Non-natural sugars, methods of synthesis and use thereof

Abstract

The application discloses a non-natural sugar and a synthesis method and application thereof. At room temperature, hydroxyl groups of an amino sugar are fully trimethylsilyl-protected, amino groups on the sugar are selectively exposed, and after coupling and conversion of the amino groups at room temperature, a non-natural sugar with orthogonal groups and full trimethylsilyl protection is obtained. The non-natural sugar with orthogonal groups and full trimethylsilyl protection is removed from trimethylsilyl protection groups, and a non-natural sugar without protection groups is obtained. The application takes into account the advantages of existing non-natural sugars, ensures that the non-natural sugars can be efficiently utilized by cells, effectively avoids S side reactions with cysteine in proteins in the metabolic process of the non-natural sugars, and simultaneously realizes efficient metabolic labeling. In a cell test, the use concentration of 1,6-bisacylated non-natural sugar is one order of magnitude lower than that of non-natural sugar without protection groups.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic chemical synthesis, in particular to a non-natural sugar and a synthesis method and application thereof. BACKGROUND

[0002] Glycosylation modification has important functions, and it is of great significance to observe glycosylation modification. In cells, metabolic labeling with chemically reported non-natural sugars has been widely used for glycan imaging and glycoproteomics analysis. In vitro, chemical enzymatic method adds chemically reported non-natural sugars to the sugar chain under the action of glycosyltransferase for further research.

[0003] Non-natural sugars are necessary for metabolic labeling and chemical enzymatic labeling. They are divided into three categories:

[0004] a. Non-natural sugars without protecting groups: can be used for metabolic labeling but require high concentration, and are also the synthesis substrates of non-natural nucleoside sugars used by chemical enzymatic method;

[0005] b. Fully acetylated sugars: developed to overcome the poor cell permeability of non-natural sugars without protecting groups, significantly reducing the required concentration for metabolic labeling. Existing fully acetylated protected non-natural sugar probes have been commercialized and are sold by companies such as Sigma-Aldrich and Click Chemical Tools.

[0006] However, in 2018, it was first discovered that fully acetylated sugars undergo non-enzymatic S side reactions with the thiol group of protein cysteine, introducing false positive signals for imaging, mass spectrometry identification, and other applications.

[0007] c. Partially protected non-natural sugars: after the discovery of S side reactions, partially acylated non-natural sugars emerged, which can easily pass through the cell membrane to achieve efficient glycometabolic labeling while not introducing S side reactions.

[0008] At present, there are mainly two methods for preparing non-natural sugars with azide at the N-acyl site and without protection of the hydroxyl group. Method one is to first synthesize sugar derivatives with halogen at the N-acyl site, and then introduce azide into the N-acyl site of the sugar by replacing halogen with azide. Method two is to first synthesize azidoacetic acid, and then couple azidoacetic acid and amino sugar to obtain non-natural sugar with azide at the N-acyl site. Non-natural sugars containing other orthogonal groups (such as alkyne) and without protection of the hydroxyl group are synthesized by the strategy of directly coupling amino sugar and small molecules containing orthogonal groups in the above method two.

[0009] But both of these two methods have disadvantages. In the first method, sodium azide is in large excess and the reaction needs to be heated, which has the risk of explosion. In the second method, the synthesis of azidoacetic acid uses iodacetic acid and excess sodium azide as raw materials. In order to improve the yield of azidoacetic acid, the aqueous solution containing excess sodium azide needs to be acidified and the azidoacetic acid needs to be extracted with an organic solvent. This operation has the risk of explosion. Moreover, azidoacetic acid has a low boiling point and is volatile, so it needs to be handled carefully and can only be synthesized in small quantities. Unprotected unnatural sugars have a large polarity, so impurities and products often co-elute when using silica gel column chromatography for purification. Therefore, repeated column chromatography is needed to obtain sufficiently pure unprotected sugars. A few reports use P-2 gel columns to purify unprotected sugars, but this operation is time-consuming and the gel column is expensive and can only be used for small-scale product purification.

[0010] Currently, partially protected unnatural sugars are divided into two categories: 1,3-bisacylated unnatural sugars and 1,6-bisacylated unnatural sugars. There are many reported examples of 1,3-bisacylated unnatural sugars, and only one example of 1,6-bisacylated unnatural sugar, 1,6-Pr2GalNAz. The synthesis of 1,3-bisacylated unnatural sugars is first to synthesize an unnatural sugar without a protecting group, which is used as a starting material. The 4 and 6 positions of the sugar are protected with propylidene, and then the 1 and 3 positions of the hydroxyl group are acylated. Finally, the propylidene protection is removed to obtain 1,3-bisacylated unnatural sugar. In this synthesis route, when synthesizing unnatural sugars without protecting groups, the above problems are faced, and when synthesizing 1,3-bisacylated unnatural sugars from unnatural sugars without protecting groups, the overall yield is low. When synthesizing 1,6-Pr2GalNAz, azidoacetic acid is needed as a raw material, and the synthesis of azidoacetic acid faces the above problems.

[0011] At present, there are two major problems in the field of chemical enzyme labeling and sugar metabolism labeling: first, some of the unnatural sugars used in metabolic labeling (such as fully acetylated sugars) will introduce false positive results, and the structure of the sugar needs to be optimized and designed. Partially protected sugars are just starting to be studied and need to be promoted urgently. Second, the existing methods for synthesizing these unnatural sugars have many disadvantages, including safety, yield, and difficulty in large-scale preparation.

[0012] For the first type of problem, all the fully acetylated unnatural sugars introduce S side reactions when used for metabolic labeling. It has been reported that partially acetylated unnatural sugars can avoid S side reactions and improve the efficiency of unnatural sugars crossing the cell membrane into the cell. In the reported data, these partially acetylated unnatural sugars include 1,3-Ac2GalNAz, 1,3-Pr2GalNAz, 1,3-Pr2ManNAz, 1,3-Pr2GlcNAz, 1,3-Pr2GlcNAl and 1,6-Pr2GalNAz. Other new types of partially acetylated sugars with important value in sugar metabolic labeling have not been reported, and their synthesis methods need to be developed, and their application research needs to be carried out.

[0013] For the second type of problem, the existing method for synthesizing unnatural sugars has many shortcomings, and new synthesis methods need to be developed to synthesize important unnatural sugars in a more economical and efficient way. For example, the synthesis of unnatural sugars containing azide requires the use of a large excess of sodium azide, which has the risk of explosion. The reaction needs to be heated, which is complicated and not safe. The product of the reaction needs to be repeatedly purified by column, which is high in cost and difficult to carry out large-scale synthesis.

[0014] The above two types of problems seriously restrict the application research related to unnatural sugars, therefore, new strategies need to be developed to solve the above two bottleneck problems. SUMMARY

[0015] The main purpose of the present application is to provide an unnatural sugar and a synthesis method and application thereof, to solve the technical problems of the prior art in the synthesis and application of unnatural sugars.

