Unnatural sugars, their synthesis and applications

Abstract

The present invention discloses unnatural sugars, their synthesis methods, and applications. Protecting the hydroxyl groups of amino sugars with tris(trimethylsilyl)silane at room temperature selectively exposes the amino groups of the sugars, and then converting them by coupling at room temperature yields unnatural sugars bearing orthogonal functional groups protected by tris(trimethylsilyl)silane. Removal of the trimethylsilane protecting group from unnatural sugars bearing orthogonal functional groups protected by tris(trimethylsilyl)silane yields unprotected unnatural sugars. Taking into account the advantages of existing unnatural sugars, the present invention not only ensures efficient cell utilization of unnatural sugars, but also effectively avoids side reactions with protein cysteines during the metabolism of unnatural sugars, thereby achieving efficient metabolic labeling. In cell experiments, the concentration of 1,6-diacylated unnatural sugars used was one order of magnitude lower than that of unprotected unnatural sugars.

Description

[Technical Field]

[0001] The present invention relates to the field of organic chemical synthesis technology, and in particular to unnatural sugars, their synthesis methods and applications. [Background technology]

[0002] Glycosylation has important functions, and monitoring glycosylation is crucial. Metabolic labeling using unnatural sugars with chemical reporters has been widely used in cells for glycoimaging and glycoproteome analysis. In vitro chemoenzymatic methods add unnatural sugars with chemical reporters to glycans under the action of glycosyltransferases for further study. Unnatural sugars are essential for metabolic and chemoenzymatic labeling. They are classified into three types: a. Unprotected unnatural sugars: These can be used for metabolic labeling but are also synthetic substrates for unnatural nucleoside sugars used in chemoenzymatic methods. b. Fully acetylated sugars: These probes were developed to overcome the poor cell permeability of unprotected unnatural sugars, significantly reducing the concentration required for metabolic labeling. Fully acetylated unnatural sugar probes are now commercially available and sold by companies such as Sigma-Aldrich and Click Chemical Tools. However, in 2018, it was first discovered that nonenzymatic S-side reactions occur between fully acetylated sugars and sulfhydryl groups of protein cysteines, resulting in false-positive signals in applications such as imaging and mass spectrometry identification. c. Partially protected unnatural sugars: After the discovery of the S side reaction, partially hydroxylated unnatural sugars were developed, which could easily cross the cell membrane without causing the S side reaction and achieve highly efficient glucose metabolic labeling.

[0003] Currently, there are two main methods for preparing unnatural sugars with an azide at the N-acyl position and unprotected hydroxyl groups. Method 1 involves first synthesizing a sugar derivative with a halogen at the N-acyl position and then replacing the halogen with azide to introduce the azide at the N-acyl position of the sugar. Method 2 involves first synthesizing an azidoacetic acid and then coupling the azidoacetic acid with an amino sugar to obtain an unnatural sugar containing an azide at the N-acyl position. Unnatural sugars containing other orthogonal functional groups (e.g., alkynyl groups) and unprotected hydroxyl groups can be synthesized by directly coupling an amino sugar with a small molecule containing bioorthogonal functional groups using Method 2 above.

[0004] However, both synthetic methods have drawbacks. Method 1 uses excess sodium azide and requires heating for the reaction, which poses a risk of explosion. Method 2 uses iodoacetic acid and excess sodium azide as raw materials to synthesize azidoacetic acid. To increase the yield, an aqueous solution containing excess sodium azide is acidified and azidoacetic acid is extracted with an organic solvent, which also poses a risk of explosion. Furthermore, azidoacetic acid has a low boiling point and is highly volatile, requiring careful handling and limited synthesis. Because unprotected unnatural sugars are highly polar, when silica gel columns are used for chromatographic purification, impurities and the product co-elute. Therefore, repeated column chromatographic purification is required to obtain sufficiently pure unprotected sugars. Several reports have used P-2 gel columns to purify unprotected sugars, but this process is time-consuming, the gel columns are expensive, and only small amounts of product can be purified.

[0005] Currently, partially protected unnatural sugars are classified into two types: 1,3-diacylated and 1,6-diacylated. While many 1,3-diacylated unnatural sugars have been reported, only one 1,6-diacylated unnatural sugar, 1,6-Pr2GalNAz, has been reported. The synthesis of 1,3-diacylated unnatural sugars begins with the synthesis of an unprotected unnatural sugar. The 4- and 6-hydroxyl groups of the starting sugar are then protected with propylidene, followed by acylation of the 1- and 3-hydroxyl groups. Finally, the propylidene protection is removed to obtain the 1,3-diacylated unnatural sugar. This synthetic process suffers from the aforementioned problems when synthesizing unprotected sugar starting materials, and also results in low overall yields when synthesizing 1,3-diacylated unnatural sugars from unprotected unnatural sugars. When synthesizing 1,6-Pr2GalNAz, it is necessary to use azidoacetic acid as a raw material, but the synthesis of azidoacetic acid has the above-mentioned problems.

[0006] Currently, there are two major problems with unnatural sugars used in chemoenzymatic labeling and metabolic labeling of sugars. First, some unnatural sugars (such as fully acetylated sugars) can lead to false-positive results in metabolic labeling, so the sugar structures need to be optimized. Research into partially protected sugars is in its infancy and requires urgent development. Second, existing methods for synthesizing these unnatural sugars have many drawbacks, including safety, yield, and difficulty in mass production.

[0007] Regarding the first issue, there are reports in the literature that fully acetylated unnatural sugars cause S side reactions when used for metabolic labeling, while partially acylated unnatural sugars can avoid S side reactions and improve the efficiency of unnatural sugars crossing the cell membrane and entering cells. According to publicly reported information, these partially acylated unnatural sugars include 1,3-Ac2GalNAz, 1,3-Pr2GalNAz, 1,3-Pr2ManNAz, 1,3-Pr2GlcNAz, 1,3-Pr2GlcNAl, and 1,6-Pr2GalNAz. Other novel partially acylated sugars with significant value in metabolic labeling of sugars have not been reported, and their synthetic methods and application research are needed.

[0008] Regarding the second problem, existing methods for synthesizing unnatural sugars have many drawbacks. To synthesize valuable unnatural sugars more economically and efficiently, new synthetic methods need to be developed. For example, when synthesizing azide-containing unnatural sugars, excessive sodium azide is used in the synthesis process, which poses an explosion risk. The reaction requires heating, making the process complicated and unsafe. Furthermore, the reaction product requires repeated purification using a column, which increases costs and makes large-scale synthesis difficult. The above two problems have significantly limited applied research related to unnatural sugars, so it is necessary to develop new strategies to solve the above two major bottlenecks. Summary of the Invention [Problem to be solved by the invention]

[0009] The main objective of the present invention is to provide unnatural sugars, methods for their synthesis and applications, to solve the technical problems of existing technologies in the synthesis and application of unnatural sugars. [Means for solving the problem]

[0010] In accordance with one aspect of the present invention, a method for synthesizing unnatural sugars is provided, which includes: The hydroxyl groups of amino sugars are protected with tris(trimethylsilyl)silane at room temperature to selectively expose the amino groups of the sugars, which are then converted by coupling at room temperature to yield unnatural sugars with orthogonal functional groups (bioorthogonal groups) protected with tris(trimethylsilyl)silane. Furthermore, removal of the trimethylsilane protecting group from the unnatural sugar having the tris(trimethylsilyl)silane-protected orthogonal functional groups (Bioorthogonal groups) yields an unnatural sugar having no protecting group. The unnatural sugar not having a protecting group is any one of GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl, and ManNAl. During the synthesis of GalNAz, GlcNAz, and ManNAz, the trimethylsilane protecting group is removed using a hydrogen ion exchange resin, and the final product precipitates from the solvent. During the synthesis of ManNAl, removal of the trimethylsilane protecting group is followed by purification by silica gel column chromatography. Furthermore, the 1- and 6-hydroxyl groups of the unnatural sugars bearing the tris(trimethylsilyl)silane-protected orthogonal functional groups (Bioorthogonal groups) are protected with hydrophobic groups to give partially acylated unnatural sugars. Furthermore, the hydrophobic group is any one of an acetyl group, a propionyl group, a butyryl group, and a valeryl group. Furthermore, in the case of unnatural sugars having orthogonal functional groups (bioorthogonal groups) protected with tris(trimethylsilyl)silane of galactose type and glucose type, pyridine is used as a solvent, and four equivalents of a carboxylic acid and an excess of the corresponding anhydride are added to exchange the trimethylsilane protecting groups at the 1st and 6th positions of the unnatural sugar having orthogonal functional groups (bioorthogonal groups) protected with tris(trimethylsilyl)silane with the corresponding ester bond. Then, the trimethylsilane protecting groups at the 3rd and 4th positions are removed with a hydrogen ion exchange resin to obtain 1,6-diacylated unnatural sugars. In the case of unnatural sugars with mannose-type tris(trimethylsilyl)silane-protected orthogonal functional groups (Bioorthogonal groups), two equivalents of ammonium acetate are added to a mixed solvent of dichloromethane and methanol to simultaneously and selectively remove the trimethylsilane protecting groups at the 1- and 6-positions, and then the ester bonds at the 1- and 6-positions are protected with pyridine. The trimethylsilane protecting groups at the 3- and 4-positions of the sugar are then removed to obtain the 1,6-diacylated mannose-type unnatural sugar derivative. Furthermore, the orthogonal functional groups (bioorthogonal groups) of the unnatural sugars protected with tris(trimethylsilyl)silane are any of azide, terminal alkyne, terminal alkene, cyclopropene, transcyclooctene, and cyclooctyne. In accordance with another aspect of the present invention, partially acylated unnatural sugars for use in metabolic labeling are disclosed, which are any of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 1,6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl, and 1,6-Pr2ManNProc. Preferably, the partially acylated unnatural sugars used in the metabolic labeling are prepared by the methods described above. The present invention also discloses a sugar metabolism labeling kit containing the partially acylated unnatural sugar. The present invention also discloses the application of partially acylated unnatural sugars in a glucose metabolism labeling kit for labeling the glucose metabolism of any of HeLa cells, 3T3 cells, CHO cells, and MCF-7 cells. [Effects of the Invention]

[0011] By utilizing the above technical solutions, the present invention has at least the following advantageous effects: The unnatural sugars, their synthesis methods, and applications provided by the present invention provide a method for efficiently synthesizing unprotected unnatural sugars in large quantities (10 gram levels). The reaction conditions are mild, the procedure is simple, and column purification is not required. These unnatural sugars include unprotected unnatural sugars and unnatural sugars with partially protected hydroxyl groups. The partially protected hydroxyl groups disclosed in the present invention take into account the advantages of existing unnatural sugars, ensuring efficient cell utilization of unnatural sugars and effectively avoiding side reactions with protein cysteines during the metabolism of unnatural sugars. At the same time, efficient metabolic labeling is achieved. In cell experiments, the concentration of 1,6-diacylated unnatural sugars used is one order of magnitude lower than that of unprotected unnatural sugars. [Brief explanation of the drawings]

[0012] In order to more clearly describe the embodiments of the present invention and the technical solutions of the prior art, the drawings necessary for describing the embodiments and the prior art are briefly introduced below. However, the drawings below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work. [Figure 1] 1 shows diagrams of reaction results of incubating 1,6-diacylated sugars with HeLa cell protein lysates and single proteins, according to one embodiment of the present invention. [Figure 2] FIG. 1 shows a diagram of results demonstrating that 1,6-diacylated sugars provide efficient metabolic labeling in HeLa cells, according to one embodiment of the present invention. [Figure 3] FIG. 1 shows an illustration of efficient metabolic labeling of 1,6-diacylated sugars in different cells according to one embodiment of the present invention. [Figure 4] FIG. 1 shows an efficient metabolic labeling diagram using 1,6-Pr2GalNAz and 1,6-Pr2ManNAz in vivo in mice according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further describes in detail the embodiments of the present invention in conjunction with the drawings in conjunction with examples. It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are intended to distinguish different entities or different parameters with the same name. Therefore, "first" and "second" are only for the convenience of description, but should not be understood as limiting the embodiments of the present invention, which will not be described point by point in the following embodiments.

