Enzymatic synthesis of unnatural sugars and applications thereof

Abstract

The application discloses an enzyme method for synthesizing unnatural sugar and application thereof, and belongs to the technical field of organic chemical synthesis. The unnatural sugar comprises an unnatural sugar obtained by introducing an acyl group into a hydroxyl group at the 6th position of a bare sugar through an acylation reaction; and the bare sugar is obtained by performing derivatization modification on an amide site of a natural sugar. The synthesis method uses a SubC enzyme as a catalyst to realize selective acylation of the hydroxyl group at the 6th position of the bare sugar, and synthesizes the unnatural sugar with an ideal yield. The unnatural sugar can be applied to glycomics metabolic labeling and mass spectrometry analysis as a marker, has excellent biological safety, specificity and labeling efficiency, can avoid non-specific labeling, and has no obvious cytotoxicity.

Description

Technical Field

[0001] This invention relates to the field of organic chemical synthesis technology, and in particular to the enzymatic synthesis of non-natural sugars and their applications. Background Technology

[0002] Glycosylation plays a crucial role in key life activities such as cell recognition, signal transduction, and immune response, and its abnormalities are closely related to the development of many major diseases. To deeply understand the dynamic glycosylation process, metabolic labeling-based non-natural sugar probe technology has become an indispensable core tool. This technology delivers non-natural sugars containing bioorthogonal groups to cells or organisms and integrates them into newly synthesized glycan chains. Subsequently, through efficient and specific click chemistry reactions, the probe is linked to tracer molecules such as fluorescent groups, thereby achieving dynamic visualization of glycosylation. Especially in mass spectrometry analysis, it provides crucial support for the precise qualitative and quantitative study of target glycoproteins.

[0003] To deliver non-natural sugar probes into cells, researchers have developed two generations of chemically synthesized probes. First-generation fully acetylated sugars (Ac4-sugars), while achieving high synthetic yields, exhibit non-specific labeling within cells, making them unsuitable for mass spectrometry analysis. Second-generation probes (such as 1,6-Pr2-sugars) overcome this non-specific labeling limitation, but their chemical synthesis routes are extremely complex, requiring cumbersome protection-deprotection processes. This results in reduced synthetic yields and is time-consuming and labor-intensive, making them unsuitable for large-scale synthesis and application.

[0004] Therefore, providing a non-natural sugar with a simple and specific synthesis method that is applicable to mass spectrometry analysis and metabolic labeling experiments has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides an enzymatic synthesis of unnatural sugars and their applications. This invention uses SubC enzymes as catalysts, resulting in unnatural sugars with high specificity and labeling efficiency, which can be used as metabolic markers in glycobiological metabolism experiments, glycoproteomics experiments, and other similar fields.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of this invention protects a non-natural sugar, including a non-natural sugar obtained by introducing an acyl group at the 6th position of a naked sugar through an acylation reaction; The naked sugar is obtained by derivatization modification of the amide sites of natural sugars.

[0008] Preferably, the natural sugar includes at least one of GlcNAc, ManNAc, and GalNAc; The structural formulas of GlcNAc, ManNAc, and GalNAc are as follows:

[0009] The modifying groups during the derivatization modification include at least one of the following: substituted or unsubstituted C2~C10 alkynyl groups, substituted or unsubstituted C0~C10 azide groups, mercapto groups, terminal alkenes, phenyl azide groups, bisacrylidine, selenylmethyl groups, ketone groups, isonitriles, norbornene, tetrazine, and biotin. The acyl group includes at least one selected from acetyl, propionyl, butyryl, valeryl, hexanoyl, heptayl, octanoyl, nonanoyl, and decanoyl.

[0010] Preferably, the naked sugar comprises any one of: GlcNAz, ManNAz, GalNAz, GlcNAl, ManNAl, GalNAl, GlcN(C3)Az, GalN(C3)Az, ManN(C3)Az, ManN(C4)Az, ManN(C5)Az, ManN(C6)Al, ManN-thiol, ManN-alkene, ManNBenzol-N3, ManNDAz, ManNSe, ManN-ketone, ManN-isonitrile, ManN-norbornene, ManNTz, and ManNtriazole-biotin; The specific structure of the naked sugar is as follows:

[0011]

[0012]

[0013]

[0014]

[0015] .

[0016] Preferably, the non-natural sugar has any one of the following structures:

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] .

[0027] A second aspect of this invention protects a method for preparing the non-natural sugar described in the first aspect, characterized by comprising the following steps: The raw sugar, solvent, acyl donor, and SubC enzyme were mixed, reacted with shaking, and then post-treated to obtain the non-natural sugar.

[0028] Preferably, the general formula of the acyl donor is R1-C(=O)-O-R2, where R1 includes hydrogen, one of C1 to C9 alkyl groups, and R2 includes one of C1 to C3 alkyl groups, C2 to C3 alkenyl groups, and p-nitrophenyl groups. The mass ratio of the acyl donor to the naked sugar is (0.5~4):(150~300). The mass ratio of the SubC enzyme to the naked sugar is (10~50):(150~300).