[0016] According to one aspect of the present application, a method for synthesizing an unnatural sugar is provided, comprising: selectively exposing the amino group on the sugar by fully trimethylsilyl protecting the hydroxyl group of the amino sugar at room temperature, and coupling and converting the amino group at room temperature to obtain an unnatural sugar with orthogonal groups protected by trimethylsilyl.

[0017] Further, the unnatural sugar without protection group is any one of GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl and ManNAl.

[0018] Further, the unnatural sugar without protection group is any one of GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl and ManNAl.

[0019] In the synthesis process of GalNAz, GlcNAz and ManNAz, the trimethylsilyl protecting group is removed by hydrogen ion exchange resin, and the final product is precipitated from the solvent.

[0020] In the synthesis of ManNAl, the trimethylsilyl protecting group is removed by silica gel column chromatography.

[0021] Further, the hydroxyl groups at the 1st and 6th positions of the per-trimethylsilyl trimethylsilyl-protected unnatural sugar with orthogonal groups are protected by hydrophobic groups to obtain a partially acylated unnatural sugar.

[0022] Further, the hydrophobic group is any one of acetyl, propionyl, butyryl and valeryl.

[0023] Further, for the per-trimethylsilyl trimethylsilyl-protected unnatural sugar with orthogonal groups of the galactose type and the glucose type, four equivalents of carboxylic acid and a large excess of the corresponding anhydride are added in pyridine, the trimethylsilyl protecting groups at the 1st and 6th positions of the per-trimethylsilyl trimethylsilyl-protected unnatural sugar are exchanged into corresponding ester bonds, and after the trimethylsilyl protecting groups at the 3rd and 4th positions are removed by a hydrogen ion exchange resin, a 1,6-biacylated unnatural sugar is obtained.

[0024] For the per-trimethylsilyl trimethylsilyl-protected unnatural sugar with orthogonal groups of the mannose type, two equivalents of ammonium acetate are added in a mixed solvent of dichloromethane and methanol to selectively remove the trimethylsilyl protecting groups at the 1st and 6th positions at the same time, ester bond protection is performed on the 1st and 6th positions in pyridine, and then the trimethylsilyl protecting groups at the 3rd and 4th positions of the sugar are removed to obtain a 1,6-biacylated unnatural sugar derivative of the mannose type.

[0025] Further, the orthogonal group of the per-trimethylsilyl trimethylsilyl-protected unnatural sugar is any one of azido, terminal alkyne, terminal alkene, cyclopropene, trans-cyclooctene and cyclooctyne.

[0026] According to another aspect of the present application, a partially acylated unnatural sugar for metabolic labeling is disclosed, which is 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.

[0027] Preferably, the partially acylated unnatural sugar for metabolic labeling is prepared by the method described above.

[0028] The present application also discloses a sugar metabolic labeling kit comprising the partially acylated unnatural sugar described above.

[0029] The application also discloses application of the partially acylated unnatural sugar in a sugar metabolism marker kit, and the sugar metabolism marker of any one of HeLa cells, 3T3 cells, CHO cells and MCF-7 cells.

[0030] By adopting the technical scheme, the application has at least the following beneficial effects:

[0031] The application provides the unnatural sugar, the synthesis method and the application, and provides a method for efficiently synthesizing the unnatural sugar without a protecting group in a large amount (10 grams). The reaction condition is mild, the operation is simple, and chromatographic column purification is not needed. The unnatural sugar includes the unnatural sugar without a protecting group and the unnatural sugar with a partially protected hydroxyl group. The unnatural sugar with a partially protected hydroxyl group has the advantages of the existing unnatural sugar, can be efficiently utilized by cells, and can effectively avoid the S side reaction with cysteine in the protein in the metabolism process of the unnatural sugar. Meanwhile, efficient metabolism labeling is realized. In the cell test, the use concentration of the 1,6-biacylated unnatural sugar is one order of magnitude lower than that of the unnatural sugar without a protecting group. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can be obtained from these drawings without creative labor.

[0033] Figure 1 A reaction result diagram of incubation of 1,6-biacylated sugar with protein lysates or single proteins of HeLa cells in an embodiment of the application is shown;

[0034] Figure 2 A result diagram of efficient metabolism labeling of 1,6-biacylated sugar in HeLa cells in an embodiment of the application is shown;

[0035] Figure 3 A diagram of efficient metabolism labeling of 1,6-biacylated sugar in different cells in an embodiment of the application is shown;

[0036] Figure 4 A diagram of efficient metabolism labeling of 1,6-Pr2GalNAz and 1,6-Pr2ManNAz in a mouse in vivo in an embodiment of the application is shown. DETAILED DESCRIPTION

[0037] In order to make the objects, technical solutions and advantages of the present application clearer, the following further describes the embodiments of the present application with reference to the accompanying drawings and in conjunction with specific embodiments.

[0038] It should be noted that all the expressions of "first" and "second" in the embodiments of the present application are used to distinguish two same name non-identical entities or non-identical parameters, and it can be seen that "first" and "second" are only for the convenience of description and should not be understood as a limitation on the embodiments of the present application. The subsequent embodiments will not be described one by one.

[0039] The embodiment of the present application discloses a method for synthesizing unnatural sugar, comprising: at room temperature, the hydroxyl group of the amino sugar is fully trimethylsilyl protected, the amino group on the sugar is selectively exposed, and after the coupling and conversion of the amino group at room temperature, the full trimethylsilyl protected unnatural sugar with orthogonal groups is obtained. The orthogonal group of the full trimethylsilyl protected unnatural sugar with orthogonal groups is any one of azide, terminal alkyne, terminal alkene, cyclopropene, trans-cyclooctene and cyclooctyne. The trimethylsilyl protecting group of the full trimethylsilyl protected unnatural sugar with orthogonal groups is removed to obtain an unnatural sugar without a protecting group. Among them, the unnatural sugar without a protecting group can be any one of GalNAz, GlcNAz, ManNAz, GalNAl, GlcNAl and ManNAl; in the synthesis process of GalNAz, GlcNAz and ManNAz, the trimethylsilyl protecting group is removed by hydrogen ion exchange resin, and the final product is precipitated from the solvent; in the synthesis process of ManNAl, the trimethylsilyl protecting group is removed by silica gel column chromatography purification.

[0040] In some embodiments of the present application, the hydroxyl groups at positions 1 and 6 of the full trimethylsilyl protected unnatural sugar with orthogonal groups are protected by a hydrophobic group to obtain a partially acylated unnatural sugar. The hydrophobic group can be any one of acetyl, propionyl, butyryl and valeryl. For full trimethylsilyl protected unnatural sugar with orthogonal groups of galactose type and glucose type, pyridine is used as the solvent, four equivalents of carboxylic acid and a large excess of the corresponding anhydride are added, the trimethylsilyl protecting groups at positions 1 and 6 of the full trimethylsilyl protected unnatural sugar are exchanged into corresponding ester bonds, and after the trimethylsilyl protecting groups at positions 3 and 4 are removed by hydrogen ion exchange resin, 1,6-bisacylated unnatural sugar is obtained; for full trimethylsilyl protected unnatural sugar with orthogonal groups of mannose type, two equivalents of ammonium acetate are added in a mixed solvent of dichloromethane and methanol to selectively remove the trimethylsilyl protecting groups at positions 1 and 6 at the same time, the ester bond protection at positions 1 and 6 is carried out in pyridine, and then the trimethylsilyl protecting groups at positions 3 and 4 of the sugar are removed to obtain 1,6-bisacylated mannose type unnatural sugar derivative (which also belongs to unnatural sugar).

