The mixture was reacted at room temperature for 12 h. Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. The temperature of the mixture was raised to 40°C to precipitate the PNIPAM compound. The supernatant was collected by centrifugation (10,000 × g). The standard peptide in the supernatant was released and desalted by C18 solid phase extraction (Sep-Pak tC18 cartridge).

[0137] After reversibly labeling, peptide samples from tumor cells were analyzed using LC / MS and database search was performed. Ultimately, 449 core fucose sites (from 326 proteins) and 732 O-GlcNAc sites (from 226 proteins) were identified in the liver cancer cell line HepG2.

[0138] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced with equivalents, without departing from the spirit and substance of the claims of the present invention; and such modifications or replacements remain within the scope of the claims of the present invention.

Claims

1. A temperature-sensitive probe represented by the following formula 1, in, n is an integer from 17 to 90.

2. The temperature-sensitive probe according to claim 1, wherein n is 87.

3. A method for preparing the temperature-sensitive probe represented by formula 1 according to claim 1 or 2, comprising the following steps: ⅰ Compound a and tert-butyl (3-aminopropyl) carbamate tert-butyl ester in the presence of a condensing agent and a solvent for condensation reaction, diluted, washed, and rotary evaporated to obtain a crude product; ii. The crude product was redissolved, purified by silica gel column, and dried to obtain compound b; iii. Compound b and the carboxylic acid-terminated poly (N-isopropylacrylamide) of Formula 3 are subjected to a condensation reaction in the presence of a condensing agent and a solvent, diluted, purified by dialysis, concentrated to obtain a crude product, and then purified by gel column and dried to obtain a temperature-sensitive probe shown in Formula 1. In Formula 3, n is as defined in claim 1 or 2.

4. The method according to claim 3, wherein: In step i, the condensing agent is O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA); and / or The solvent is selected from one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and acetonitrile (MeCN); In step i, the reaction molar ratio of compound a and tert-butyl (3-aminopropyl) carbamate is 1:1-1:5; and / or The reaction temperature is room temperature and the reaction time is 4-8h.

5. The method according to claim 3, wherein: In step iii, the condensing agent is O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA); and / or The solvent is selected from one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and acetonitrile (MeCN); In step iii, the reaction molar ratio of carboxylic acid-terminated poly(N-isopropylacrylamide) and compound b is 1:1-1:5; and / or The reaction temperature is room temperature and the reaction time is 4-8h.

6. A method for labeling core fucosylation and / or O-GlcNAc glycosylation for non-diagnostic or therapeutic purposes, comprising the following steps: S01, Marking: Adding a sugar chain probe compound represented by the following formula 2 and an ENGase mutant selective for core fucose and / or an ENGase mutant selective for O-GlcNAc to a buffer containing a glycosylated peptide segment with core fucosylation and / or O-GlcNAc glycosylation to carry out a reaction; Wherein, the ENGase mutant selective for core fucose is selected from one or more of EndoF3-D165A and EndoF2-D124A; The ENGase mutant selective for O-GlcNAc is selected from one or more of EndoCC-N180H and EndoM-N75Q; S02, Enrichment: Adding the temperature-sensitive probe represented by formula 1 according to claim 1 or 2 to the labeled system to carry out a reaction, removing unlabeled miscellaneous peptide segments, and obtaining a solid; S03, Release: The washed solid is dissolved in a buffer solution, and an ENGase selective for core fucose and / or an ENGase selective for O-GlcNAc is added to react. After the reaction is completed, sodium chloride is added, and the mixture is heated for a period of time and then separated to obtain the supernatant, thereby obtaining the labeled peptide fragments. Wherein, the ENGase selective for core fucose is selected from one or more of EndoF3 and EndoF2; The ENGase selective for O-GlcNAc is selected from one or more of EndoCC and EndoM.

7. The method according to claim 6, wherein: In step S01, the reaction temperature is 25-37° C., and the reaction time is 1-3 h.

8. The method according to claim 6, wherein: In step S02, the reaction temperature is 20-30°C and the reaction time is 1-3h; and / or Removing unlabeled miscellaneous peptide segments includes: adding sodium chloride, heating for a period of time to produce a precipitate, discarding the supernatant, dissolving the precipitate in water in an ice bath, continuing to add sodium chloride, heating for a period of time to produce a precipitate, discarding the supernatant, and repeating the process; and / or In step S03, the reaction temperature is 4-30°C and the reaction time is 3-12h; and / or In step S03, the heating temperature is 40-60°C and the heating time is 5-10 minutes.

9. The method according to claim 8, wherein In step S02, the heating temperature for removing unlabeled miscellaneous peptide segments is 40-60°C, and the heating time is 5-10 minutes.

10. A method for detecting core fucosylation and / or O-GlcNAc glycosylation in vitro for non-diagnostic or therapeutic purposes, comprising: The peptides are labeled according to the method of any one of claims 6 to 9, and then the labeled peptides are detected and analyzed.

11. The method according to claim 10, wherein: The analysis is performed by liquid chromatography, mass spectrometry, or liquid chromatography / mass spectrometry.

12. An in vitro detection kit for core fucosylation and / or O-GlcNAc glycosylation, comprising: The temperature-sensitive probe shown in Formula 1 below: Wherein, n is an integer from 17 to 90; The sugar chain probe compound is represented by the following formula 2: ENGase mutants selective for core fucose and / or ENGase mutants selective for O-GlcNAc; ENGase selective for core fucose and / or ENGase selective for O-GlcNAc; and buffer, Wherein, the ENGase mutant selective for core fucose is selected from one or more of EndoF3-D165A and EndoF2-D124A; The ENGase mutant selective for O-GlcNAc is selected from one or more of EndoCC-N180H and EndoM-N75Q; The ENGase selective for core fucose is selected from one or more of EndoF3 and EndoF2; The ENGase selective for O-GlcNAc is selected from one or more of EndoCC and EndoM; The buffer solution includes PBS buffer solution.

13. The in vitro detection kit according to claim 12, wherein In Formula 1, n is 87.

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