[0029] Preferably, the mass-to-volume ratio of the naked sugar to the solvent (mg / ml) is (150~300):(10~50). The solvent includes at least one of 2-methyl-2-butanol, N,N-dimethylformamide, dimethyl sulfoxide, water, ethyl acetate, acetone, methanol, acetonitrile, 1,4-dioxane, and pyridine. The oscillation reaction is carried out at a temperature of 30~37℃ for a duration of not less than 4 hours and at a frequency of 220 rpm. The post-processing includes purification.

[0030] Preferably, the naked sugar comprises ManNAz, and the preparation method of ManNAz includes the following steps: S1. Mix glucosamine hydrochloride, CmCDA enzyme, NahK enzyme, buffer B and water to obtain mixture I. Shake at 220 rpm for 10-14 h at 30-40℃ to obtain reaction solution I. S2. Heat reaction solution I to 93-98℃ and keep it at that temperature for 1-15 min. Cool it down to 2-6℃, centrifuge and collect the supernatant. Add UTP and MgCl2 to the supernatant, adjust the pH to 7.5-8.5, add water to obtain mixture II, then add AGX2 enzyme, PmPpA enzyme and ecNeuC enzyme to obtain mixture III. React overnight at 35-39℃ with shaking at 220 rpm to obtain reaction solution II. Mix reaction solution II with an equal volume of methanol, precipitate at -85~-20℃ for 2-6 h, centrifuge and collect the supernatant for purification to obtain ManNAz. In step S1: The mass ratio of glucosamine hydrochloride to CmCDA enzyme is 20:(0.5~5). The mass ratio of glucosamine hydrochloride to NahK enzyme is 20:(0.5~5). The buffer solution B comprises: 200 mM Tris-HCl, 0.2 M azideacetic acid, 60 mM ATP, 50 mM MgCl2, and pH 8.0. The mass-to-volume ratio of glucosamine hydrochloride to buffer B is 100:(0.5~5). The concentration of glucosamine hydrochloride in the mixture I is 5~15 mg / ml; In step S2: The UTP concentration in the mixture II is 40~100 mM; The concentration of MgCl2 in the mixture II is 30~100 mM; The concentration of AGX2 enzyme in the mixture III is 0.35~0.5 mg / ml; The concentration of PmPpA enzyme in the mixture III is 0.35~0.5 mg / ml; The concentration of ecNeuC enzyme in the mixture III is 0.35~0.5 mg / ml.

[0031] Preferably, the naked sugar comprises GalNAz, and the preparation method of GalNAz includes the following steps: (1) Mix glucosamine hydrochloride, CmCDA enzyme, NahK enzyme, buffer B and water to obtain mixture IV. Shake at 220 rpm for 10-14 h at 30-40℃ to obtain reaction solution III; (2) Heat the reaction solution III to 93~98℃ and keep it warm for 1~15 min, cool it down to 2~6℃, centrifuge and take the supernatant. Add UTP and MgCl2 to the supernatant, adjust the pH to 7.5~8.5, add water to get mixture V, then add AGX2 enzyme, PmPpA enzyme, WbgU enzyme and thGTA enzyme to get mixture VI. Shake the mixture at 220 rpm at 35~39℃ overnight to get reaction solution IV. Mix reaction solution IV with an equal volume of methanol, precipitate at -85~-20℃ for 2~6 h, centrifuge and take the supernatant for purification to get GalNAz. In step (1): The mass ratio of glucosamine hydrochloride to CmCDA enzyme is 20:(0.5~1.5). The mass ratio of glucosamine hydrochloride to NahK enzyme is 20:(0.5~1.5). The buffer solution B comprises: 200 mM Tris-HCl, 0.2 M azideacetic acid, 60 mM ATP, 50 mM MgCl2, and pH 8.0. The volume ratio of glucosamine hydrochloride to buffer B is 100:(0.5~5) mg / ml. The concentration of glucosamine hydrochloride in the mixture IV is 5-15 mg / ml; In step (2): The UTP concentration in the mixture V is 40~100 mM; The MgCl2 concentration in the mixture V is 30~100 mM; The concentration of AGX2 enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of PmPpA enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of WbgU enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of thGTA enzyme in the mixture VI is 0.35~0.5 mg / ml.

[0032] The third aspect of this invention protects the use of a non-natural sugar as described in the first aspect, and / or a non-natural sugar synthesized by the synthesis method described in the second aspect, in the preparation of metabolic markers; the metabolic markers include: metabolic markers for glycobiological metabolism, bioimaging tracing, glycoproteomics research, and confocal microscopy.

[0033] The beneficial technical effects of this invention are as follows: This invention utilizes SubC enzyme as a catalyst to achieve selective acylation of the hydroxyl group at position 6 of naked sugars, synthesizing non-natural sugars in ideal yields. Furthermore, this invention uses readily available and inexpensive GlcN as a starting material to synthesize naked sugars ManNAz and GalNAz in high yields, realizing the total enzymatic synthesis of non-natural sugars in this invention. The above reactions are carried out under mild conditions, without requiring harsh conditions such as anhydrous and oxygen-free environments, avoiding the protection-deprotection process, and are simple to operate and easy to scale up for industrial production.