[0041] Some embodiments of the present application also disclose a partially acylated unnatural sugar for metabolic labeling, the unnatural sugar being a six-carbon sugar and a pyranose structure, and having acyl modifications on the hydroxyl groups at the 1st and 6th positions of the sugar. The unnatural sugar is a 1-hydroxyl and 6-hydroxyl protected unnatural sugar with a hydrophobic group, wherein the unnatural sugar is generally a derivative of a partially hydroxyl-protected six-carbon sugar. The hydrophobic group is an acetyl group, a propionyl group, a butyryl group, or a valeryl group. The above-mentioned partially acylated unnatural sugar for metabolic labeling is made by the above-mentioned synthesis method, i.e., the hydroxyl groups of the sugar are fully trimethylsilyl (TMS) protected, and the amino group on the sugar is selectively exposed; the amino group is coupled and chemically converted to obtain a fully TMS-protected unnatural sugar with a orthogonal group. Starting from the fully TMS-protected unnatural sugar with a orthogonal group, the unprotected unnatural sugar and the partially acylated unnatural sugar can be efficiently synthesized. The unprotected unnatural sugar is GalNAz, GlcNAz, ManNAz, ManNAl, and ManNProc. The partially acylated unnatural sugar is 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.

[0042] Some embodiments of the present application also disclose a sugar metabolic labeling kit comprising the above-mentioned partially acylated unnatural sugar. The 1st and 6th hydroxyl groups of the partially acylated unnatural sugar are protected. The partially acylated unnatural sugar is a 1st and 6th position-protected six-carbon sugar analog. The partially acylated unnatural sugar is a 1st and 6th position hydroxyl group-protected unnatural sugar with a hydrophobic group. It can achieve sugar metabolic labeling of any one of HeLa cells, 3T3 cells, CHO cells, and MCF-7 cells.

[0043] The above-mentioned embodiments of the present application are based on a full TMS (trimethylsilyl) protection strategy, and a method for efficiently synthesizing a large amount of unprotected unnatural sugar (target compound category 1) and 1,6-bis-acylated unnatural sugar (target compound category 2) is developed.

[0044] In general, embodiments of the present application start from amino sugars containing six carbon atoms, protect all hydroxyl groups on the sugar with TMS, selectively expose the amino group on the sugar, and further couple and transform to obtain a fully TMS-protected unnatural sugar with orthogonal groups. The selective exposure of the amino group in the fully TMS-protected method can avoid the participation or interference of the hydroxyl groups on the sugar in the subsequent coupling reaction, and on the other hand, the reaction can be carried out in a solvent without hydroxyl groups, avoiding the participation or interference of the hydroxyl groups in the solvent in the coupling reaction (when the hydroxyl groups are not protected by TMS, the amino sugar can only be dissolved in a solvent containing hydroxyl groups, such as water and methanol). Therefore, in the fully TMS-protected method, the amount of molecules required for coupling with the amino group on the sugar is small (one or slightly more than one equivalent), the coupling efficiency is high (almost quantitative), and the solvent used is easy to remove by reduced pressure (usually dichloromethane is used as the coupling solvent). The fully TMS-protected unnatural sugar with orthogonal groups is an important intermediate for the synthesis of target compound category 1 and target compound category 2 in this patent.

[0045] The fully TMS-protected unnatural sugar obtained by the above method can be purified without column chromatography, and the TMS protecting group can be removed by hydrogen ion exchange resin to obtain an unnatural sugar without protecting group (target compound category 1, GalNAz, GlcNAz, ManNAz and ManNAl have been successfully synthesized in large quantities). It should be particularly pointed out that when this synthesis method is used to synthesize GalNAz, GlcNAz and ManNAz, there is no chromatographic purification step in the middle, and the final product is precipitated from ethanol. When synthesizing ManNAl, precipitation cannot be used, but it can be quickly and cleanly purified by one-time silica gel column chromatography.

[0046] In addition, the fully TMS-protected unnatural sugar obtained by the above method can be purified by one-time simple silica gel column, and then obtained 1,6-bisacylated unnatural sugar through two simple reactions. Two technical routes are developed for different sugars:

[0047] For fully TMS-protected unnatural sugars of galactose type and glucose type, four equivalents of carboxylic acid and a large excess of corresponding anhydride are added in pyridine as solvent, and the TMS at positions 1 and 6 of the fully TMS-protected unnatural sugar is exchanged into corresponding ester bond, and after removing the TMS protecting groups at positions 3 and 4 by hydrogen ion exchange resin, 1,6-bisacylated unnatural sugar is obtained;

[0048] For the full TMS-protected non-natural sugar of the mannose type, two equivalents of ammonium acetate are added to selectively remove the TMS protecting groups at positions 1 and 6 of the sugar simultaneously in a mixed solvent of dichloromethane and methanol, and the ester bond protection is carried out at positions 1 and 6 in pyridine, followed by removal of the TMS protecting groups at positions 3 and 4 of the sugar to obtain the 1,6-bisacylated non-natural sugar derivative of the mannose type.

[0049] Based on the above synthesis strategy, the 1,6-bisacylated non-natural sugar (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.

[0050] It should be particularly pointed out that there are documents that synthesize 1,6-Pr2GalNAz from full TMS-protected GalNAz, but when synthesizing full TMS-protected GalNAz, azidoacetic acid needs to be used as a raw material. As described above, the synthesis of azidoacetic acid has the risk of explosion. The synthesis method disclosed in the embodiments of the present application avoids the need to synthesize azidoacetic acid and is systematically expanded to other sugars.

[0051] From a technical point of view, compared with the existing synthesis method, the advantages of the synthesis method disclosed in the embodiments of the present application are that the full TMS-protected sugar derivative has smaller polarity and has good solubility in many low-polarity solvents (such as dimethylformamide, dichloromethane, ethyl acetate, petroleum ether, acetonitrile, etc.), which can facilitate various chemical transformations (such as replacing halogen with azide) to obtain various full TMS-protected non-natural sugars containing orthogonal groups. In the above synthesis method, all reactions are room temperature reactions, there is no process of chromatographic purification, and large-scale synthesis (10 grams) can be carried out. In the synthesis of non-natural sugars containing azide, only one equivalent of sodium azide is needed, the amount used is small, and the operation is safe.