[0014] The present invention discloses a method for synthesizing unnatural sugars by protecting the hydroxyl groups of amino sugars with tris(trimethylsilyl)silane at room temperature, selectively exposing the amino groups of the sugars, and then converting the amino groups by coupling at room temperature to obtain unnatural sugars having orthogonal functional groups protected with tris(trimethylsilyl)silane. The orthogonal functional groups of the unnatural sugars having orthogonal functional groups protected with tris(trimethylsilyl)silane are azides, terminal alkynes, terminal alkenes, cyclopropenes, transcyclooctene, or cyclooctynes. Removal of the trimethylsilane protecting groups from the unnatural sugars having orthogonal functional groups protected with tris(trimethylsilyl)silane yields unprotected unnatural sugars. Among these, unnatural sugars without protecting groups are GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl, and ManNAl. In the synthesis of GalNAz, GlcNAz, and ManNAz, the trimethylsilane protecting group is removed using a hydrogen ion exchange resin, and the final product is precipitated from the solvent. In the synthesis of ManNAl, the trimethylsilane protecting group is removed and the product is purified by silica gel column chromatography.

[0015] In some embodiments of the present invention, the 1- and 6-hydroxyl groups of the unnatural sugar having the tris(trimethylsilyl)silane-protected orthogonal groups (bioorthogonal groups) are protected with a hydrophobic group to obtain a partially acylated unnatural sugar, where the hydrophobic group is an acetyl group, a propionyl group, a butyryl group, or a valeryl group. In the case of unnatural sugars with orthogonal functional groups protected with tris(trimethylsilyl)silane of galactose type and glucose type, four equivalents of carboxylic acid and an excess of the corresponding anhydride are added to pyridine as a solvent, and the trimethylsilane protecting groups at the 1st and 6th positions of the unnatural sugars with orthogonal functional groups protected with tris(trimethylsilyl)silane are exchanged with the corresponding ester bond. The trimethylsilane protecting groups at the 3rd and 4th positions are then removed by hydrogen ion exchange resin to obtain 1,6-diacylated unnatural sugars. However, in the case of unnatural sugars with orthogonal functional groups protected with tris(trimethylsilyl)silane of mannose type, the trimethylsilane protecting groups at the 3rd and 4th positions are exchanged with the corresponding ester bond. In the case of unnatural sugars with 1,6-diacylated mannose-type unnatural sugar derivatives (which also belong to unnatural sugars), two equivalents of ammonium acetate are added to a mixed solvent of dichloromethane and methanol to simultaneously and selectively remove the trimethylsilane protecting groups at the 1st and 6th positions, and then the ester bonds at the 1st and 6th positions are protected with pyridine. The trimethylsilane protecting groups at the 3rd and 4th positions of the sugar are then removed to give 1,6-diacylated mannose-type unnatural sugar derivatives (which also belong to unnatural sugars).

[0016] Some embodiments of the present invention also disclose partially acylated unnatural sugars for metabolic labeling. These unnatural sugars are hexoses with a pyranose structure and acylation modifications at the 1- and 6-hydroxyl groups of the sugar. These unnatural sugars have the 1- and 6-hydroxyl groups protected with hydrophobic groups, and unnatural sugars are typically hexose derivatives in which some of the hydroxyl groups are protected. The hydrophobic groups are acetyl, propionyl, butyryl, or valeryl. The partially acylated unnatural sugars for metabolic labeling are prepared by the synthetic method described above. Specifically, the hydroxyl groups of the sugar are protected with tris(trimethylsilyl)silane (TMS), selectively exposing the amino groups of the sugar, which can then be conjugated or chemically transformed to yield unnatural sugars bearing fully TMS-protected orthogonal functional groups (bioorthogonal groups). The system's fully TMS-protected orthogonal functional groups (bioorthogonal groups) enable the efficient synthesis of unprotected and partially acylated unnatural sugars. The unprotected unnatural sugars are GalNAz, GlcNAz, ManNAz, ManNAl, and ManNProc. The partially acylated unnatural sugars are 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 1,6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl, and 1,6-Pr2ManNProc.

[0017] Some embodiments of the present invention also disclose a glucose metabolism labeling kit comprising the partially acylated unnatural sugar. The hydroxyl groups at positions 1 and 6 of the partially acylated unnatural sugar are protected. The partially acylated unnatural sugar is a hexose analogue in which the hydroxyl groups at positions 1 and 6 are protected with hydrophobic groups. This kit can be used to label the glucose metabolism of any cell, including HeLa cells, 3T3 cells, CHO cells, and MCF-7 cells.

[0018] The above-described embodiments of the present invention have developed a large-scale and efficient synthesis of unprotected unnatural sugars (target compound category 1) and an efficient method for the synthesis of 1,6-diacylated unnatural sugars (target compound category 2) based on complete TMS (trimethylsilane) protection.

[0019] Overall, embodiments of the present invention utilize amino sugars containing six carbon atoms, and protect all hydroxyl groups on the sugar with TMS to selectively expose the sugar amino groups for further coupling and transformation to obtain fully TMS-protected unnatural sugars bearing orthogonal functional groups (bioorthogonal groups). The selective exposure of amino groups by complete TMS protection prevents the sugar hydroxyl groups from participating in or interfering with subsequent coupling reactions without TMS protection. On the other hand, coupling can be performed in a hydroxyl-free solvent, thereby avoiding the possibility of solvent hydroxyl groups participating in or interfering with the coupling reaction (without TMS protection, amino sugars are soluble only in hydroxyl-containing solvents such as water or methanol). Therefore, complete TMS protection allows for low amounts of molecules to be coupled to the sugar amino groups (one equivalent or slightly more than one equivalent is sufficient), high coupling efficiency (near-quantitative reactions), and solvent evaporation under reduced pressure (typically dichloromethane is used as the coupling solvent). Unnatural sugars equipped with fully TMS-protected orthogonal functional groups (Bioorthogonal groups) are key intermediates for the synthesis of target compound category 1 and target compound category 2 of this patent.

[0020] The fully TMS-protected unnatural sugars obtained by the above method can be removed with a hydrogen ion exchange resin without the need for column chromatography purification, to obtain unprotected unnatural sugars (target compound category 1, for which GalNAz, GlcNAz, ManNAz, ManNAl, etc. have been successfully synthesized in large quantities). It is particularly noteworthy that when synthesizing GalNAz, GlcNAz, and ManNAz using this synthetic method, no chromatographic purification step is required, and the final products are precipitated from ethanol. When synthesizing ManNAl, precipitation is not possible, but it can be rapidly purified by silica gel column chromatography.

[0021] Furthermore, the fully TMS-protected unnatural sugar obtained by the above method can be purified through a simple silica gel column, followed by a simple two-step reaction to obtain the 1,6-diacylated unnatural sugar. Two technical processes have been developed for different sugars, namely: In the case of fully TMS-protected unnatural sugars of the galactose and glucose types, pyridine is used as the solvent, and four equivalents of a carboxylic acid and an excess of the corresponding anhydride are added to exchange the TMS at positions 1 and 6 of the fully TMS-protected unnatural sugar with the corresponding ester bonds. The TMS protecting groups at positions 3 and 4 are then removed using a hydrogen ion exchange resin to obtain the 1,6-diacylated unnatural sugar. In the case of a fully TMS-protected unnatural sugar of the mannose type, two equivalents of ammonium acetate are added to a mixed solvent of dichloromethane and methanol to simultaneously and selectively remove the TMS protecting groups at positions 1 and 6 of the sugar, and the ester bonds at positions 1 and 6 are protected with pyridine, followed by removal of the TMS protecting groups at positions 3 and 4 of the sugar to obtain a 1,6-diacylated unnatural sugar derivative of the mannose type.

[0022] Based on the above synthesis mechanism, 1,6-diacylated unnatural sugars (target compound category 2), including 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 1,6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl, and 1,6-Pr2ManNProc, can be synthesized with high efficiency.

[0023] In particular, there is a literature report on the synthesis of 1,6-Pr2GalNAz based on fully TMS-protected GalNAz, but this method requires the use of azidoacetic acid as a starting material when synthesizing fully TMS-protected GalNAz. As mentioned above, the synthesis of azidoacetic acid carries the risk of explosion. The synthetic method disclosed in the present embodiment avoids the problem of synthesizing azidoacetic acid and is systematically extended to other sugars.

[0024] From a technical perspective, the advantages of the synthetic method disclosed in the embodiments of the present invention over existing synthetic methods are that the fully TMS-protected sugar derivatives have low polarity and good solubility in many low-polarity solvents (e.g., dimethylformamide, dichloromethane, ethyl acetate, petroleum ether, acetonitrile, etc.), allowing for easy chemical transformations (e.g., replacing halogens with azides) to obtain unnatural sugars bearing various types of fully TMS-protected orthogonal functional groups (bioorthogonal groups). In the above synthetic method, all reactions are performed at room temperature and no chromatographic purification step is required, making large-scale synthesis (10 gram level) possible. The synthesis of azide-containing unnatural sugars requires only one equivalent of sodium azide, allowing for safe operation in small quantities.