[0034] The non-natural sugar synthesized in this invention can be used as a marker (such as a biological probe) for glycobiological metabolic labeling and mass spectrometry analysis. It has excellent biosafety, specificity and labeling efficiency, can avoid non-specific labeling and has no obvious cytotoxicity. Compared with the current Ac4-ManNAz and 1,6-Pr2-ManNAz probes, it has significantly higher glycoproteomics application value. Attached Figure Description

[0035] Figure 1 The non-natural sugar H-GlcNAz(1e) prepared in Example 3 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0036] Figure 2 The non-natural sugar H-GlcNAz(1e) prepared in Example 3 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0037] Figure 3 The non-natural sugar H-ManNAz(2e) prepared in Example 4 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0038] Figure 4 The non-natural sugar H-ManNAz(2e) prepared in Example 4 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0039] Figure 5 The non-natural sugar H-GalNAz(3e) prepared in Example 5 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0040] Figure 6 The non-natural sugar H-GalNAz(3e) prepared in Example 5 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0041] Figure 7The non-natural sugar H-GlcNAl(1j) prepared in Example 6 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0042] Figure 8 The non-natural sugar H-GlcNAl(1j) prepared in Example 6 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0043] Figure 9 The non-natural sugar H-ManNAl(2j) prepared in Example 7 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0044] Figure 10 The non-natural sugar H-ManNAl(2j) prepared in Example 7 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0045] Figure 11 The non-natural sugar H-GalNAl(3j) prepared in Example 8 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0046] Figure 12 The non-natural sugar H-GalNAl(3j) prepared in Example 8 of this invention 13 C NMR (101 MHz, MeOD) spectrum.

[0047] Figure 13 The non-natural sugar H-ManN-norbornene (2v) prepared in Example 9 of this invention 1 H NMR (400MHz, MeOD) spectrum.

[0048] Figure 14 The non-natural sugar H-ManN-norbornene (2v) prepared in Example 9 of this invention 13 C NMR (101MHz, MeOD) spectrum.

[0049] Figure 15 The non-natural sugar H-ManNTz (2w) prepared in Example 10 of this invention 1 H NMR (400 MHz, MeOD) spectrum.

[0050] Figure 16 The non-natural sugar H-ManNTz (2w) prepared in Example 10 of this invention 13C NMR (101 MHz, MeOD) spectrum.

[0051] Figure 17 The non-natural sugar H-ManNtriazole-biotin (2x) prepared in Example 11 of this invention 1 1H NMR (400 MHz, DMSO-d6) spectrum.

[0052] Figure 18 The non-natural sugar H-ManNtriazole-biotin (2x) prepared in Example 11 of this invention 13 C NMR (101 MHz, DMSO-d6) spectrum.

[0053] Figure 19 This is a safety evaluation diagram of non-natural sugars in the test examples of this invention.

[0054] Figure 20 The image shows the test results of the non-natural sugar labeling signal intensity in the invention test example.

[0055] Figure 21 The figure shows the results of the non-natural sugar label specificity test in the invention test examples.

[0056] Figure 22 This is a diagram showing the results of a glycoproteomics study based on non-natural sugar labels in the invention test case.

[0057] Figure 23 This is a confocal microscopy image of a non-natural sugar in an invention test example. Detailed Implementation

[0058] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0059] A non-natural sugar comprising an acylation reaction to introduce an acyl group at the 6th position of a naked sugar; wherein the naked sugar is obtained by derivatization modification of the amide site of a natural sugar.

[0060] The natural sugars include at least one of GlcNAc, ManNAc, and GalNAc.

[0061] The modifying groups during the derivatization modification include at least one of the following: substituted or unsubstituted C2-C10 alkynyl groups, substituted or unsubstituted C0-C10 azide groups, mercapto groups, terminal alkenes, phenyl azide groups, bisacrylidine, selenylmethyl groups, ketone groups, isonitriles, norbornene, tetrazine, and biotin.

[0062] Preferably, the modifying group during the derivatization modification includes at least one of the following: substituted or unsubstituted C2-C6 alkynyl group, substituted or unsubstituted C0-C6 azide group, mercapto group, terminal alkene group, phenyl azide group, bisacrylidine group, selenylmethyl group, ketone group, isonitrile group, norbornene group, tetrazine group, and biotin group.

[0063] The SubC enzyme (CAS: 9014-01-1) used in this invention can be obtained by purchase.

[0064] All raw materials used in the following embodiments of the present invention can be purchased.

[0065] The naked sugar described in this invention can be synthesized by the synthesis method described in this invention, or it can be obtained by purchasing.