[0052] From an application point of view, the synthesized 1,6-bisacylated sugar is easy to pass through the cell membrane and enter the cell, can effectively avoid the S side reaction in the sugar metabolism labeling test, and can also maintain a high metabolic efficiency.

[0053] In summary, the main purpose of the present application is to provide a method for synthesizing non-natural sugars in large quantities and with high efficiency, and to carry out application research. The newly reported 1,6-bisacylated non-natural sugar in the present application can improve the efficiency of sugar metabolism labeling while not introducing the S side reaction.

[0054] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0055] Non-natural sugar: Chemical modification of natural monosaccharide, connecting azide, alkyne and other bio-orthogonal groups. Non-natural sugar can be taken up by cells, integrated into the sugar chain through the natural sugar metabolic pathway, and then labeled, imaged or enriched through bio-orthogonal reaction.

[0056] Bio-orthogonal reaction: refers to those chemical reactions that can be carried out in living cells or tissues without interfering with the biochemical reactions of the organism itself. It is used for the study of nucleic acids, proteins, sugars or lipids and other biological macromolecules.

[0057] The present application solves the problem of chemical synthesis of non-natural sugar reported in the sugar chemical enzyme labeling and metabolic labeling, and discloses a novel 1,6-bisacylated non-natural sugar.

[0058] The present application studies and improves the existing method for synthesizing non-natural sugar, develops a strategy based on full TMS protection, and develops a method for synthesizing a large amount of non-natural sugar without protection group; at the same time, starting from full TMS protected non-natural sugar, 1,6-bisacylated non-natural sugar can be synthesized with high efficiency. 1,6-bisacylated non-natural sugar can easily pass through the cell membrane, enter the cell, effectively avoid S side reaction in sugar metabolic labeling test, and also maintain high metabolic efficiency.

[0059] Specifically, the present application is from the amino sugar containing six carbon atoms, all the hydroxyl groups on the sugar are protected by TMS, and the amino group on the sugar is selectively exposed. After coupling with various small molecules, various full TMS protected acyl group substituted amino sugar derivatives are obtained. The full TMS protected sugar derivative has small polarity and good solubility in many low polarity solvents (such as dimethylformamide, dichloromethane, ethyl acetate, petroleum ether, acetonitrile, etc.), which can be subjected to various chemical transformations (such as substitution of halogen with azide), to obtain various full TMS protected non-natural sugars (the full TMS protected non-natural sugar contains azide or alkyne and other orthogonal groups). In methanol, TMS protecting group is removed by using hydrogen ion exchange resin, filtered, concentrated, and then ethanol is added, and the solid separated is non-natural sugar without protection group. In the process of synthesizing non-natural sugar without protection group by the above method, all reactions are room temperature reactions, there is no process of chromatographic purification, and large-scale synthesis (10 grams) can be carried out. In the process of synthesizing non-natural sugar containing azide, only one equivalent of sodium azide is needed, the amount is small, and the operation is safe. Based on the TMS protection strategy, the applicant has synthesized GalNAz, GlcNAz, ManNAz and ManNAl in large quantities.

[0060] In the synthesis of unprotected unnatural sugars above, the full TMS protected unnatural sugar containing orthogonal groups is an important intermediate, from which 1,6-bisacylated unnatural sugars can be obtained in two simple steps. For the full TMS protected unnatural sugars of galactosamine and glucosamine types, in pyridine, four equivalents of carboxylic acid and excess anhydride are added, the TMS groups at 1 and 6 positions of the full TMS protected unnatural sugars are exchanged to the corresponding ester bonds, and after removal of the TMS groups at 3 and 4 positions, 1,6-bisacylated unnatural sugars are obtained; for the full TMS protected unnatural sugar of mannosamine type, in a mixture of dichloromethane and methanol, two equivalents of ammonium acetate are used to selectively remove the TMS groups at 1 and 6 positions of the sugar, and in pyridine, the ester bonds at 1 and 6 positions are protected, and then the TMS groups at 3 and 4 positions are removed, to obtain 1,6-bisacylated unnatural sugar derivatives of mannosamine type.

[0061] In the metabolic labeling experiments, it is found that 1,6-bisacylated sugars do not have S side reactions with proteins; at the same time, 1,6-bisacylated sugars have high metabolic labeling efficiency. In addition, compared with unprotected sugars, different ester bond protecting groups have different effects on the metabolic labeling efficiency, for example, acetyl group has little effect on the efficiency compared with propionyl group, but butyryl modification and propionyl modification can significantly improve the metabolic efficiency of unnatural sugars. Finally, 1,6-bispropionylated sugars can also be used for efficient metabolic labeling in mice in vivo.

[0062] In some embodiments of the present application, in the synthesis of unprotected unnatural sugars based on the full TMS strategy, the orthogonal groups contained are not limited to azide and alkyne groups, and unprotected unnatural sugars containing other orthogonal groups can also be synthesized by this strategy. Other orthogonal groups include, but are not limited to, terminal alkene, cyclopropene, trans-cyclooctene and cyclooctyne. Therefore, the synthesis of these unprotected unnatural sugars is also protected by this patent.

[0063] In some embodiments of the present application, in the synthesis of unprotected unnatural sugars based on the full TMS strategy, the substrates used are not limited to galactosamine derivatives, glucosamine derivatives and mannosamine derivatives, and other sugars containing amino groups can also be used as substrates for this type of synthesis. Therefore, the synthesis of these unprotected unnatural sugars is also protected by this patent.

[0064] In some embodiments of the present application, in the synthesis of partially acylated unnatural sugars based on the full TMS strategy, the acyl groups used are not limited to acetyl group, propionyl group and butyryl group, and other acyl groups can also be introduced by the method of this application. Therefore, other acylated partially acylated unnatural sugars are also protected by this patent.

[0065] In the first typical embodiment of the present application, a simple and efficient method is provided for synthesizing non-natural sugars without a protecting group, specifically GalNAz, GlcNAz, ManNAz and ManNAl, and synthesizing the above-mentioned partially acylated non-natural sugars.

[0066] In the second typical embodiment of the present application, a sugar metabolism labeling kit is provided, which comprises any of the above-mentioned partially acylated non-natural sugars.

[0067] In the third typical embodiment of the present application, the above-mentioned partially acylated non-natural sugar or the above-mentioned kit is applied in sugar metabolism labeling. The use of the partially acylated non-natural sugar of the present application in metabolism labeling not only has a higher metabolic efficiency, but also does not have a side reaction with cysteine in a protein. In a preferred embodiment, the above-mentioned application comprises sugar metabolism labeling of any of the following cells: HeLa cells, MCF-7 cells, CHO cells and 3T3 cells. In a preferred embodiment, 1,6-Pr2GalNAz and 1,6-Pr2ManNAz are used for in vivo metabolism labeling of mice.