[0025] From an application perspective, the synthesized 1,6-diacylated sugars can easily cross the cell membrane and enter cells, effectively avoiding S side reactions and maintaining high metabolic efficiency in glucose metabolic labeling studies. In summary, the main purpose of this invention is to provide a method for the efficient and large-scale synthesis of unnatural sugars and to promote their application research. The newly reported 1,6-diacylated unnatural sugars in this invention do not induce S side reactions and can improve the efficiency of metabolic labeling of sugars. It should be specifically stated that, unless there is a contradiction, the embodiments and features of the embodiments of the present application can be combined with each other.

[0026] Unnatural sugars: Natural monosaccharides are chemically modified to attach bioorthogonal groups, such as azide or alkynyl groups. Unnatural sugars are taken up by cells and incorporated into glycans through natural sugar metabolic pathways, allowing subsequent labeling, imaging, and enrichment of glycans through bioorthogonal reactions. Bioorthogonal reactions: Chemical reactions that can be carried out within living cells and tissues without interfering with the biochemical reactions of the organism itself. They are used in the study of biological macromolecules such as nucleic acids, proteins, sugars, and lipids.

[0027] The present invention solves the problems of chemical synthesis of unnatural sugars reported in chemoenzymatic and metabolic labeling of sugars, and discloses 1,6-diacylated unnatural sugars with novel structures.

[0028] This application studies and improves existing methods for synthesizing unnatural sugars, developing a mechanism based on complete TMS protection to develop a method for large-scale synthesis of unprotected unnatural sugars. At the same time, 1,6-diacylated unnatural sugars can be synthesized with high efficiency based on fully TMS-protected unnatural sugars. 1,6-diacylated unnatural sugars can easily cross cell membranes and enter cells, effectively avoiding S side reactions and maintaining high metabolic efficiency in glucose metabolic labeling tests.

[0029] Specifically, this application uses an amino sugar containing six carbon atoms, and protects all hydroxyl groups of the sugar with TMS to selectively expose the sugar's amino group. Coupling of the amino group with a small molecule yields a variety of fully TMS-protected acyl-substituted amino sugar derivatives. These fully TMS-protected sugar derivatives have low polarity and good solubility in many low-polarity solvents (e.g., dimethylformamide, dichloromethane, ethyl acetate, petroleum ether, acetonitrile, etc.). Chemical transformations (e.g., halogen substitution with azide) can be performed to obtain a variety of fully TMS-protected unnatural sugars (fully TMS-protected unnatural sugars containing orthogonal functional groups such as azide and alkynyl groups). The TMS protecting groups are removed using a hydrogen ion exchange resin in methanol, filtered, and the filtrate is concentrated. Adding ethanol results in a precipitated solid, which is the unprotected unnatural sugar. The process for synthesizing unnatural sugars without protecting groups using the above method allows for large-scale synthesis (10 gram level) because all reactions are performed at room temperature and do not require a chromatographic purification step. The synthesis of azide-containing unnatural sugars requires only one equivalent of sodium azide, allowing for safe operation in small quantities. Based on this TMS protection mechanism, the applicant has successfully synthesized GalNAz, GlcNAz, ManNAz, and ManNAl in large quantities.

[0030] In the above process for synthesizing unnatural sugars without protecting groups, fully TMS-protected unnatural sugars containing orthogonal functional groups (Bioorthogonal groups) are key intermediates. Based on these intermediates, 1,6-diacylated unnatural sugars can be obtained by two simple reactions. For fully TMS-protected unnatural sugars of the galactose and glucose types, pyridine is used as the solvent, and four equivalents of a carboxylic acid and an excess of the corresponding anhydride are added to convert the TMS at positions 1 and 6 of the fully TMS-protected unnatural sugar to the corresponding ester bonds. After the TMS protecting groups at positions 3 and 4 are removed, 1,6-diacylated unnatural sugars are obtained. For fully TMS-protected unnatural sugars of the mannose type, two equivalents of ammonium acetate are used in a mixed solvent of dichloromethane and methanol to simultaneously and selectively remove the TMS protecting groups at positions 1 and 6 of the sugar, and then pyridine is used to protect the sugar at positions 1 and 6 with ester bonds. The TMS protecting groups at positions 3 and 4 of the sugar are then removed to obtain 1,6-diacylated unnatural sugar derivatives of the mannose type.

[0031] In carbohydrate metabolic labeling studies, we found that none of the 1,6-diacylated sugars caused S-side reactions with proteins, and that the 1,6-diacylated sugars exhibited high metabolic labeling efficiency. Furthermore, compared with unprotected sugars, different ester-linked protecting groups improved metabolic labeling efficiency to different degrees. For example, while the acetyl group did not significantly improve efficiency compared with the propionyl group, but butyryl and propionyl modifications significantly improved the metabolic efficiency of unnatural sugars. Finally, 1,6-dipropionylated sugars can also be used for efficient metabolic labeling in vivo in mice.

[0032] In some embodiments of the present invention, when unprotected unnatural sugars are synthesized based entirely on the TMS mechanism, the orthogonal functional groups contained therein are not limited to azide or alkynyl groups, and unprotected unnatural sugars containing other orthogonal functional groups can also be synthesized using this mechanism. Other orthogonal functional groups include, but are not limited to, terminal alkenes, cyclopropene, trans-cyclooctene, and cyclooctyne. Therefore, the synthesis of these unprotected unnatural sugars is covered by this patent.

[0033] In some embodiments of the present invention, when unprotected unnatural sugars are synthesized based entirely on the TMS mechanism, applicable substrates are not limited to galactosamine, glucosamine, and mannosamine derivatives, and other amino-containing sugars can be used as substrates for such synthesis. Therefore, the synthesis of these unprotected unnatural sugars is covered by this patent.

[0034] In some embodiments of the present invention, when synthesizing partially acylated unnatural substances based entirely on the TMS mechanism, the acyl groups used are not limited to acetyl, propionyl, or butyryl groups; other acyl groups can be introduced using the methods described herein. Therefore, other acylated partially acylated unnatural sugars are also covered by this patent.

[0035] In Example 1 of the present application, we provide a simple and efficient method for synthesizing unnatural sugars without protecting groups, specifically GalNAz, GlcNAz, ManNAz, and ManNAl, as well as the partially acylated unnatural sugars described above. Example 2 of the present application provides a sugar metabolism labeling kit containing any of the above partially acylated unnatural sugars. Example 3 of the present application provides an application of any of the above partially acylated unnatural sugars and the above kits in metabolic labeling of sugars. The use of the partially acylated unnatural sugar metabolic labels of the present application not only achieves higher metabolic efficiency but also prevents side reactions with protein cysteines. In a preferred embodiment, the above application includes metabolic labeling of sugars in cells such as HeLa cells, MCF-7 cells, CHO cells, and 3T3 cells. In a preferred embodiment, metabolic labeling is performed in vivo in mice using 1,6-Pr2GalNAz and 1,6-Pr2ManNAz.

[0036] When synthesizing unnatural sugars containing partially protected azides using existing techniques, sodium azide is required, posing a risk of explosion, especially when synthesizing large quantities at the 10-gram level. The synthesis method for 1,6-Pr2GalNAz according to the present invention is more efficient and safer than previously reported methods. Compared with other reported 1,3-diacylated unnatural sugars, the 1,6-diacylated unnatural sugars of the present invention are a better choice based on the mechanism of S-glycosylation side reactions on protein sulfhydryl groups and the stability of partially acylated sugars. Furthermore, the synthesis of 1,6-diacylated sugars according to the present invention is simpler and more efficient than fully protected sugars. The beneficial effects of the present invention will be further explained below in conjunction with examples. [Example]

[0037] Example 1: Synthesis of GalNAz, GlcNAz and ManNAz. For the synthesis of GalNAz, GlcNAz, and ManNAz, refer to Synthesis Process 1. [ka] The conditions for steps I, II, III, and IV are as follows: GalN hydrochloride (compound 1a, 100 mmol, 21.5 g) was dispersed in 200 mL of anhydrous acetonitrile, and 40.3 g (250 mmol) of HMDS was added in portions. The mixture was allowed to react at room temperature for 3 hours. After filtration, the filtrate was concentrated in vacuo to give compound 1b as an oil, which was used directly in the next step without further purification.

[0038] 27.6 g (200 mmol) of bromoacetic acid and 25.3 g (220 mmol) of HOSU were dissolved in 220 mL of dichloromethane, and the solution was cooled to -5°C. A dichloromethane solution (50 mL) containing 45.3 g (220 mmol) of DCC was added dropwise to the above solution over 1 hour. After the addition was complete, the reaction system was warmed to room temperature and reacted for 2 hours. After filtration, the filtrate was concentrated in vacuo to obtain a residue. This residue was treated with 200 mL of n-hexane and thoroughly stirred to obtain a mixture. The mixture was filtered, washed with n-hexane, and dried in vacuo to obtain 36.0 g of crude 2,5-dioxopyrrolidin-1-yl 2-bromoacetate (4a). Compound 4a was used directly in the next reaction without purification. All of the compound 1b obtained in the previous step was dissolved in 200 mL of dichloromethane and cooled in an ice-water bath. 33.3 g (140 mmol) of 4a was then added. The reaction mixture was stirred at room temperature for 2 hours, concentrated in vacuo to a constant weight, and 200 mL of normal hexane and 20 mL of ethyl acetate were added and stirred. The mixture was precipitated and filtered, and the filtrate was spin-dried to obtain compound 1c as a yellow oil. 1c was used directly in the next step without further purification.

[0039] 1c obtained in the previous step was dissolved in 150 mL of DMF, and 6.8 g (100 mmol) of sodium azide was added. The mixture was stirred overnight at room temperature. The mixture was diluted with 200 mL of ethyl acetate, and the organic phase was washed with water. The aqueous phase was extracted once with 100 mL of ethyl acetate. The organic phase was washed twice with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under vacuum to give 1d as a yellow oil, which was used directly in the next step without further purification.

[0040] 1d was dissolved in 150 ml of methanol, 10 g of hydrogen ion exchange resin (Dowex) was added, and the mixture was stirred at room temperature for 2 hours. TLC showed the reaction was complete. The reaction was filtered, the filtrate was spin-dried, 200 ml of ethanol was added, and the mixture was stirred and filtered. The resulting mixture was collected and dried under vacuum to give 11.6 g of product 1e. The yield over the four steps was 44%, but the product was in the α-structure.

[0041] The nuclear magnetic resonance detection data for compound 1e (GalNAz) is 1 H NMR (500 MHz, DMSO-d6) δ7.78 (d, J = 8.7 Hz, 1H), 6.43 (dd, J = 4.4, 1.2 Hz, 1H), 4.95 (t, J = 3.9 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 4.3 Hz, 1H), 4.45 - 4.40 (m, 2H), 4.01 (ddd, J = 11.5, 8.7, 3.4 Hz, 1H), 3.85 (d, J = 15.4 Hz, 1H), 3.83 - 3.79 (m, 1H), 3.80 (d, J = 15.4 Hz, 1H), 3.76 - 3.70 (m, 1H), 3.68 - 3.58 (m, 1H), 3.58 - 3.50 (m, 1H), 3.43 (dd, J= 10.7, 6.2 Hz, 1H). 13 C NMR (126 MHz, DMSO-d) δ 167.7, 90.9, 70.6, 68.3, 67.4, 60.7, 50.7, 50.6.