[0066] Example 1 The synthesis method of the naked sugar ManNAz includes the following steps: S1. Mix 100 mg GlcN·HCl, 5 mg CmCDA enzyme, 5 mg NahK enzyme, and 5 ml buffer B (200 mM Tris-HCl, 0.2 M azidoacetic acid, 60 mM ATP, 50 mM MgCl2, pH 8.0), add ddH2O to make up to 10 ml to obtain mixture I. Shake at 220 rpm for 12 h at 37℃ to obtain reaction solution I.

[0067] S2. Heat reaction solution I to 95℃ and keep warm for 10 min. After cooling to 4℃, centrifuge at 8000 g and collect the supernatant. Add 60 mM UTP and 50 mM MgCl2 to the supernatant, adjust the pH to 8.0, and add water to make up to 12 ml to obtain mixture II. Add 5 mg AGX2 enzyme, 5 mg PmPpA enzyme, and 5 mg ecNeuC enzyme to obtain mixture III. React at 37℃ with shaking at 220 rpm overnight to obtain reaction solution II. Mix reaction solution II with an equal volume of methanol and precipitate at -80℃ for 4 h. Centrifuge and collect the supernatant for column chromatography purification (DCM:MeOH approximately 4:1). Anisaldehyde is used for color development to obtain ManNAz with a yield of 64%.

[0068] The reaction pathway described above is as follows:

[0069] Example 2 The synthesis method of the naked sugar GalNAz includes the following steps: S1. Mix 100 mg GlcN·HCl, 5 mg CmCDA enzyme, 5 mg NahK enzyme, and 5 ml buffer B (200 mM Tris-HCl, 0.2 M azidoacetic acid, 60 mM ATP, 50 mM MgCl2, pH 8.0), add ddH2O to make up to 10 ml to obtain mixture IV. Shake at 220 rpm for 12 h at 37℃ to obtain reaction solution III.

[0070] S2. Heat reaction solution III to 95℃ and keep warm for 10 min. After cooling to 4℃, centrifuge at 8000 g and collect the supernatant. Add 60 mM UTP and 50 mM MgCl2 to the supernatant, adjust the pH to 8.0, and add water to make up to 12 ml to obtain mixture V. Add 5 mg AGX2 enzyme, 5 mg PmPpA enzyme, 5 mg WbgU enzyme, and 5 mg thGTA enzyme to obtain mixture VI. React overnight at 37℃ with shaking at 220 rpm to obtain reaction solution IV. Mix reaction solution IV with an equal volume of methanol, precipitate at -80℃ for 4 h, centrifuge and collect the supernatant for column chromatography purification (DCM:MeOH approximately 4:1). Develop with anisaldehyde to obtain GalNAz with a yield of 55%.

[0071] The reaction pathway described above is as follows:

[0072] Example 3 The method for synthesizing the non-natural sugar H-GlcNAz(1e) includes the following steps: 200 mg of GlcNAz was weighed and dissolved in a mixed solution of 10 ml 2-methyl-2-butanol and 4 ml DMF. After sonication to aid dissolution, 2.2 ml vinyl hexanoate and 30 mg SubC enzyme were added. The reaction system was placed at 37°C and shaken at 220 rpm for 4 h. The reaction product was monitored by TLC (EA:MeOH=30:1), and anisaldehyde was observed as a colorimetric indicator. After the reaction was completed, the product was purified by column chromatography (DCM:MeOH) and then purified by preparative RP-HPLC to obtain approximately 230 mg of H-GlcNAz as a white solid, with a yield of 86%.

[0073] Example 4 The synthesis method of the non-natural sugar H-ManNAz (2e) is basically the same as that in Example 3, except that GlcNAz is replaced with ManNAz obtained in Example 1, the shaking reaction temperature is 30°C, and the yield of H-ManNAz is 90%.

[0074] Example 5 The method for synthesizing the non-natural sugar H-GalNAz (3e) is basically the same as that in Example 3, except that GlcNAz is replaced with GalNAz obtained in Example 2, and the yield of H-GalNAz is 76%.

[0075] Example 6 The synthesis method of the non-natural sugar H-GlcNAl (1j) is basically the same as that in Example 3, except that GlcNAz is replaced with GlcNAl, the shaking reaction time is 6 h, and the yield of H-GlcNAl is 84%.

[0076] Example 7 The synthesis method of non-natural sugar H-ManNAl (2j) is basically the same as that in Example 3, except that GlcNAz is replaced with ManNAl, the shaking reaction time is 6 h, and the yield of H-ManNAl is 87%.

[0077] Example 8 The synthesis method of non-natural sugar H-GalNAl (3j) is basically the same as that in Example 3, except that GlcNAz is replaced with GalNAl, the shaking reaction time is 6 h, and the yield of H-GalNAl is 85%.

[0078] Example 9 The synthesis method of the non-natural sugar H-ManN-norbornene (2v) is basically the same as that in Example 3, except that GlcNAz is replaced with ManN-norbornene, the shaking reaction time is 12 h, and the yield of H-ManN-norbornene is 68%.