[0068] In the prior art, sodium azide is used in the synthesis of partially hydroxyl-protected non-natural sugar containing azide, which has an explosion risk, especially in large-scale synthesis, such as 10 grams, which has a higher explosion risk. The method for synthesizing 1,6-Pr2GalNAz in the present application is more efficient and safer than the synthesis method reported in the literature. Compared with other 1,3-bis-acylated non-natural sugars that have been reported, the 1,6-bis-acylated non-natural sugar of the present application is a better choice based on the mechanism of S-glycosylation modification side reaction on the protein thiol group and the stability of the partially acylated sugar. In the present application, 1,6-bis-acylated sugar is synthesized from a fully TMS-protected sugar, and the synthesis is simpler and more efficient.

[0069] The beneficial effects of the present application will be further illustrated in conjunction with specific examples.

[0070] Example 1: Synthesis of GalNAz, GlcNAz and ManNAz.

[0071] The synthesis of GalNAz, GlcNAz and ManNAz is shown in Synthesis Route I:

[0072]

[0073] The conditions of steps I, II, III and IV are as follows:

[0074] GalN hydrochloride (compound la, 100 mmol, 21.5 g) was dissolved in 200 mL of anhydrous acetonitrile and 40.3 g of HMDS (250 mmol) was added in portions. The reaction was stirred at room temperature for 3 hours. The mixture was filtered and the filtrate was concentrated under vacuum to give compound lb as an oil, which was used in the next step without purification.

[0075] 27.6 g of bromoacetic acid (200 mmol) and 25.3 g of HOSU (220 mmol) were dissolved in 220 mL of dichloromethane and the solution was cooled to -5°C. A solution of 45.3 g (220 mmol) of DCC in 50 mL of dichloromethane was added dropwise to the above solution over a period of 1 hour. After the addition was complete, the reaction was warmed to room temperature and stirred for 2 hours. The mixture was filtered and the filtrate was concentrated under vacuum to give a residue, which was treated with 200 mL of n-hexane and stirred thoroughly to give a mixture. The mixture was filtered and the filter cake was washed with n-hexane to give a white solid, which was dried under vacuum to give 36.0 g of crude 2-bromoacetate 2,5-dioxopyrrolidin-l-yl ester (4a). Compound 4a was used in the next step without purification. The entire amount of compound lb obtained in the previous step was dissolved in 200 mL of dichloromethane and the solution was cooled in an ice-water bath. Then, 33.3 g of 4a (140 mmol) was added. The reaction was stirred at room temperature for 2 hours, concentrated under vacuum to constant weight, and treated with 200 mL of n-hexane and 20 mL of ethyl acetate and stirred. The precipitate was filtered and the filtrate was evaporated to give a yellow oil, which was compound lc. Compound lc was used in the next step without purification.

[0076] Compound lc 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 at room temperature overnight. The mixture was diluted with 200 mL of ethyl acetate and the organic phase was washed with water. The aqueous phase was extracted with 100 mL of ethyl acetate once. The organic phase was washed with saturated brine twice and dried over anhydrous sodium sulfate. The mixture was filtered and the filtrate was concentrated under vacuum to give a yellow oil, which was compound Id. Compound Id was used in the next step without purification.

[0077] Compound Id was dissolved in 150 mL of methanol and 10 g of a hydrogen ion exchange resin (Dowex) was added. The mixture was stirred at room temperature for 2 hours and TLC showed that the reaction was complete. The reaction was filtered and the filtrate was evaporated. Then, 200 mL of ethanol was added and the mixture was stirred. The filter cake was collected and dried under vacuum to give 11.6 g of product le in a yield of 44% over four steps. The product was in the alpha configuration.

[0078] The nuclear magnetic resonance data of compound le (GalNAz) are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.78 (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-d6) δ 167.7, 90.9, 70.6, 68.3, 67.4, 60.7, 50.7, 50.6.

[0079] Compound 2e (GlcNAz, 9.6 grams) was obtained following the procedure described above for the synthesis of GalNAz in Example 1 starting from 21.5 grams (100 mmol) of GlcN hydrochloride. Four steps yield 37%. The product is a mixture of anomers, a / β, 5 / 1.

[0080] NMR spectroscopic data for compound 2e (GlcNAz) are: 1 H NMR (a anomer, 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.

[0081] Compound 3e (ManNAz, 10.8 grams) was obtained following the procedure described above for the synthesis of GalNAz in Example 1 starting from 21.5 grams (100 mmol) of ManN hydrochloride. Four steps yield 41%. The product is a mixture of anomers, a / β, 2 / 1.

[0082] NMR data for compound 3e (ManNAz): 1 H NMR (500 MHz, Methanol-d4) δ 5.03 (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.

[0083] Example 2: Synthesis of ManNAI.

[0084] Synthesis of ManNAI is shown in Scheme II:

[0085]

[0086] The conditions of I, II steps are as follows:

[0087] 2.16 g (22 mmol) of pent-4-ynoic acid and 2.78 g (24.2 mmol) of HOSU were dissolved in 50 mL of dichloromethane, and the solution was cooled to -5 °C. To the above solution, 4.98 g (24.4 mmol) of DCC in 20 mL of dichloromethane was added dropwise over a period of 20 min. The reaction was warmed to room temperature and stirred for another 2 h. After completion of the reaction, the mixture was filtered and the filtrate was concentrated to give the product 4b (2,5-dioxopyrrolidin-l-yl pent-4-ynoate). The product 4b was used directly in the next step without purification.

[0088] According to the method in Example 1, 18.7 mmol of ManN hydrochloride (3a) was used to synthesize compound 3b. The compound 3b was dissolved in 50 mL of dichloromethane, and the solution was cooled to 0 °C in an ice water bath. To the above solution, 4b obtained in the previous step was added dropwise. The reaction was stirred at room temperature for 2 h. After completion of the reaction, the mixture was concentrated under vacuum to give a residue. To the residue, 100 mL of n-hexane was added, and the mixture was stirred and filtered. The filtrate was concentrated under vacuum to give yellow oil 3f, which was used directly in the next step without purification.

[0089] 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 was stirred at room temperature for 2 hours, and the reaction was complete. Filtration, and the filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel column to obtain 3.06 g of compound 3g (ManNAI). The three-step yield was 64%. The product was an end group isomer mixture, a / β, 3 / 1.

[0090] The nuclear magnetic detection data of compound 3g are as follows: 1 H NMR (500 MHz, Methanol-d4) δ 5.01 (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 CNMR (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.

[0091] Example 3: Synthesis of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz and 1,6-Bu2GlcNAz.

[0092] The synthesis of 1,6-Ac2GalNAz, 1,6-Pr2GalNAz, 1,6-Bu2GalNAz, 6-Ac2GlcNAz, 1,6-Pr2GlcNAz and 1,6-Bu2GlcNAz is shown in synthesis route three:

[0093]

[0094] The conditions of steps I and II are as follows:

[0095] Step I, General procedure for synthesis of If, Ig, Ih, 2f, 2g and 2h: 0.5 mmol of Id or 2d was dissolved in 1 mL of pyridine, 0.75 mL of acid anhydride and 2.0 mmol of corresponding carboxylic acid were added under nitrogen protection. The reaction was stirred at room temperature for 20 h, the reaction mixture was diluted with ethyl acetate, and the organic phase was washed with 1 M aqueous HC1, saturated NaHC03solution and saturated brine successively. The organic phase was dried over anhydrous Na2S04, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography to give the corresponding product.