[0042] Compound 2e (GlcNAz, 9.6 g) was obtained using 21.5 g (100 mmol) of GlcN hydrochloride as the starting material according to the synthesis method of GalNAz described in Example 1. The yield over the four steps was 37%. The product was an anomeric mixture, α / β, 5 / 1. The nuclear magnetic resonance data for compound 2e (GlcNAz) were as follows: 1H NMR (α isomer, 500 MHz, methanol-d4) δ 5.11 (d, J = 3.5 Hz, 1H), 3.95 (d, J = 15.8 Hz, 1H), 3.92 (s, 1H), 3.87 (dd, J = 10.7, 3.7 Hz, 1H), 3.83 - 3.76 (m, 2H), 3.74 - 3.67 (m, 2H), 3.38 (t, J = 9.2 Hz, 1H). 13 C NMR (126 MHz, Methanol-d4) δ 170.9, 170.4, 96.8, 92.5, 78.1, 75.8, 73.2, 72.8, 72.4, 72.2, 62.9, 62.8, 59.0, 55.9, 53.2, 52.9.

[0043] Compound 3e (ManNAz, 10.8 g) was obtained from 21.5 g (100 mmol) of ManN hydrochloride according to the synthesis of GalNAz described in Example 1. The yield over the four steps was 41%. The product was an anomeric mixture, α / β, 2 / 1. The nuclear magnetic resonance detection data for compound 3e (ManNAz) is as follows: 1 H NMR (500 MHz, Methanol-d4) δ5.03 (d, J = 1.6 Hz, 1H), 4.29 (dd, J = 4.7, 1.7 Hz, 1H), 4.02 (dd, J = 9.7, 4.7 Hz, 1H), 3.93 (s, 1H), 3.89 (d, J = 15.8 Hz, 1H), 3.86 - 3.74 (m, 3H), 3.55 (t, J = 9.6 Hz, 1H). 13 C NMR (151 MHz, Methanol-d4) δ 171.5, 170.6, 94.6, 94.5, 78.1, 74.1, 73.2, 70.2, 68.3, 68.0, 62.0, 61.9, 55.7, 55.0, 52.6, 52.4, 49.6.

[0044] Example 2: Synthesis of ManNAl. For the synthesis of ManNAl, see Synthesis Scheme 2. [ka] The conditions for steps I and II are as follows: 2.16 g (22 mmol) of pentynic acid and 2.78 g (24.2 mmol) of HOSU were dissolved in 50 mL of dichloromethane. The solution was cooled to -5°C, and 4.98 g (24.4 mmol) of DDC in 20 mL of dichloromethane was added dropwise over 20 minutes. The reaction was allowed to warm to room temperature and stirred for 2 hours to complete the reaction. After filtration, the filtrate was concentrated to give product 4b (2,5-dioxopyrrolidin-1-yl pent-4-ynoate), which was used directly in the next step without further purification.

[0045] Crude compound 3b was synthesized using 18.7 mmol of ManN hydrochloride (3a) according to the method of Example 1. The crude 3b was dissolved in 50 mL of dichloromethane and cooled to 0°C in an ice-water bath. 4b, obtained in the previous reaction, was added dropwise to the solution. The reaction mixture was stirred at room temperature for 2 hours, and upon completion, the mixture was concentrated in vacuo to obtain a residue. 100 mL of normal hexane was added to the residue, which was stirred and filtered. The filtrate was concentrated in vacuo to obtain a yellow oil 3f, which was used directly in the next step without further purification.

[0046] The 3f obtained in the previous step was dissolved in 50 ml of methanol and 2 g of hydrogen ion exchange resin was added. The reaction mixture was stirred at room temperature for 2 hours to allow the reaction to proceed completely. After filtration, the filtrate was concentrated in vacuo to give a residue, which was purified on a silica gel column to give 3.06 g of compound 3g (ManNAl). The yield over the three steps was 64%. The product was an anomeric mixture, α / β, 3 / 1.

[0047] The nuclear magnetic resonance detection data for compound 3g is as follows: 1H NMR (500 MHz, Methanol-d4) δ5.01 (d, J = 1.6 Hz, 1H), 4.29 (dd, J = 4.6, 1.7 Hz, 1H), 4.00 (dd, J = 9.7, 4.7 Hz, 1H), 3.87 - 3.74 (m, 3H), 3.58 (t, J= 9.7 Hz, 1H), 3.35 (s, 1H), 2.59 - 2.42 (m, 4H), 2.26 (t, J = 2.3 Hz, 1H). 13 C NMR (126 MHz, Methanol-d4) δ 175.5, 174.5, 94.8, 94.7, 83.8, 83.5, 78.0, 74.3, 73.2, 70.4, 70.0, 69.9, 68.3, 67.9, 62.0, 61.9, 55.6, 54.9, 35.9, 35.72, 35.67, 15.42, 15.40.

[0048] Example 3: Synthesis of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz and 1,6-Bu2GlcNAz. For the synthesis of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz, and 1,6-Bu2GlcNAz, see Synthesis Process 3. [ka] The conditions for steps I and II are as follows:

[0049] Step I: General method for synthesizing 1f, 1g, 1h, 2f, 2g, and 2h: 0.5 mmol of 1d or 2d was dissolved in 1 mL of pyridine. Under nitrogen protection, 0.75 mL of anhydride and 2.0 mmol of the corresponding carboxylic acid were added. After reacting at room temperature for 20 hours, the reaction mixture was diluted with ethyl acetate, and the organic phase was washed sequentially with 1 mol / L dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo, and the residue was purified on a silica gel column to give the corresponding product.

[0050] 1d. 1d is an intermediate from 1a to 1e and can be purified by flash chromatography on a silica gel column using petroleum ether and ethyl acetate as washings. 20 mmol of 1a can be used to synthesize 4.1 g of 1d, a 37% yield over three steps. The product is in the α-configuration. The nuclear magnetic resonance detection data for compound 1d is as follows: 1 H NMR (500 MHz, Chloroform-d) δ 6.29 (d, J = 8.0 Hz, 1H), 5.16 - 5.10 (m, 1H), 4.10 (dd, J= 10.4, 2.8 Hz, 1H), 3.92 (s, 2H), 3.87 (d, J= 2.8 Hz, 1H), 3.68 - 3.62 (m, 1H), 3.62 - 3.55 (m, 1H), 3.55 - 3.49 (m, 1H), 3.47 - 3.43 (m, 1H), 0.13 (s, 9H), 0.12 (s, 9H), 0.12 (s, 9H), 0.11 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 166.70, 94.63, 75.17, 71.24, 70.63, 61.04, 57.42, 53.18, 0.76, 0.44, 0.30, -0.40.

[0051] 2d. The synthesis and purification of 2d was carried out in a similar manner to that of 1d, but starting from 20 mmol of 2a, 5.91 g of 2d was synthesized, a 54% yield over three steps. The product is in the α-configuration. The nuclear magnetic resonance detection data for compound 2d is as follows: 1 H NMR (400 MHz, Chloroform-d) δ 6.36 (d, J = 10.1 Hz, 1H), 5.01 (d, J = 3.5 Hz, 1H), 4.07 - 3.94 (m, 3H), 3.76 - 3.64 (m, 3H), 3.63 - 3.56 (m, 2H), 0.17 (s, 9H), 0.15 (s, 9H), 0.12 (s, 9H), 0.09 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 166.24, 92.59, 74.04, 72.71, 72.07, 61.75, 54.53, 53.11, 1.05, 0.90, -0.13, -0.21.

[0052] 1f. 1f was synthesized according to the general method described in Example 3 above. The yield is 86%. The product is an anomeric mixture, α / β, 1 / 1.56. The detected data from 1f nuclear magnetic resonance is 1 H NMR (β isomer, 500 MHz, Chloroform-d) δ 6.20 (d, J = 8.8 Hz, 1H), 5.90 (d, J = 8.6 Hz, 1H), 4.18 (d, J = 2.0 Hz, 1H), 4.17 - 4.13 (m, 1H), 4.04 (dt, J= 10.5, 8.7 Hz, 1H), 3.94 (dd, J = 11.0, 2.7 Hz, 3H), 3.83 (d, J = 2.7 Hz, 1H), 3.78 (td, J = 6.3, 0.9 Hz, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 0.14 (s, 9H), 0.14 (s, 9H). 1H NMR (α anomer mixture, 500 MHz, Chloroform-d) δ 6.16 (d, J = 3.7 Hz, 1H), 6.03 (d, J = 9.4 Hz, 1H), 4.63 (td, J = 10.0, 3.6 Hz, 1H), 4.13 (d, J = 6.4 Hz, 2H), 4.05 (d, J = 16.8 Hz, 1H), 3.99 (d, J = 16.6 Hz, 1H), 3.98 (t, J = 6.3 Hz, 1H), 3.89 (d, J = 1.8 Hz, 1H), 3.81 (dd, J = 10.6, 2.6 Hz, 1H), 2.15 (s, 3H), 2.07 (s, 3H), 0.18 (s, 9H), 0.15 (s, 9H). 13 C NMR (α / β, 126 MHz, Chloroform-d) δ 170.73, 170.69, 169.7, 169.2, 166.8, 166.3, 92.3, 92.1, 73.55, 71.9, 71.2, 71.1, 70.8, 69.6, 63.2, 63.2, 52.9, 52.8, 52.8, 48.3, 21.1, 21.0, 20.9, 0.63, 0.55, 0.4, 0.36.

[0053] 1g. 1g was synthesized according to the general method described in Example 3 above. The yield is 89%. The product is an anomeric mixture, α / β, 1.25 / 1. The nuclear magnetic resonance detection data for compound 1g is as follows: 1H NMR (α and β anomer mixture, 500 MHz, Chloroform-d) δ 6.21 (d, J = 8.9 Hz, 1H), 6.18 (d, J = 3.6 Hz, 1H), 6.04 (d, J = 9.5 Hz, 1H), 5.90 (d, J = 8.6 Hz, 1H), 4.64 (td, J = 10.1, 3.6 Hz, 1H), 4.19 (d, J = 6.3 Hz, 2H), 4.15 (d, J = 6.5 Hz, 2H), 4.11 - 4.02 (m, 2H), 4.02 - 3.96 (m, 2H), 3.94 (d, J = 5.4 Hz, 2H), 3.91 - 3.88 (m, 1H), 3.85 - 3.80 (m, 2H), 3.80 - 3.76 (m, 1H), 2.46 - 2.30 (m, 8H), 1.23 - 1.09 (m, 12H), 0.18 (s, 9H), 0.15 (s, 19H), 0.14 (s, 8H). 13 C NMR (α and β anomer mixture, 126 MHz, Chloroform-d) δ 174.2, 174.2, 173.2, 172.6, 166.8, 166.2, 92.3, 91.9, 73.6, 72.0, 71.2, 71.1, 70.8, 69.7, 63.0, 62.9, 52.9, 52.8, 52.8, 48.3, 27.8, 27.6, 27.55, 27.52, 9.2, 9.17, 9.14, 8.9, 0.63, 0.56, 0.4, 0.3.