[0079] Example 10 The synthesis method of the non-natural sugar H-ManNTz (2w) is basically the same as that in Example 3, except that GlcNAz is replaced with ManNTz, the shaking reaction time is 24 h, and the yield of H-ManNTz is 11%.

[0080] Example 11 The synthesis method of the non-natural sugar H-ManNtriazole-biotin (2x) is basically the same as that in Example 3, except that GlcNAz is replaced with ManNtriazole-biotin, the shaking reaction time is 24 h, and the yield of H-ManNtriazole-biotin is 21%.

[0081] Example 12 The synthesis method of the non-natural sugar H-ManN(C3)Az(2k) is basically the same as that in Example 3, except that GlcNAz is replaced with ManN(C3)NAz, the shaking reaction time is 6 h, and the yield of H-ManN(C3)NAz is 90%.

[0082] Test case 1. Safety evaluation of non-natural sugars The non-natural sugar H-ManNAz synthesized in Example 4, the commercially available non-natural sugar Ac4-ManNAz, and 1,6-Pr2-ManNAz were subjected to CCK8 cytotoxicity tests in HeLa, MCF7, SKOV3, HEK293T, and CHO cells. Taking MCF7 cells as an example, the test method is as follows: (1) Take adherent cells from a 10 cm culture dish, gently rinse twice with PBS buffer, add 1 mL of trypsin and digest at 37°C for 4 min.

[0083] (2) Subsequently, 2 mL of cell culture medium was added to resuspend the cells to prepare a cell suspension. 10 μL of this suspension was taken for cell counting, and then, based on the counting results, cells containing 10 × 10⁻⁶ cells were aspirated. 6 A suspension of cells was diluted in 12 mL of culture medium and shaken thoroughly. (3) Spread the well-mixed cell suspension into a 96-well plate at a volume of 100 μL per well, and mix the cells in the well by shaking in a cross-shaped motion after adding the sample.

[0084] (4) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 12 h.

[0085] (5) Take out the previously prepared and frozen 100 mM three non-natural sugars and DMSO reagent and thaw them.

[0086] (6) Use a suction pump to remove the supernatant culture medium from each well of the 96-well plate. Add 100 μl of complete culture medium solution containing non-natural sugars at preset concentration gradients (100 μM, 200 μM, 500 μM) to each well as the experimental group. Add DMSO: culture medium (v / v) = 200:1 as the control group. Continue to incubate in a 37℃, 5% CO2 incubator for 48 h. Finally, use a commercial CCK8 kit to evaluate the toxicity of the cells.

[0087] Cell safety evaluation results as follows Figure 19 The results showed that Ac4-ManNAz exhibited significant cytotoxicity at a concentration of 500 μM, while other non-natural sugars generally showed high cellular safety and had no significant adverse effects on cell proliferation.

[0088] 2. Non-natural sugar labeling signal intensity test The testing method is as follows: (1) The non-natural sugar H-ManNAz synthesized in Example 4, the commercially available non-natural sugar Ac4-ManNAz, and 1,6-Pr2-ManNAz were prepared into a 100 mM concentrated stock solution of cell-grade DMSO and stored at -20°C for later use. (2) When the cells in the 6-well plate reached 30% confluence, 5 μM H-ManNAz, 20 μM H-ManNAz, 100 μM H-ManNAz, 100 μM Ac4-ManNAz, and 100 μM 1,6-Pr2-ManNAz were added to different wells as experimental groups, and 2 μl DMSO solvent was added as control group. After the addition was completed, the plate was incubated in a 37℃, 5% CO2 incubator for 48 h. (3) After incubation, gently rinse the cells twice with PBS buffer, add trypsin and digest at 37°C for 4 min, then centrifuge to collect the cell pellet. (4) The cell pellet was washed three times with PBS buffer, and then resuspended in RIPA buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (w / v) and protease inhibitor, and placed on ice. (5) After placing the cells in an ice bath, the cells were sonicated and centrifuged (4℃, 14000 g) for 10 min to collect the supernatant. The protein concentration was determined using a BCA protein detection kit and standardized to 2 mg / ml. (6) 50 μM CuSO4-100 μM BTTAA complex and 100 μM alkynyl-Cy5 were premixed in cell lysate, and 2.5 mM freshly prepared sodium ascorbate was added. The click chemical reaction was completed by shaking at 25°C for 2 h. (7) After the reaction is complete, add 5× denatured protein loading buffer containing disulfitol (DTT) to the sample and heat at 95°C for 10 min. (8) The processed samples were electrophoresed on a 12% SDS-PAGE gel and imaged using a ChemiDoc XRS+ imager. The gel was then stained with Coomassie Brilliant Blue (CBB) as a control for the amount of sample loaded. The image data were analyzed using Image Lab software.

[0089] Test results are as follows Figure 20 As shown, according to Figure 20It can be seen that the labeling intensity of H-ManNAz increases with increasing concentration. At the same concentration of 100 µM, the three probes, H-ManNAz, commercially available non-natural sugar Ac4-ManNAz, and 1,6-Pr2-ManNAz, showed similar fluorescence signals, indicating that they have nearly equal labeling capabilities.