[0096] 1d. 1d is an intermediate for the synthesis of 1e from 1a, which can be purified by silica gel column chromatography with petroleum ether and ethyl acetate as eluent. Starting from 20 mmol of 1a, 4.1 g of 1d can be synthesized with a three-step yield of 37%. The product configuration.

[0097] The NMR detection data of compound 1d are 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 166.70, 94.63, 75.17, 71.24, 70.63, 61.04, 57.42, 53.18, 0.76, 0.44, 0.30, -0.40.

[0098] 2d. The synthesis and purification of 2d were carried out by a similar method to 1d. Starting from 20 mmol of 2a, 5.91 g of 2d can be synthesized with a three-step yield of 54%. The product configuration.

[0099] The NMR detection data of compound 2d are 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). 13C 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.

[0100] 1f. Synthesized according to the general procedure described in Example 3 above. Yield 86%. The product was a mixture of anomers, a / β, 1 / 1.56.

[0101] NMR data for 1f are: 1 H NMR (β anomer, 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). 1 H NMR (a anomer, 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 (a / 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.

[0102] 1g. 1g was synthesized according to the general procedure described in Example 3 above. Yield 89%. The product was a mixture of anomers, a / ß, 1.25 / 1.

[0103] NMR data for compound 1g are: 1 H NMR (mixture of a and ß anomers, 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 (mixture of a and ß anomers, 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.

[0104] 1h. 1h was synthesized according to the general procedure described in Example 3 above. Yield 70%. The product was a mixture of anomers, a / ß, 2.55 / 1.

[0105] NMR data for compound 1h 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 CNMR (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.

[0106] 2f. 2f was synthesized according to the general procedure described in Example 3 above. Yield 62%. Alpha isomer.

[0107] NMR spectral data for compound 2f are: 1 H NMR (500 MHz, Methanol-d4) δ 5.97 (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). 13CNMR (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.

[0108] 2g. 2g was synthesized according to the general procedure described in Example 3 above. Yield 62%. Alpha isomer.

[0109] NMR data for compound 2g are: 1 H 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.

[0110] 2h. 2h was synthesized according to the general procedure described in Example 3 above. Yield 44%. Alpha isomer.

[0111] NMR data for compound 2h are: 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, 1.2.

[0112] General procedure for synthesis of 1i, 1j, 1k, 2i, 2j and 2k: To a solution of 1f, 1g, 1h, 2f, 2g or 2h in methanol was added an appropriate amount of hydrogen ion exchange resin and stirred at room temperature. After TLC showed the reaction was complete, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column to give the corresponding product.

[0113] 1i. 1i was synthesized according to the general procedure described above in Example 3. Yield 52%, a / β, 2 / 1.

[0114] NMR and mass spectrometry data for compound 1i are: 1 H NMR (a isomer, 500 MHz, Methanol-d4) δ 6.15 (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). 13C NMR (α and β isomer mixture, 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) Calcd for C 12 H 19 N4O8[M+H] + 347.12029, observed mass 347.11959.

[0115] 1j. 1j was synthesized according to the general procedure described above in Example 3. Yield 83%, a / β, 1.45 / 1.

[0116] NMR and mass spectrometry data for compound 1j are: 1 H NMR (a isomer, 500 MHz, Methanol-d4) δ 6.16 (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 CNMR (126 MHz, Methanol-d4) δ 175.71, 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) Calcd for C 14 H 23 N4O8[M+H] + 375.15159, observed mass 375.15076.

[0117] 1k. 1k was synthesized according to the general procedure described above in Example 3. Yield 69%, a / β, 2.67 / 1.

[0118] NMR and mass spectrometry data for compound 1k are: 1 H NMR (500 MHz, Methanol-d4) δ 6.17 (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) calculated mass of molecular ion C 16 H 27 N4O8[M+H] + 403.18289, detected mass 403.18234.

[0119] 2i. 2i was synthesized according to the general procedure described in Example 3 above. Yield 69%. Alpha isomer.

[0120] NMR and mass spectrometry data for compound 2i are: 1 H 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). 13C 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) calculated for C 12 H 19 N4O8[M+H] + 347.12029, observed mass 347.11922.

[0121] 2j. 2j was synthesized according to the general procedure described above in Example 3. Yield 79%. Alpha isomer.

[0122] NMR and mass spectrometry data for compound 2j are: 1 H 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) δ 175.8, 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) calculated for C 14 H 23 N4O8[M+H] + 375.15159, observed mass 375.15102.

[0123] 2k. 2k was synthesized according to the general procedure described above in Example 3. Yield 78%. Alpha isomer.

[0124] NMR and mass spectrometry data for compound 2k are: 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, 1.2 Hz, 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 CNMR (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, 13.68. HRMS (ESI) calculated molecular weight C 16 H 27 N4O8[M+H] + 403.18289, detected molecular weight 403.18225.

[0125] Example 4: Synthesis of 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc.

[0126] The synthesis of 1,6-Ac2ManNAz, 1,6-Pr2ManNAz, 1,6-Bu2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc is shown in Scheme IV:

[0127]

[0128] The conditions for steps I and II are as follows:

[0129] 3d. The synthesis and purification of 3d was carried out using a similar method to that of 1d, starting from 20 mmol of 3a to synthesize 4.1 g of 3d with a three-step yield of 37%. Alpha isomer.

[0130] The NMR detection data of compound 3d are as follows: 1H NMR (500 MHz, Chloroform-d) δ 6.43 (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.

[0131] 3f. 3.95 g (8.44 mmol, 1.0 eq) of 3b was dissolved in 50 mL of anhydrous dichloromethane, and 1.65 g (8.44 mmol, 1.0 eq) of 2,5-dioxopyrrolidin-l-yl pent-4-ynoate (4b) was added. The reaction was stirred at room temperature for 2 hours, TLC showed that the reaction was complete, and the solvent was rotary evaporated to obtain a residue, which was purified by silica gel column to obtain 4.40 g of product 3f. Yield 95%. Alpha isomer.

[0132] The nuclear magnetic test results of compound 3f are as follows: 1 H 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.

[0133] 3g. 3.95 g (8.44 mmol, 1.0 eq) of 3b was dissolved in 50 mL of a mixture of anhydrous dichloromethane and pyridine (volume ratio, 1 / 1), and the reaction was cooled to 0 °C with an ice water bath. 1.0 g (8.44 mmol, 1.0 eq) of prop-2-yn-1-yl carbonochloridate (4c) was added. The reaction was allowed to warm to room temperature over 2 h, and stirred overnight. TLC showed that the reaction was complete. After concentration under vacuum, 4.59 g of the product of 3g was obtained by purification on a silica gel column. Yield 99%. Alpha isomer.