[0054] 1h. 1h was synthesized according to the general method described in Example 3 above. The yield was 70%. The product is an anomeric mixture, α / β, 2.55 / 1. The nuclear magnetic resonance detection data for compound 1h (GalNAz) are 1H NMR (500 MHz, Chloroform-d) δ 6.18 (d, J = 3.7 Hz, 1H), 6.02 (d, J = 9.5 Hz, 1H), 4.63 (td, J = 10.0, 3.6 Hz, 1H), 4.03 (d, J = 16.9 Hz, 1H), 4.00 (s, 1H), 3.98 - 3.95 (m, 1H), 3.92 (d, J= 4.2 Hz, 1H), 3.90 (d, J = 2.6 Hz, 1H), 3.83 (dd, J = 4.9, 2.6 Hz, 1H), 2.36 (t, J = 7.3 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H), 1.73 - 1.66 (m, 2H), 1.66 - 1.59 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H), 0.17 (s, 9H), 0.14 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 173.1, 173.0, 172.0, 171.4, 166.5, 166.0, 91.8, 91.5, 73.2, 71.8, 71.0, 70.9, 70.5, 69.4, 62.8, 62.5, 52.6, 52.4, 52.4, 48.0, 36.0, 35.8, 35.8, 35.4, 18.2, 18.2, 18.0, 17.9, 13.40, 13.37, 13.3, 13.2, 0.33, 0.26, 0.1, 0.01.

[0055] 2f. 2f was synthesized according to the general method described in Example 3 above. The yield was 62%. It is the α-isomer. The nuclear magnetic resonance detection data for compound 2f is as follows: 1H NMR (500 MHz, Methanol-d4) δ5.97 (d, J = 3.9 Hz, 1H), 4.37 (dd, J = 12.1, 2.3 Hz, 1H), 4.18 (dd, J = 10.1, 3.9 Hz, 1H), 4.07 (dd, J = 12.1, 4.3 Hz, 1H), 3.89 - 3.84 (m, 3H), 3.82 (dd, J= 10.1, 8.1 Hz, 1H), 3.69 (dd, J = 9.7, 8.1 Hz, 1H), 2.16 (s, 3H), 2.08 (s, 3H), 0.19 (s, 9H), 0.17 (s, 9H). 13 C NMR (126 MHz, Methanol-d4) δ 172.6, 171.4, 170.8, 92.4, 75.1, 74.0, 73.4, 64.4, 54.5, 53.0, 21.1, 21.0, 1.4, 1.2.

[0056] 2g. 2g was synthesized according to the general method described in Example 3 above. The yield was 62%. It is the alpha isomer. The nuclear magnetic resonance detection data for compound 2g is as follows: 1H NMR (500 MHz, Chloroform-d) δ 6.35 (d, J= 9.9 Hz, 1H), 6.06 (d, J = 3.5 Hz, 1H), 4.39 (dd, J = 12.1, 2.7 Hz, 1H), 4.28 (td, J = 9.6, 3.5 Hz, 1H), 4.10 (dd, J = 12.1, 4.8 Hz, 1H), 4.06 (d, J = 16.8 Hz, 1H), 4.00 (d, J = 16.8 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.75 (dd, J= 9.3, 7.6 Hz, 1H), 3.68 (dd, J = 8.5, 7.6 Hz, 1H), 2.42 (qd, J = 7.5, 0.9 Hz, 2H), 2.36 (qd, J = 7.6, 2.9 Hz, 2H), 1.19 (t, J = 7.5 Hz, 3H), 1.14 (t, J = 7.5 Hz, 3H), 0.16 (s, 9H), 0.16 (s, 9H). 13 C NMR (151 MHz, Chloroform-d) δ 173.8, 172.2, 166.0, 90.4, 73.2, 72.9, 71.3, 62.2, 52.4, 51.8, 27.4, 27.2, 8.82, 8.78.

[0057] 2h. 2h was synthesized according to the general method described in Example 3 above. The yield was 44%. It is the α-isomer. The nuclear magnetic resonance detection data for compound 2h is as follows: 1H NMR (500 MHz, Chloroform-d) δ 6.32 (d, J = 9.9 Hz, 1H), 6.07 (d, J = 3.5 Hz, 1H), 4.40 (dd, J = 12.1, 2.7 Hz, 1H), 4.28 (td, J = 9.7, 3.6 Hz, 1H), 4.08 (dd, J = 12.1, 4.9 Hz, 1H), 4.06 (d, J = 16.8 Hz, 1H), 4.00 (d, J = 16.8 Hz, 1H), 3.83 (ddd, J = 7.8, 4.8, 2.6 Hz, 1H), 3.75 (dd, J = 9.4, 7.7 Hz, 1H), 3.67 (dd, J = 8.7, 7.7 Hz, 1H), 2.37 (t, J = 7.3 Hz, 2H), 2.31 (td, J = 7.4, 3.7 Hz, 2H), 1.72 - 1.63 (m, 4H), 0.99 (t, J = 7.4 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H), 0.17 (s, 9H), 0.16 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 172.4, 170.8, 165.5, 89.8, 72.6, 70.8, 61.6, 51.8, 51.3, 35.3, 35.2, 17.6, 17.5, 12.8, 12.7, 1.4, It is 1.2.

[0058] General synthetic method for synthesizing 1i, 1j, 1k, 2i, 2j, and 2k: To a methanol solution of 1f, 1g, 1h, 2f, 2g, or 2h, an appropriate amount of hydrogen ion exchange resin was added and stirred at room temperature. After TLC showed the reaction was complete, it was filtered, the filtrate was concentrated in vacuo, and the residue was purified on a silica gel column to give the corresponding product.

[0059] 1i. 1i was synthesized according to the general method described in Example 3 above. The yield was 52%, with a 2 / 1 α / β ratio. The nuclear magnetic resonance and mass spectrometry detection data for compound 1i are as follows: 1H NMR (α isomer, 500 MHz, Methanol-d4) δ6.15 (d, J = 3.7 Hz, 1H), 4.42 (dd, J = 11.1, 3.7 Hz, 1H), 4.22 (d, J = 3.3 Hz, 1H), 4.21 (d, J = 1.5 Hz, 1H), 4.11 (ddd, J = 6.8, 5.1, 1.3 Hz, 1H), 3.94 (dd, J = 3.2, 1.3 Hz, 1H), 3.93 - 3.81 (m, 4H), 2.12 (s, 3H), 2.04 (s, 3H). 13 C NMR (mixture of α and β isomers, 126 MHz, methanol-d4) δ 171.2, 169.9, 169.7, 169.6, 169.5, 92.8, 91.0, 73.6, 70.78, 70.77, 68.4, 69.0, 67.3, 63.4, 63.3, 51.8, 51.6, 51.3, 49.1, 19.4, 19.3. HRMS (ESI) calculated molecular weight for C12H19N4O8 [M+H]+: 347.12029, detected molecular weight: 347.11959.

[0060] 1j. 1j was synthesized according to the general method described in Example 3 above. The yield was 83% with an α / β ratio of 1.45 / 1. The nuclear magnetic resonance and mass spectrometry detection data for compound 1j are as follows: 1H NMR (α isomer, 500 MHz, Methanol-d4) δ6.16 (d, J = 3.7 Hz, 1H), 4.41 (dd, J = 11.1, 3.7 Hz, 1H), 4.10 (ddd, J = 6.8, 5.1, 1.3 Hz, 1H), 3.94 (dd, J = 3.3, 1.3 Hz, 1H), 3.90 (d, J = 15.8 Hz, 1H), 3.88 (dd, J = 11.1, 3.2 Hz, 1H), 3.83 (d, J = 15.8 Hz, 1H), 2.42 (qd, J = 7.6, 1.9 Hz, 2H), 2.33 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H), 1.10 (t, J = 7.6 Hz, 3H). 13 C NMR (126 MHz, Methanol-d4) δ175.71, 175.68, 174.4, 174.2, 170.7, 170.6, 94.0, 92.0, 74.8, 72.0, 71.9, 69.6, 69.1, 68.6, 64.4, 64.2, 53.0, 52.8, 52.5, 50.4, 28.0, 28.0, 27.97, 27.94, 9.2, 9.14, 9.06, 8.9. HRMS(ESI) theoretical molecular weight is C 14 H 23 N4O8[M+H] + 375.15159 and the detected molecular weight is 375.15076.

[0061] 1k. 1k was synthesized according to the general method described in Example 3 above. The yield was 69% with an α / β ratio of 2.67 / 1. The nuclear magnetic resonance and mass spectrometry detection data for compound 1k are as follows: 1H NMR (α isomer, 500 MHz, Methanol-d4) δ6.17 (d, J = 3.7 Hz, 1H), 4.41 (dd, J = 11.1, 3.7 Hz, 1H), 4.34 - 4.19 (m, 3H), 4.09 (ddd, J = 7.4, 4.7, 1.3 Hz, 1H), 3.94 (dd, J = 3.2, 1.3 Hz, 1H), 3.92 - 3.80 (m, 5H), 2.39 (t, J= 7.3 Hz, 2H), 2.35 - 2.27 (m, 4H), 1.72 - 1.57 (m, 6H), 1.00 - 0.91 (m, 9H). 13 C NMR (126 MHz, Methanol-d4) δ 173.7, 173.6, 172.3, 172.2, 169.5, 169.4, 92.7, 90.7, 73.6, 70.90, 70.86, 68.5, 68.0, 67.4, 63.4, 63.0, 51.8, 51.6, 51.3, 49.2, 35.5, 35.42, 35.41, 18.00, 17.96, 17.8, 12.5, 12.5, 12.4. HRMS (ESI) theoretical molecular weight is C 16 H 27 N4O8[M+H] + 403.18289 and the detected molecular weight is 403.18234.