[0090] 3. Specificity test for non-natural sugar markers The testing method is as follows: (1) Collect cells that have grown to 90% confluence in a 6-well plate and wash them three times with PBS; (2) Cells were lysed by sonication in an ice bath, centrifuged (14000 g, 4℃) for 5 minutes, and the protein concentration in the supernatant was determined using a BCA protein detection kit and standardized to 2 mg / ml. (3) The standardized samples were added with 100 μM non-natural sugar H-ManNAz, commercially available non-natural sugar Ac4-ManNAz, and 1,6-Pr2-ManNAz as experimental groups, and 2 μl of DMSO as control group. They were incubated at 37°C for 2 hours. (4) Then add 150 μl methanol, 37.5 μl chloroform and 100 μl ultrapure water to precipitate the protein; (5) The precipitate was resuspended in PBS buffer containing 1% SDS; (6) Prepare a Click reaction solution containing 10 μl 50 mM CuSO4, 20 μl 5 mM alkynyl-Cy5, and 100 μl 15 mg / ml freshly prepared sodium ascorbate. Add 8 μl of the reaction solution to each sample tube. In both the experimental and control groups, some groups were additionally given 25 mM iodoacetamide (“+” group). All samples were shaken and reacted at room temperature in the dark for 2 hours. (7) After the reaction is complete, add 5× denatured protein loading buffer containing DTT to each sample tube and heat at 95°C for 10 minutes. (8) The processed samples were electrophoresed on a 12% SDS-PAGE gel and imaged using a ChemiDoc XRS+ imager. The gel was then stained with Coomassie Brilliant Blue as a control for the amount of sample loaded. The image data were analyzed using Image Lab software.

[0091] Test results are as follows Figure 21 As shown, according to Figure 21As can be seen, Ac4-ManNAz exhibits a very strong fluorescence signal in the absence of iodoacetamide ("-" lane), indicating that nonspecific labeling has occurred. When iodoacetamide is added ("+" lane), the nonspecific signal of Ac4-ManNAz is significantly weakened, which proves that nonspecific labeling mainly occurs on cysteine ​​residues of proteins that can be blocked by iodoacetamide.

[0092] In contrast, the lanes ("-" lanes) of H-ManNAz and 1,6-Pr2-ManNAz showed almost no fluorescence signal, indicating that these two sugar probes do not cause nonspecific labeling and have excellent specificity.

[0093] 4. Glycoproteomics studies based on non-natural sugar labels The experimental method is as follows: (1) Cells labeled with non-natural sugar metabolism were collected into 1.5 ml Eppendorf tubes, centrifuged at 800 g for 3 min and washed with 500 μl DPBS, centrifuged at 800 g for 3 min to collect the precipitate. The cell lysate prepared by sonication (60 W, 3 s on, 3 s off) was prepared by adding Ripa lysis buffer (100 μl Ripa lysis buffer, 1X protease inhibitor) and incubating with 500 μM premixed CuSO4-250 μM BTTAA, 10 mM PC-biotin-PEG3-alkynyl, and 5 mM freshly prepared sodium ascorbate at 25 °C for 3 h.

[0094] (2) The mixture obtained in step (1) was precipitated overnight with pre-cooled methanol at -80°C.

[0095] (3) After washing twice with pre-cooled methanol, the protein was resuspended in 50 mM NH4HCO3 containing 4 M urea (2 mL of solvent for every 10 mg of protein), incubated with 10 mM DTT at 37°C for 1 hour, and then incubated with 20 mM iodoacetamide in the dark at 25°C for 30 minutes.

[0096] (4) Dilute the obtained solution with 50 mM NH4HCO3 to a urea concentration of 1 M, and incubate it with mass spectrometry grade trypsin (enzyme:substrate ratio of 1:50) at 37 °C for 16 h. Gently rotate and incubate the obtained solution with streptavidin-coated agarose beads (150 μL of agarose beads for every 40 mg of protein) at 25 °C for 3 h.

[0097] (5) Wash the agarose beads 5 times with PBS, wash them 5 times with ultrapure water, then resuspend them in 350 μl of ultrapure water, and then irradiate them 3 times with a 365 nm UV lamp (CL-1000, UVP) for 5 minutes each time.

[0098] (6) After centrifuging at 3500 g for 3 min, collect the supernatant, add 350 μl of ultrapure water, and repeat the above operation once.

[0099] (7) Combine the supernatants obtained from the two processes, evaporate them in a vacuum centrifuge, and then perform LC-MS / MS analysis.

[0100] (8) Glycopeptide samples were analyzed using a 50 cm C18 column with a dissociation voltage of 2.1 kV, a capillary temperature of 320°C, and HCD fragmentation mode was used for data acquisition.

[0101] (9) The obtained data were analyzed using pGlyco3 software.