[0134] The NMR detection data of compound 3g are as follows: 1 H NMR (500 MHz, Chloroform-d) δ 5.17 (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.

[0135] 3h. 16.80 g of 3d (30.49 mmol, 1.0 eq) was dissolved in 125 mL of a mixture of dichloromethane and 125 mL of methanol, and 4.70 g (60.98 mmol, 2.0 eq) of ammonium acetate was added. After the reaction was stirred at room temperature for 16 h, it was concentrated under vacuum, treated with 100 mL of ethyl acetate, filtered, and the filtrate was concentrated and purified on a silica gel column to obtain 7.69 g of 3h. Yield 62%. The product was an endo isomer, alpha / beta, 1.0 / 0.3.

[0136] The NMR detection data of compound 3h are 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) calculated molecular weight C 14 H 31 N4O6Si2[M+H] + 407.59400, detected molecular weight 407.59736.

[0137] 3i. 3i was synthesized from 3f, the synthesis method refers to the synthesis of 3h in Example 4. The yield was 67%. The product was an end group isomer, a / β, 4 / 1.

[0138] The nuclear magnetic detection data of compound 3i are as follows: 1 H 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). 13C 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.

[0139] 3j. 3j was synthesized from 3g in 63% yield. The product was a mixture of anomers, a / β, 1.0 / 0.11.

[0140] The NMR data of compound 3j were 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). 13 C 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.

[0141] 3k. 550 mg (1.35 mmol, 1.0 eq) of 3h was dissolved in 10 mL of pyridine, cooled to 0 °C in an ice water bath, and 552 mg (5.41 mmol, 4.0 eq) of acetic anhydride was added under nitrogen protection. The reaction was gradually warmed to room temperature and reacted for 18 hours. 218 μL of methanol was added to quench the reaction, and stirred at room temperature for 1 hour. The reaction was concentrated to constant weight under vacuum. The residue was dissolved in 50 mL of methanol, 5 g of hydrogen ion exchange resin was added, and stirred at room temperature for 2 hours. TLC showed that the reaction was complete. Filtration, the filtrate was concentrated under vacuum, and the residue was purified by silica gel column to give 331 mg of compound 3k. The two-step yield was 68%. a / β, 1.0 / 0.28.

[0142] The NMR data of compound 3k were as follows: 1H NMR (500 MHz, Methanol-d4, a 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, a 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. 1 H 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) Calcd. for C 12 H 19 N4O8[M+H] + 347.12029, found 347.11914.

[0143] 3l. 3l was prepared according to the synthetic route of 3k in Example 4, using 3h and propionic anhydride as starting materials. The yield of two steps was 81%. a isomer.

[0144] The NMR data of compound 3l were as follows: 1H NMR (600 MHz, D20) 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, D20) 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) Calcd. for C 14 H 23 N4O8[M+H] + 375.15159, found 375.15095.

[0145] 3m. 3m was prepared according to the synthetic route of 3k in Example 4, using 3h and butyric anhydride as starting materials. The overall yield was 79%. a / β, 1.35 / 1.

[0146] The NMR data of 3m is as follows: a isomer, 1 H 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). 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 H 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). 13 C 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, 13.6. HRMS (ESI) calculated mass of molecular ion C 16 H 27 N4O8[M+H] + 403.18289, observed mass 403.18199.

[0147] 3n. 3n was synthesized from 3i in two steps, the synthesis method was referred to the synthesis of 3l in Example 4. The overall yield of two steps was 83%. a / β, 1.0 / 0.33.

[0148] The nuclear magnetic detection data of compound 3n were as follows: 1H NMR (600 MHz, D20) δ 5.94 (d, J = 1.8 Hz, 1H, a-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 + a-H-2 + a-H-6a), 4.35 - 4.31 (m, 0.33H, β-H-6b), 4.28 (dd, J = 12.2, 6.3 Hz, 1H, a-H-6b), 4.14 (dd, J = 9.8, 4.9 Hz, 1H, a-H-3), 3.98 - 3.92 (m, 1.33H, a-H-5-a + β-H-3), 3.81 - 3.70 (m, 1.33H, β-H-5 + a-H-4), 3.63 (t, J = 9.8 Hz, 0.33H, β-H-4), 2.67 - 2.35 (m, 11.97H, 2xCOC H2 CH2CCH + 2xCOC H2 CH3), 1.11 (m, 7.98H, 2xCOC H3 )。 13 C NMR (151 MHz, D20) δ 177.29 (β- C OCH2CH3-6), 177.26 (a- C OCH2CH3-6), 175.91 (β-NH C O), 175.44 (a-NH C O), 175.18 (a- C OCH2CH3-1), 174.95 (β- C OCH2CH3-1), 92.22 (a-C-1), 91.77 (β-C-1), 83.58, 83.35, 74.87 (β-C-5), 72.04 (a-C-5), 70.90, 70.22, 70.11, 68.42 (a-C-3), 66.84 (β-C-4), 66.67 (a-C-4), 63.31 (a-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.

[0149] 3o. 3o was synthesized from 3j in two steps, the synthetic method was referred to the synthesis of 3l in Example 4, the total yield of two steps was 79%. a / β, 1.0 / 0.15.

[0150] The nuclear magnetic detection data of compound 3o are as follows:1 H NMR (500 MHz, D20) δ 6.02 (s, 1.0H, a-H-1), 5.88 (d, J = 1.4 Hz, 0.15H, β-H-1), 4.78 - 4.69 (m, 2.30H, NHCOOC H2 CCH), 4.49 (m, 1.15H, a-H-6a + β-H-6a), 4.37 - 4.26 (m, 1.30H, β-H-2 + a-H-6b + β-H-6b), 4.21 (d, J = 3.6 Hz, 1.0H, a-H-2), 4.14 (dd, J = 9.7, 4.7 Hz, 1.0H, a-H-3), 3.97 (ddd, J = 8.7, 6.5, 2.1 Hz, 1.15H, a-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, a-H-4), 3.60 (t, J = 9.8 Hz, 0.15H, β-H-4), 3.10 - 2.93 (m, 1.15H, NHCOOCH2CC H ), 2.57 - 2.43 (m, 4.6H, 2 x CH3C H2 COO), 1.20 - 1.12 (m, 6.9H, 2 x C H3 CH2COO). 13 C NMR (126 MHz, D20) δ 177.40 (β-CH3CH2 C OO-6), 177.38 (a-CH3CH2 C OO-6), 175.25 (a-CH3CH2 C OO-1), 174.98 (β-CH3CH2 C OO-1), 158.33 (β-NH C OOCH2CCH), 157.61 (a-NHCOOCH2 C CH), 92.62 (a-C-1), 92.04 (β-C-1), 78.59 (β-NHCOOCH2 C CH), 78.50 (a-NHCOOCH2 C CH), 75.93 (a-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),66.8 2(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).

[0151] 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, 1,6-Bu2ManNAz with proteins.