[0062] 2i. 2i was synthesized according to the general method described in Example 3 above. The yield was 69%. It is the α-isomer. The nuclear magnetic resonance and mass spectrometry detection data for compound 2i are as follows: 1H NMR (500 MHz, Methanol-d4) δ 6.10 (d, J= 3.7 Hz, 1H), 4.33 (dd, J = 12.1, 2.3 Hz, 1H), 4.23 (dd, J = 12.1, 5.2 Hz, 1H), 4.04 (dd, J = 10.8, 3.6 Hz, 1H), 3.90 (d, J = 15.8 Hz, 1H), 3.87 - 3.80 (m, 2H), 3.73 (dd, J= 10.8, 8.8 Hz, 1H), 3.45 (dd, J = 10.2, 8.8 Hz, 1H), 2.13 (s, 3H), 2.06 (s, 3H). 13 C NMR (126 MHz, Methanol-d4) δ 172.7, 171.1, 170.8, 91.9, 73.5, 72.0, 71.8, 64.4, 54.4, 52.6, 20.7, 20.6. HRMS (ESI) theoretical molecular weight is C 12 H 19 N4O8[M+H] + 347.12029 and the detected molecular weight is 347.11922.

[0063] 2j. 2j was synthesized according to the general method described in Example 3 above. The yield was 79%. It is the α-isomer. The nuclear magnetic resonance and mass spectrometry detection data for compound 2j are as follows: 1H NMR (500 MHz, Methanol-d4) δ 6.12 (d, J = 3.6 Hz, 1H), 4.35 (dd, J = 12.0, 2.2 Hz, 1H, C6-Ha), 4.23 (dd, J = 12.0, 5.4 Hz, 1H, C6-Hb), 4.04 (dd, J = 10.8, 3.7 Hz, 1H), 3.90 (d, J = 15.9 Hz, 1H, CH2a), 3.87 - 3.80 (m, 2H, C5-H), 3.73 (dd, J = 10.8, 8.8 Hz, 1H), 3.45 (dd, J = 10.1, 8.8 Hz, 1H), 2.45 (qd, J = 7.5, 1.1 Hz, 2H), 2.36 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 6.8 Hz, 3H), 1.12 (t, 3H). 13 C NMR (126 MHz, Methanol-d4) δ175.8, 174.2, 170.6, 91.6, 73.3, 71.9, 71.6, 64.1, 54.3, 52.4, 28.0, 9.2, 9.0. HRMS (ESI) theoretical molecular weight is C 14 H 23 N4O8[M+H] + 375.15159 and the detected molecular weight is 375.15102.

[0064] 2k. 2k was synthesized according to the general method described in Example 3 above. The yield was 78%. It is the α-isomer. The nuclear magnetic resonance and mass spectrometry detection data for compound 2k are as follows: 1H NMR (500 MHz, Methanol-d4) δ 6.12 (d, J = 3.6 Hz, 1H), 4.37 (dd, J = 11.9, 2.2 Hz, 1H), 4.21 (dd, J = 12.0, 5.7 Hz, 1H), 4.03 (dd, J = 10.8, 3.7 Hz, 1H), 3.90 (d, J = 15.8 Hz, 1H), 3.85 - 3.80 (m, 2H), 3.73 (dd, J= 10.8, 8.8 Hz, 1H), 3.43 (dd, J = 10.1, 8.8 Hz, 1H), 2.40 (td, J = 7.2, 1.2Hz, 2H), 2.32 (t, J = 7.3 Hz, 2H), 1.72 - 1.66 (m, 2H), 1.67 - 0.96 (m, 8H), 0.98 (t, J= 7.4 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). 13 C NMR (126 MHz, Methanol-d4) δ 174.9, 173.3, 170.5, 91.4, 73.4, 71.9, 71.7, 64.1, 54.3, 52.4, 36.6, 36.6, 19.2, 19.1, 13.72, It is 13.68. HRMS (ESI) theoretical molecular weight is C 16 H 27 N4O8[M+H] + 403.18289 and the detected molecular weight is 403.18225.

[0065] Example 4: Synthesis of 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc. For the synthesis of 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl, and 1,6-Pr2ManNProc, refer to Synthesis Process 4. JPEG2025535890000005.jpg41170The conditions for steps I and II are as follows:

[0066] 3d. The synthesis and purification of 3d was carried out in a similar manner to 1d, but based on 20 mmol of 3a. 4.1 g of 3d was synthesized in 37% yield over three steps. This is the alpha isomer. The nuclear magnetic resonance detection data for compound 3d is as follows: 1 H NMR (500 MHz, Chloroform-d) δ6.43 (d, J = 7.9 Hz, 1H), 5.10 (d, J = 1.5 Hz, 1H), 4.15 - 4.06 (m, 2H), 4.00 (d, J= 16.5 Hz, 1H), 3.95 (d, J = 16.3 Hz, 1H), 3.77 (dd, J = 11.3, 3.4 Hz, 1H), 3.72 - 3.60 (m, 3H), 0.16 (s, 9H), 0.16 (s, 9H), 0.15 (s, 9H), 0.12 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 166.77, 93.39, 72.77, 70.10, 68.85, 61.65, 55.60, 53.00, 0.74, 0.37, -0.04, -0.25.

[0067] 3f. 3.95 g (8.44 mmol, 1.0 equiv.) of 3b was dissolved in 50 mL of anhydrous dichloromethane, and 1.65 g (8.44 mmol, 1.0 equiv.) of 2,5-dioxopyrrolidin-1-yl pent-4-ynoate (4b) was added. The reaction was stirred at room temperature for 2 h. TLC indicated the reaction was complete. The solvent was spin-evaporated to give a residue, which was purified through a silica gel column to give 4.40 g of product 3f. The yield was 95%. This is the α isomer. The nuclear magnetic resonance detection data for compound 3f is as follows: 1H NMR (500 MHz, Chloroform-d) δ 5.78 (d, J = 7.1 Hz, 1H), 5.14 (d, J= 1.4 Hz, 1H), 4.11 - 4.05 (m, 2H), 3.73 (dd, J = 11.4, 4.2 Hz, 1H), 3.69 (dd, J = 11.4, 2.1 Hz, 1H), 3.65 (ddd, J = 9.6, 4.2, 2.1 Hz, 1H), 3.55 (t, J = 9.0 Hz, 1H), 2.55 - 2.48 (m, 2H), 2.46 - 2.40 (m, 2H), 2.01 (t, J = 2.6 Hz, 1H), 0.15 (s, 18H), 0.13 (s, 9H), 0.11 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 171.14, 93.38, 83.12, 72.51, 70.08, 69.54, 69.06, 61.87, 55.71, 35.55, 14.84.

[0068] 3g. 3.95 g (8.44 mmol, 1.0 equiv.) of 3b was dissolved in 50 mL of a mixture of anhydrous dichloromethane and pyridine (1 / 1 volume ratio). The reaction was cooled to 0 °C in an ice-water bath, and 1.0 g (8.44 mmol, 1.0 equiv.) of prop-2-yn-1-yl carbonochloridate (4c) was added. The reaction was allowed to warm to room temperature over 2 hours and stirred overnight, at which point TLC indicated completion. The residue was concentrated in vacuo and purified on a silica gel column to give 4.59 g of product 3g. The yield was 99%. This is the α isomer. The nuclear magnetic resonance detection data for compound 3g is as follows: 1H NMR (500 MHz, Chloroform-d) δ5.17 (d, J = 1.8 Hz, 1H), 4.99 (d, J= 7.5 Hz, 1H), 4.68 (d, J = 2.6 Hz, 2H), 4.05 (dd, J = 9.1, 4.7 Hz, 1H), 3.79 (ddd, J = 7.0, 4.7, 1.7 Hz, 1H), 3.69 (d, J = 3.3 Hz, 2H), 3.64 (dt, J = 9.3, 3.2 Hz, 1H), 3.49 (t, J = 9.2 Hz, 1H), 2.48 (t, J = 2.5 Hz, 1H), 0.15 (s, 9H), 0.14 (d, J = 2.0 Hz, 18H), 0.10 (s, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 155.51, 93.46, 78.27, 74.68, 72.70, 70.19, 68.94, 61.88, 57.33, 52.58, 0.76, 0.28, -0.10, -0.27.

[0069] 3h. 16.80 g (30.49 mmol, 1.0 equiv.) of 3b was dissolved in 125 mL of a mixture of dichloromethane and 125 mL of methanol, and 4.70 g (60.98 mmol, 2.0 equiv.) of ammonium acetate was added. The reaction mixture was stirred at room temperature for 16 hours, concentrated in vacuo, treated with 100 mL of ethyl acetate, filtered, and the filtrate was concentrated and purified on a silica gel column to give 7.69 g of 3h in a 62% yield. This product is an anomeric mixture, α / β, 1.0 / 0.3. The nuclear magnetic resonance detection data for compound 3h is as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.75 (d, J = 8.8 Hz, 1H), 7.42 (d, J= 10.0 Hz, 0.3H), 6.66 (d, J = 4.5 Hz, 1H), 6.60 (d, J = 6.9 Hz, 0.3H), 4.85 (dd, J = 4.5, 1.3 Hz, 1H), 4.79 (dd, J = 6.9, 1.3 Hz, 0.3H), 4.42 - 4.37 (m, 1.3H), 4.20 (dd, J= 10.0, 3.1 Hz, 0.3H), 4.09 - 4.00 (ddd, J =8.8, 4.7, 1.5 Hz, 1H), 3.94 - 3.81 (m, 3.6H), 3.69 - 3.54 (m, 3.6H), 3.54 - 3.42 (m, 1.6H), 3.18 - 3.13 (m, 0.3H). 13 C NMR (126 MHz, DMSO-d6) δ 167.92, 167.66, 92.90, 92.49, 76.83, 73.29, 72.68, 70.17, 68.63, 68.51, 60.79, 60.71, 54.49, 54.16, 50.90, 50.64, 40.02, 39.86, 39.69, 39.52, 39.35, 39.19, 39.02, 0.81, 0.77, 0.19, 0.16. HRMS (ESI) theoretical molecular weight is C 14 H 31 N4O6Si2[M+H] + 407.59400 and the detected molecular weight is 407.59736.

[0070] 3i. 3i was synthesized from 3F, but the synthesis method was the same as that of 3h in Example 4. The yield was 67%. This product was an anomeric mixture, α / β, 4 / 1. The nuclear magnetic resonance detection data for compound 3i is as follows: 1H NMR (500 MHz, Methanol-d4) δ 4.96 (d, J= 1.6 Hz, 1H), 4.27 (dd, J = 4.6, 1.7 Hz, 1H), 4.03 (dd, J = 8.2, 4.6 Hz, 1H), 3.81 - 3.74 (m, 4H), 3.73 - 3.69 (m, 1H), 2.54 - 2.42 (m, 5H), 2.26 - 2.22 (m, 1H), 0.18 - 0.14 (m, 22H). 13 C NMR (126 MHz, Methanol-d4) δ 175.19, 95.96, 84.61, 74.69, 72.91, 71.31, 70.90, 62.97, 56.60, 37.03, 16.97, 1.93, 1.42.