[0102] Test results are as follows Figure 22 As shown, according to Figure 22 As can be seen, the experiment using the 1,6-Pr2-ManNAz probe for labeling only identified 239 glycosylation sites. In contrast, the two experiments using the H-ManNAz probe prepared in Example 4 and the H-ManN(C3)Az probe prepared in Example 12 identified 2117 and 2277 complete glycosylation sites, respectively, demonstrating its high labeling and identification efficiency. This proves the superiority of the non-natural sugar synthesized in this invention in glycoproteomics research.

[0103] 5. Confocal microscopy imaging of non-natural sugars The experimental method is as follows: (1) Resuspend the cells in a 10 cm culture dish with 2 mL of serum and seed them into an 8-well slide at a density of 4 μL / 200 μL.

[0104] (2) 100 μM of non-natural sugars H-ManNAz, ManNAz, commercially available non-natural sugars Ac4-ManNAz, and 1,6-Pr2-ManNAz were added to different wells as experimental groups, and 2 μl of solvent (DMSO) was added as control group.

[0105] (3) Gently wash the cells with 200 μL of DPBS.

[0106] (4) Prepare a Click reaction system containing 50 μM CuSO4-100 μM BTTAA complex, 15 mg / mL sodium ascorbate and 5 mM MAlk-488, and react on ice for 10 minutes.

[0107] (5) Add 200 μL (chamber) of freshly thawed 4% paraformaldehyde solution to each well and let stand at room temperature for 10 min.

[0108] (6) Add 100 μL of Click reaction solution per well and react on ice in the dark for 10 minutes.

[0109] (7) Wash twice with 200 μL / well of PBS.

[0110] (8) Prepare 40 μM Hoechst nuclear dye: Add 2.5 μL of Hoechst stock solution to each 1 mL of 1×PBS, vortex, and centrifuge briefly.

[0111] (9) Add 200 μL of dye to each well and incubate at room temperature in the dark for 10 minutes.

[0112] (10) Wash three times with 200 μL / well of PBS. (11) Immerse the sample in PBS and image it using an Olympus IXplore SpinSR10 rotary confocal microscope with a 63x oil immersion lens and a 10x objective lens.

[0113] Test results are as follows Figure 23 As shown in the figure: green fluorescence (AF488) represents successfully labeled glycoproteins, mainly distributed on the cell membrane; blue fluorescence (Hoechst) indicates the location of the cell nucleus.

[0114] according to Figure 23 As can be seen, the control group (Vehicle), which only added solvent, did not show green fluorescence, proving that the entire chemical labeling process did not generate background noise. The ManNAz group showed weak green fluorescence, indicating that its labeling efficiency was relatively low. The H-ManNAz group showed the strongest green fluorescence signal, with labeling efficiency comparable to or even better than that of the classic probes Ac4-ManNAz and 1,6-Pr2-ManNAz, and significantly better than the weak fluorescence of the ManNAz group. This indicates that H-ManNAz can be effectively taken up by cells and integrated into glycoproteins on the cell surface, and can show glycosylation modification on the cell membrane.

[0115] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A non-natural sugar, characterized in that, This includes non-natural sugars obtained by introducing an acyl group at the 6th position of the hydroxyl group of naked sugar through an acylation reaction; The naked sugar is obtained by derivatization modification of the amide sites of natural sugars.

2. The non-natural sugar according to claim 1, characterized in that, The natural sugar includes at least one of GlcNAc, ManNAc, and GalNAc; The structural formulas of GlcNAc, ManNAc, and GalNAc are as follows: The modifying groups during the derivatization modification include at least one of the following: substituted or unsubstituted C2~C10 alkynyl groups, substituted or unsubstituted C0~C10 azide groups, mercapto groups, terminal alkenes, phenyl azide groups, bisacrylidine, selenylmethyl groups, ketone groups, isonitriles, norbornene, tetrazine, and biotin. The acyl group includes at least one selected from acetyl, propionyl, butyryl, valeryl, hexanoyl, heptayl, octanoyl, nonanoyl, and decanoyl.

3. The non-natural sugar according to claim 1 or 2, characterized in that, The naked sugar includes: GlcNAz, ManNAz, GalNAz, GlcNAl, ManNAl, GalNAl, GlcN(C3)Az, GalN(C3)Az, ManN(C3)Az , any one of ManN(C4)Az, ManN(C5)Az, ManN(C6)Al, ManN-thiol, ManN-alkene, ManNBenzol-N3, ManNDAz, ManNSe, ManN-ketone, ManN-isonitrile, ManN-norbornene, ManNTz, ManNtriazole-biotin; The specific structure of the naked sugar is as follows: 。 4. The non-natural sugar according to claim 1, characterized in that, The non-natural sugar has any of the following structures: 。 5. A method for preparing the non-natural sugar according to any one of claims 1 to 4, characterized in that, Includes the following steps: The raw sugar, solvent, acyl donor, and SubC enzyme were mixed, reacted with shaking, and then post-treated to obtain the non-natural sugar.