[0152] To verify that nine partially acylated non-natural 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) cannot spontaneously react with protein cysteine, fully acetylated non-natural sugars (Ac4GalNAz, Ac4GlcNAz, and Ac4ManNAz) were used as controls. These partially acylated non-natural sugars were incubated with HeLa cell lysates or single proteins at 37°C for 2 hours. The azide signal on the proteins was then detected by electrophoresis. The reaction results are shown below. Figure 1 .

[0153] Example 6: Metabolic labeling effect 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 in living cells.

[0154] To verify the metabolic labeling effect of the above 1,6-biacylated unnatural sugars in living cells, the above different compounds were used to metabolically label HeLa cells. The specific operation steps are as follows:

[0155] 200 μM of 1,6-biacylated unnatural sugars and 2 mM unprotected sugars were added to the culture medium of HeLa cells for culture, and then the HeLa cells treated in different ways were subjected to in-gel fluorescence detection by bioorthogonal reaction with Cy5 fluorescently labeled substances. The detection results are shown in Figure 2 ( Figure 3 CBB in the figure represents Coomassie brilliant blue staining to show the loading amount control).

[0156] As can be seen from Figure 2 , the labeling efficiency of 200 μM 1,6-Pr2GalNAz or 1,6-Pr2ManNAz is close to or slightly stronger than that of 2 mM unprotected sugar compared with GalNAz or ManNNAz. The labeling efficiency of 1,6-Bu2GalNAz or 1,6-Bu2ManNAz is comparable to that of 1,6-Pr2GalNAz or 1,6-Pr2ManNAz, but the labeling efficiency of 1,6-Ac2GalNAz or 1,6-Ac2ManNAz is much lower than that of 1,6-Pr2GalNAz or 1,6-Pr2ManNAz Figure 2 a and b). For MCF-7 (MCF7-T2A-GFP) cells expressing GFP, GlcNAz and 1,6-biacylated GlcNAz treatment did not have obvious labeling Figure 2 c), by overexpressing the mutant enzyme AGX2F383G which can convert GlcNAz-P- into UDP-GlcNAz, in MCF7-AGX2F383G-T2A-GFP cells, GlcNAz and its three 1,6-biacylated derivatives can produce obvious labeling Figure 2 c). The above data well illustrate that 1,6-biacylated azidosugars can achieve metabolic labeling under conditions of much lower concentration than unprotected azidosugars.

[0157] Example 7: 1,6-Pr2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc for metabolic labeling in living cells.

[0158] Different cells were metabolically labeled with 200 μΜ of 1,6-Pr2ManNAz, 1,6-Pr2ManNAl and 1,6-Pr2ManNProc for 48 hours. After lysis, the Cy5-labeled probe was added to the lysate to react with the glycoproteins with orthogonal groups. After SDS-PAGE separation, the in-gel fluorescence analysis was performed. The results are shown in Figure 3 ( Figure 3 CBB indicates Coomassie blue staining to show the loading control.

[0159] Example 8: 1,6-Pr2GalNAz and 1,6-Pr2ManNAz for in vivo metabolic labeling in mice.

[0160] In the in vivo experiment in mice, B6D2F1 / J mice were injected intraperitoneally with 1,6-Pr2GalNAz or 1,6-Pr2ManNAz (500 mg / kg) every day for 3 or 7 days. The mice were euthanized on the 4th or 8th day, and different organs were homogenized. The azide-modified glycoproteins were click-labeled and subjected to SDS-PAGE separation. The in-gel fluorescence analysis showed that the glycoproteins from the heart, lung and spleen could be well labeled, indicating that 1,6-Pr2GalNAz and 1,6-Pr2ManNAz could be well used for in vivo metabolic labeling Figure 4 ).

[0161] It should be particularly pointed out that each component or step in each of the above embodiments can be crossed, replaced, added, deleted, and therefore, the combinations formed by these reasonable permutations and combinations should also belong to the protection scope of the present application, and the protection scope of the present application should not be limited to the above embodiments.

[0162] The above is the exemplary embodiments disclosed by the present application, and the sequence of the above embodiments disclosed by the present application is only for description, and does not represent the advantages and disadvantages of the embodiments. However, it should be noted that the discussion of any of the above embodiments is only exemplary, and is not intended to imply that the scope of the embodiments disclosed by the present application (including the claims) is limited to these examples, and various changes and modifications can be made without departing from the scope defined by the claims. The functions, steps and / or acts of the method claims described herein need not be performed in any particular order. Furthermore, although the elements of the embodiments disclosed by the present application can be described or claimed in individual form, they can also be understood as plural unless explicitly limited as singular. The embodiments disclosed by the present application are not limited to the above examples, and any changes, equivalent replacements, improvements, etc. within the scope of the present application should also belong to the protection scope of the present application.

[0163] Those skilled in the art should understand that the above discussion of any embodiment is only exemplary, and is not intended to mean that the scope of the embodiments disclosed by the present application (including claims) is limited to these examples; under the idea of the embodiments of the present application, the above embodiments or technical features among different embodiments can also be combined, and there are many other changes of different aspects of the embodiments of the present application as described above, which are not provided in details for the sake of brevity. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of the embodiments of the present application shall be included in the protection scope of the embodiments of the present application.

Claims

1. A method of synthesizing 1,6-bisacylated mannose-type unnatural sugar derivatives, characterized by, The application relates to a method for synthesizing a 1,6-bisacylated mannose type unnatural sugar derivative. The hydroxyl groups of an amino sugar are fully trimethylsilyl-protected at room temperature, the amino group on the sugar is selectively exposed, the amino group is coupled and converted at room temperature to obtain a full trimethylsilyl-protected unnatural sugar with orthogonal groups; In a mixed solvent of dichloromethane and methanol, two equivalents of ammonium acetate are added to selectively remove the trimethylsilyl protecting groups at the 1st and 6th positions of the full trimethylsilyl-protected unnatural sugar with orthogonal groups, the 1st and 6th positions are ester bond-protected in pyridine, and then the trimethylsilyl protecting groups at the 3rd and 4th positions of the sugar are removed to obtain a 1,6-bisacylated mannose type unnatural sugar derivative; The synthesis route of the 1,6-bisacylated mannose type unnatural sugar derivative is as follows: Wherein, n=0, 1 or 2, and the values of n in the formula are the same.

2. The method of claim 1, wherein, In the process of obtaining the full trimethylsilyl-protected unnatural sugar with orthogonal groups based on the method, no chromatographic purification process is needed.

3. The method of claim 1, wherein, The hydroxyl groups at the 1st and 6th positions of the full trimethylsilyl-protected unnatural sugar with orthogonal groups are protected by a hydrophobic group, which is any one of acetyl, propionyl and butyryl.

4. The method of claim 1, wherein, The orthogonal group of the full trimethylsilyl-protected unnatural sugar with orthogonal groups is azide.

5. The method of claim 1, wherein, The 1,6-bisacylated mannose type unnatural sugar derivative includes any one of 1,6-Ac2ManNAz, 1,6-Pr2ManNAz and 1,6-Bu2ManNAz.

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