[0071] 3j. 3j was synthesized from 3g using the synthesis method of 3h in Example 4 with a yield of 63%. This product is an anomeric mixture, α / β, 1.0 / 0.11. The nuclear magnetic resonance detection data for compound 3j is as follows: 1 H NMR (500 MHz, Chloroform-d) δ 5.34 (s, 1H), 5.16 (d, J = 6.4 Hz, 1H), 4.69 (d, J= 2.5 Hz, 2H), 4.06 (dt, J = 10.3, 5.1 Hz, 1H), 3.92 (ddd, J = 6.6, 4.7, 1.7 Hz, 1H), 3.85 (ddd, J = 9.4, 4.8, 2.5 Hz, 1H), 3.77 (dd, J = 11.8, 2.6 Hz, 1H), 3.69 (dd, J = 11.7, 5.4 Hz, 1H), 3.58 - 3.49 (m, 1H), 3.37 - 3.26 (m, 1H), 2.49 (t, J= 2.4 Hz, 1H), 0.16 (s, 9H), 0.15 (s, 9H). 13C NMR (126 MHz, Chloroform-d) δ 155.72, 93.12, 77.72, 75.33, 74.96, 70.00, 68.80, 61.85, 55.95, 53.42, 0.71, 0.23.

[0072] 3k. 550 mg (1.35 mmol, 1.0 equiv.) of 3h was dissolved in 10 mL of pyridine and cooled to 0 °C in an ice-water bath. Under nitrogen protection, 552 mg (5.41 mmol, 4.0 equiv.) of acetic anhydride was added. The reaction was allowed to warm to room temperature for 18 hours, after which 218 μL of methanol was added to quench the reaction. The mixture was stirred at room temperature for 1 hour and then concentrated in vacuo to a constant weight. The residue was dissolved in 50 mL of methanol, 5 g of hydrogen ion exchange resin was added, and the mixture was stirred at room temperature for 2 hours. TLC indicated the reaction was complete. After filtration, the filtrate was concentrated in vacuo, and the residue was purified on a silica gel column to give 331 mg of compound 3k. The yield for the two steps was 68%. α / β ratio was 1.0 / 0.28. The nuclear magnetic resonance detection data for compound 3k is as follows: 1 H NMR (500 MHz, Methanol-d4, α isomer) δ 5.95 (d, J = 1.8 Hz, 1H), 4.33 (dd, J = 11.9, 2.3 Hz, 1H), 4.29 (dd, J = 4.8, 1.9 Hz, 1H), 4.25 (dd, J = 12.0, 6.5 Hz, 1H), 3.97 (dd, J = 9.6, 4.7 Hz, 1H), 3.95 (s, 1H), 3.94 (s, 1H), 3.81 (ddd, J = 10.1, 6.4, 2.4 Hz, 1H), 3.58 (t, J = 9.7 Hz, 1H), 2.12 (s, 3H), 2.05 (s, 3H). 13 C NMR (151 MHz, Methanol-d4, α isomer) δ 172.6, 170.8, 170.1, 101.1, 93.2, 73.7, 69.8, 68.2, 64.6, 53.3, 52.3, 20.50, 20.47. 1H NMR (500 MHz, Methanol-d4, β isomer) δ 5.79 (d, J = 1.8 Hz, 1H), 4.58 (dd, J = 4.6, 1.8 Hz, 1H), 4.42 (dd, J = 12.0, 2.2 Hz, 1H), 4.29 (dd, J = 11.9, 7.0 Hz, 1H), 3.99 (s, 1H), 3.99 (s, 1H), 3.80 (dd, J = 9.3, 4.6 Hz, 1H), 3.61 (ddd, J = 9.3, 7.0, 2.3 Hz, 1H), 3.48 (t, J = 9.5 Hz, 1H), 2.08 (s, 3H), 2.06 (s, 3H). 13 C NMR (126 MHz, Methanol-d4, β isomer) δ 170.9, 169.4, 168.5, 91.2, 75.0, 71.0, 66.7, 63.0, 51.4, 50.8, 18.84, 18.77. HRMS (ESI) theoretical molecular weight is C 12 H 19 N4O8[M+H] + 347.12029 and the detected molecular weight is 347.11914.

[0073] 3l. 3l was obtained by reacting 3h with propionic anhydride as raw materials following the synthesis process of 3k in Example 4. The yield over the two steps was 81%. It is the α-isomer. The nuclear magnetic resonance detection data for compound 3l is as follows: 1H NMR (600 MHz, D2O) δ 5.96 (s, 1H), 4.47 (dd, J = 4.7, 1.3 Hz, 1H), 4.42 (dd, J = 12.2, 2.0 Hz, 1H), 4.30 (dd, J = 12.2, 6.2 Hz, 1H), 4.15 (dd, J = 9.8, 4.8 Hz, 1H), 4.11 (s, 2H), 3.98 - 3.94 (m, 1H), 3.70 (t, J = 9.9 Hz, 1H), 2.51 (qd, J= 7.5, 1.9 Hz, 2H), 2.44 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H), 1.11 (t, J = 7.6 Hz, 3H). 13 C NMR (151 MHz, D2O) δ 177.3, 175.2, 171.1, 92.0, 72.1, 68.5, 66.6, 63.3, 51.71, 51.58, 27.21, 8.32, 8.15. HRMS(ESI) theoretical molecular weight is C 14 H 23 N4O8[M+H] + 375.15159 and the detected molecular weight is 375.15095.

[0074] 3m. 3m was obtained by reacting 3h with butyric anhydride as raw materials, following the synthesis process of 3k in Example 4. The yield for the two steps was 79%. The α / β ratio was 1.35 / 1. The 3m nuclear magnetic resonance detection data shows that the α isomer, 11H NMR (500 MHz, Methanol-d4) δ 5.98 (d, J = 1.8 Hz, 1H), 4.37 (dd, J = 11.9, 2.2 Hz, 1H), 4.29 (dd, J = 4.9, 1.9 Hz, 1H), 4.25 (dd, J = 11.9, 7.0 Hz, 1H), 4.00 - 3.96 (m, 2H), 3.94 (d, J = 15.9 Hz, 1H), 3.81 (ddd, J = 9.5, 6.9, 2.2 Hz, 1H), 3.57 (t, J = 9.7 Hz, 1H), 2.39 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.68 (q, J = 7.3 Hz, 2H), 1.63 (q, J = 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H). 13 13C NMR (126 MHz, Methanol-d4) δ 175.0, 172.6, 170.7, 92.9, 73.8, 69.9, 68.3, 64.4, 53.3, 52.4, 36.63, 36.59, 19.2, 19.1, 13.72, 13.66. β isomer, 1 1H NMR (500 MHz, Methanol-d4) δ 5.79 (d, J = 1.8 Hz, 1H), 4.56 (dd, J = 4.6, 1.8 Hz, 1H), 4.39 (dd, J = 11.9, 2.3 Hz, 1H), 4.32 (dd, J = 12.0, 7.1 Hz, 1H), 3.97 (s, 1H), 3.97 (s, 1H), 3.79 (dd, J = 9.4, 4.5 Hz, 1H), 3.59 (ddd, J = 9.6, 7.1, 2.4 Hz, 1H), 3.45 (t, J = 9.4 Hz, 1H), 2.35 - 2.32 (m, 2H), 2.32 - 2.28 (m, 2H), 1.63 (dh, J = 11.7, 7.4 Hz, 4H), 0.94 (t, J = 7.4 Hz, 6H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, Methanol-d4) δ 175.0, 172.6, 171.0, 92.7, 76.7, 72.8, 68.6, 64.4, 53.2, 52.5, 36.6, 36.5, 19.2, 18.8, 13.7, It is 13.6. HRMS (ESI) theoretical molecular weight is C 16 H 27 N4O8[M+H] + The detected molecular weight is 403.18289 and 403.18199.

[0075] 3n. 3n was synthesized in two steps from 3i. The synthesis method is similar to that of 3l in Example 4. The yield over the two steps was 83%. The α / β ratio was 1.0 / 0.33. The nuclear magnetic resonance detection data for compound 3n is as follows: 1 H NMR (600 MHz, D2O) δ 5.94 (d, J = 1.8 Hz, 1H, α-H-1), 5.86 (d, J = 1.8 Hz, 0.33H, β-H-1), 4.63 (dd, J = 4.6, 1.8 Hz, 0.33H, β-H-2), 4.44 (m, 2.33H, β-H-6a + α-H-2 +α-H-6a), 4.35 - 4.31 (m, 0.33H, β-H-6b), 4.28 (dd, J = 12.2, 6.3 Hz, 1H, α-H-6b), 4.14 (dd, J = 9.8, 4.9 Hz, 1H, α-H-3), 3.98 - 3.92 (m, 1.33H, α-H-5-a+β-H-3), 3.81 - 3.70 (m, 1.33H, β-H-5+α-H-4), 3.63 (t, J = 9.8 Hz, 0.33H, β-H-4), 2.67 - 2.35 (m, 11.97H, COC H 2CH2CCH+ 2 × COC H 2CH3), 1.11 (m, 7.98H, 2 × COCH2C H 3). 13 C NMR (151 MHz, D2O) δ 177.29 (β- C OCH2CH3-6), 177.26 (α-C OCH2CH3-6), 175.91 (β-NH C O), 175.44 (α-NH C O), 175.18 (α- C OCH2CH3-1), 174.95 (β- C OCH2CH3-1), 92.22 (α-C-1), 91.77 (β-C-1), 83.58, 83.35, 74.87 (β-C-5), 72.04 (α-C-5), 70.90, 70.22, 70.11, 68.42 (α-C-3), 66.84 (β-C-4), 66.67 (α-C-4), 63.31 (α-C-6), 63.29 (β-C-5), 51.77 (β-C-2), 51.63, 34.32, 34.08, 27.23, 27.21, 27.19, 27.15, 14.48, 14.43, 8.33, 8.29, 8.16, 7.97.