6. The synthesis method according to claim 5, characterized in that, The general formula of the acyl donor is R1-C(=O)-O-R2, where R1 includes hydrogen and one of C1 to C9 alkyl groups, and R2 includes one of C1 to C3 alkyl groups, C2 to C3 alkenyl groups, and p-nitrophenyl groups. The mass ratio of the acyl donor to the naked sugar is (0.5~4):(150~300). The mass ratio of the SubC enzyme to the naked sugar is (10~50):(150~300).

7. The synthesis method according to claim 5, characterized in that, The mass-to-volume ratio of the naked sugar to the solvent (mg / ml) is (150~300):(10~50); The solvent includes at least one of 2-methyl-2-butanol, N,N-dimethylformamide, dimethyl sulfoxide, water, ethyl acetate, acetone, methanol, acetonitrile, 1,4-dioxane, and pyridine. The oscillation reaction is carried out at a temperature of 30~37℃ for a duration of not less than 4 hours and at a frequency of 220 rpm. The post-processing includes purification.

8. The synthesis method according to claim 5, characterized in that, The naked sugar includes ManNAz, and the preparation method of ManNAz includes the following steps: S1. Mix glucosamine hydrochloride, CmCDA enzyme, NahK enzyme, buffer B and water to obtain mixture I. Shake at 220 rpm for 10-14 h at 30-40℃ to obtain reaction solution I. S2. Heat reaction solution I to 93-98℃ and keep it at that temperature for 1-15 min. Cool it down to 2-6℃, centrifuge and collect the supernatant. Add UTP and MgCl2 to the supernatant, adjust the pH to 7.5-8.5, add water to obtain mixture II, then add AGX2 enzyme, PmPpA enzyme and ecNeuC enzyme to obtain mixture III. React overnight at 35-39℃ with shaking at 220 rpm to obtain reaction solution II. Mix reaction solution II with an equal volume of methanol, precipitate at -85~-20℃ for 2-6 h, centrifuge and collect the supernatant for purification to obtain ManNAz. In step S1: The mass ratio of glucosamine hydrochloride to CmCDA enzyme is 20:(0.5~5). The mass ratio of glucosamine hydrochloride to NahK enzyme is 20:(0.5~5). The buffer solution B comprises: 200 mM Tris-HCl, 0.2 M azideacetic acid, 60 mM ATP, 50 mM MgCl2, and pH 8.0; The mass-to-volume ratio of glucosamine hydrochloride to buffer B is 100:(0.5~5). The concentration of glucosamine hydrochloride in the mixture I is 5~15 mg / ml; In step S2: The UTP concentration in the mixture II is 40~100 mM; The concentration of MgCl2 in the mixture II is 30~100 mM; The concentration of AGX2 enzyme in the mixture III is 0.35~0.5 mg / ml; The concentration of PmPpA enzyme in the mixture III is 0.35~0.5 mg / ml; The concentration of ecNeuC enzyme in the mixture III is 0.35~0.5 mg / ml.

9. The synthesis method according to claim 5, characterized in that, The naked sugar includes GalNAz, and the preparation method of GalNAz includes the following steps: (1) Mix glucosamine hydrochloride, CmCDA enzyme, NahK enzyme, buffer B and water to obtain mixture IV. Shake at 220 rpm for 10-14 h at 30-40℃ to obtain reaction solution III; (2) Heat the reaction solution III to 93~98℃ and keep it warm for 1~15 min, cool it down to 2~6℃, centrifuge and take the supernatant. Add UTP and MgCl2 to the supernatant, adjust the pH to 7.5~8.5, add water to get mixture V, then add AGX2 enzyme, PmPpA enzyme, WbgU enzyme and thGTA enzyme to get mixture VI. Shake the mixture at 220 rpm at 35~39℃ overnight to get reaction solution IV. Mix reaction solution IV with an equal volume of methanol, precipitate at -85~-20℃ for 2~6 h, centrifuge and take the supernatant for purification to get GalNAz. In step (1): The mass ratio of glucosamine hydrochloride to CmCDA enzyme is 20:(0.5~1.5). The mass ratio of glucosamine hydrochloride to NahK enzyme is 20:(0.5~1.5). The buffer solution B comprises: 200 mM Tris-HCl, 0.2 M azideacetic acid, 60 mM ATP, 50 mM MgCl2, and pH 8.0; The volume ratio of glucosamine hydrochloride to buffer B is 100:(0.5~5) mg / ml. The concentration of glucosamine hydrochloride in the mixture IV is 5-15 mg / ml; In step (2): The UTP concentration in the mixture V is 40~100 mM; The MgCl2 concentration in the mixture V is 30~100 mM; The concentration of AGX2 enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of PmPpA enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of WbgU enzyme in the mixture VI is 0.35~0.5 mg / ml; The concentration of thGTA enzyme in the mixture VI is 0.35~0.5 mg / ml.

10. The use of a non-natural sugar according to any one of claims 1 to 4, and / or a non-natural sugar synthesized by the synthetic method according to any one of claims 5 to 9, in the preparation of metabolic markers; said metabolic markers comprising: Metabolic markers for glycobiological metabolism, bioimaging tracing, glycoproteomics research, and confocal microscopy.

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