[0076] 3o. 3o was synthesized in two steps from 3j. The synthesis method is similar to that of 3l in Example 4. The yield over the two steps was 79%. The α / β ratio was 1.0 / 0.15. The nuclear magnetic resonance detection data for compound 3o is as follows: 1 H NMR (500 MHz, D2O) δ 6.02 (s, 1.0H, α-H-1), 5.88 (d, J = 1.4Hz, 0.15 H, β-H-1), 4.78 - 4.69 (m, 2.30H, NHCOOC H2CCH), 4.49 (m, 1.15H, α-H-6a+β-H-6a), 4.37 - 4.26 (m, 1.30H, β-H-2+α-H-6b+β-H-6b), 4.21 (d, J = 3.6 Hz, 1.0H, α-H-2), 4.14 (dd, J = 9.7, 4.7 Hz, 1.0H, α-H-3), 3.97 (ddd, J = 8.7, 6.5, 2.1 Hz, 1.15H, α-H-5+β-H-3), 3.78 (ddd, J = 9.2, 6.7, 2.1 Hz, 0.15H, β-H-5), 3.69 (t, J = 9.9 Hz, 1.0H, α-H-4), 3.60 (t, J = 9.8 Hz, 0.15H, β-H-4), 3.10 - 2.93 (m, 1.15 H, NHCOOCH2CC H ), 2.57 - 2.43 (m, 4.6H, 2 × CH3C H 2COO), 1.20 - 1.12 (m, 6.9H, 2 × C H 3CH2COO). 13 C NMR (126 MHz, D2O) δ 177.40 (β-CH3CH2 C OO-6), 177.38 (α-CH3CH2 C OO-6), 175.25 (α-CH3CH2 C OO-1), 174.98 (β-CH3CH2 C OO-1), 158.33 (β-NH C OOCH2CCH), 157.61 (α-NHCOOCH2 C CH), 92.62 (α-C-1), 92.04 (β-C-1), 78.59 (β-NHCOOCH2 C CH), 78.50 (α-NHCOOCH2 C CH), 75.93 (α-NHCOOCH2C C H), 75.87 (β-NHCOOCH2C CH), 74.92 (β-C-5), 72.21 (α-C-5), 71.00 (β-C-3), 68.73 (α-C-3), 66.98 (β-C-4), 66.82 (α-C-4), 63.53 (α-C-6), 63.46 (β-C-6), 54.05 (β-C-2), 53.43 (α-C-2), 53.18 (NHCOO C H2CCH), 27.35 (CH3 C H2COO), 27.30 (CH3 C H2COO), 27.26 (CH3 C H2COO), 8.45 ( C H3CH2COO), 8.41 ( C H3CH2OCOO), 8.30 ( C H3CH2COO), 8.13 ( C H3CH2COO).

[0077] Example 5: In vitro reaction of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, and 1,6-Bu2ManNAz with proteins. To verify that the nine partially acylated unnatural sugars, 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, and 1,6-Bu2ManNAz, do not spontaneously react with protein cysteines, we incubated these partially acylated unnatural sugars with HeLa cell lysates or single proteins, using fully acetylated unnatural sugars (Ac4GalNAz, Ac4GlcNAz, and Ac4ManNAz) as controls, for 2 hours at 37°C, and then detected the azide signals of the proteins on an electrophoretic gel. The reaction results are shown in Figure 1.

[0078] Example 6: Use of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz for metabolic labeling of live cells. To verify the metabolic labeling effect of the above 1,6-diacylated unnatural sugars in live cells, we used the above different compounds to metabolically label HeLa cells. HeLa cells were cultured with 200 μM of 1,6-diacylated unnatural sugars and 2 mM of unprotected sugars, and then bioorthogonal reactions were performed using Cy5 fluorescently labeled substances. The HeLa cells were then subjected to in-gel fluorescence detection. The detection results are shown in Figure 2 (CBB in Figure 3 indicates Coomassie Brilliant Blue staining, as a control for sample loading).

[0079] As can be seen in Figure 2, the labeling efficiency of 200 μM 1,6-Pr2GalNAz and 1,6-Pr2ManNAz is close to or slightly stronger than that of 2 mM unprotected sugars compared with GalNAz and ManNNAz. Compared with 1,6-Pr2GalNAz and 1,6-Pr2ManNAz, the labeling efficiency of 1,6-Bu2GalNAz and 1,6-Bu2ManNAz is comparable, but the labeling efficiency of 1,6-Ac2GalNAz and 1,6-Ac2ManNAz is much lower (Figure 2a and b). In MCF-7 cells expressing GFP (MCF7-T2A-GFP), treatment with GlcNAz and 1,6-diacylated GlcNAz did not produce significant labeling (Figure 2c). However, by overexpressing the mutant AGX2 enzyme AGX2F383G, this enzyme converts GlcNAz-P- to UDP-GlcNAz, and in MCF7-AGX2F383G-T2A-GFP cells, GlcNAz and its three corresponding 1,6-diacylated derivatives can produce significant labeling (Figure 2c). The above data clearly demonstrate that 1,6-diacylated azido sugars can achieve metabolic labeling at much lower concentrations than unprotected azido sugars.

[0080] Example 7: Use of 1,6-Pr2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc for metabolic labeling of live cells. Different cell lines were metabolically labeled with 200 μM 1,6-Pr2ManNAz, 1,6-Pr2ManNAl, and 1,6-Pr2ManNProc for 48 hours. Then, the cells were lysed and the lysates were reacted with a Cy5 fluorescently labeled probe and glycoproteins bearing orthogonal functional groups to perform bioorthogonal reactions. After SDS-PAGE separation, in-gel fluorescence analysis was performed. The results are shown in Figure 3 (CBB in Figure 3 indicates Coomassie Brilliant Blue staining, indicating sample loading control).

[0081] Example 8: Use of 1,6-Pr2GalNAz and 1,6-Pr2ManNAz for metabolic labeling in vivo in mice. In vivo mouse studies, B6D2F1 / J mice were intraperitoneally administered 1,6-Pr2GalNAz or 1,6-Pr2ManNAz (both at 500 mg / kg) daily for 3 or 7 consecutive days. Mice were euthanized on days 4 or 8, and different organs were selected and homogenized. azide-modified glycoproteins were click-labeled and separated by SDS-PAGE. In-gel fluorescence analysis of proteins demonstrated that glycoproteins from the heart, lung, and spleen could be successfully labeled, demonstrating that 1,6-Pr2GalNAz and 1,6-Pr2ManNAz are well suited for in vivo metabolic labeling (Figure 4).

[0082] It is particularly important to note that the components and steps in the above embodiments can be mutually interchanged, substituted, added or deleted, and therefore, combinations formed by these reasonable substitutions, combinations and transformations are also included within the scope of protection of the present invention, and the scope of protection of the present invention should not be limited to the described embodiments.

[0083] The above are exemplary embodiments of the present invention. However, the order of the disclosed embodiments of the present invention is for illustrative purposes only and does not represent the merits or demerits of the embodiments. However, the above discussion of any embodiment is merely illustrative, and does not imply that the scope of the disclosed embodiments of the present invention (including the claims) is limited to these examples. However, it should be noted that various changes and modifications can be made without departing from the limited scope of the claims. The method of the embodiments disclosed herein does not require the functions, steps, and / or actions of the claims to be performed in any particular order. Furthermore, elements disclosed in the embodiments of the present invention may be described or claimed individually, but may also be understood in the plural unless expressly limited to the singular.

[0084] Those skilled in the art will understand that the discussion of any of the above embodiments is merely illustrative and does not mean that the disclosure scope of the embodiments of the present invention (including the claims) is limited to these examples. Based on the concept of the embodiments of the present invention, the technical features of the above embodiments or different embodiments can be combined, and there are many other modifications in different aspects of the above embodiments of the present invention, which are not described in detail for simplicity. Therefore, it should be understood that any omissions, modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the embodiments of the present invention should be included in the protection scope of the embodiments of the present invention.

Claims

1. A method for synthesizing unnatural sugars, comprising protecting the hydroxyl groups of amino sugars with tris(trimethylsilyl)silane at room temperature to selectively expose the amino groups of the sugars, and then converting the amino groups by coupling at room temperature to obtain unnatural sugars with orthogonal functional groups (bioorthogonal groups) protected with tris(trimethylsilyl)silane.

2. 2. The method of claim 1, wherein removal of the trimethylsilane protecting group from the unnatural sugar having tris(trimethylsilyl)silane-protected orthogonal functional groups (Bioorthogonal groups) yields an unprotected unnatural sugar.

3. the unnatural sugar having no protecting group is any one of GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl, and ManNAl; During the synthesis of GalNAz, GlcNAz, and ManNAz, the trimethylsilane protecting group is removed using a hydrogen ion exchange resin, and the final product precipitates from the solvent. The method according to claim 2, characterized in that during the synthesis of ManNAl, the trimethylsilane protecting group is removed and purified by silica gel column chromatography.

4. The method of claim 1, wherein the 1- and 6-hydroxyl groups of the unnatural sugar having the tris(trimethylsilyl)silane-protected orthogonal functional groups (bioorthogonal groups) are protected with hydrophobic groups to obtain a partially acylated unnatural sugar.

5. 5. The method according to claim 4, wherein the hydrophobic group is any one of an acetyl group, a propionyl group, a butyryl group, and a valeryl group.

6. In the case of unnatural sugars with galactose-type and glucose-type bioorthogonal groups protected with tris(trimethylsilyl)silane, pyridine is used as a solvent, and four equivalents of a carboxylic acid and an excess of the corresponding anhydride are added to exchange the trimethylsilane protecting groups at the 1st and 6th positions of the unnatural sugar with the bioorthogonal groups protected with tris(trimethylsilyl)silane with the corresponding ester bond. The trimethylsilane protecting groups at the 3rd and 4th positions are then removed using a hydrogen ion exchange resin to obtain 1,6-diacylated unnatural sugars. For unnatural sugars with mannose-type tris(trimethylsilyl)silane-protected orthogonal functional groups (bioorthogonal groups), the method of claim 4 is characterized in that two equivalents of ammonium acetate are added to a mixed solvent of dichloromethane and methanol to simultaneously and selectively remove the trimethylsilane protecting groups at the 1- and 6-positions, and then the ester bonds at the 1- and 6-positions are protected with pyridine, followed by removing the trimethylsilane protecting groups at the 3- and 4-positions of the sugar to obtain a 1,6-diacylated mannose-type unnatural sugar derivative.

7. The method of claim 1, wherein the orthogonal functional groups of the unnatural sugar having the tris(trimethylsilyl)silane-protected orthogonal functional groups are any of azide, terminal alkyne, terminal alkene, cyclopropene, transcyclooctene, and cyclooctyne.

8. any one of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 1,6-Ac2GlcNAz, 1,6-Pr2GlcNAz, 1,6-Bu2GlcNAz, 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc, A partially acylated unnatural sugar for metabolic labeling, preferably prepared using the method according to any one of claims 1 to 7.

9. A sugar metabolism labeling kit comprising the partially acylated unnatural sugar of claim 8.

10. 9. The use of the partially acylated unnatural sugar in the sugar metabolism labeling kit described in claim 8, characterized in that the sugar metabolism of any of HeLa cells, 3T3 cells, CHO cells, and MCF-7 cells is labeled.

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