阅读说明：本技术 氨基酸衍生物及其制备方法 (Amino acid derivatives and process for preparing the same ) 是由 谭忠平 裴湘尧 李耀豪 冯高超 蒋明兴 于 2021-07-01 设计创作，主要内容包括：本发明公开了一种氨基酸衍生物及其制备方法。其中该氨基酸衍生物由氨基酸和糖链通过连接基团连接得到,其中所述氨基酸包含-SH和/或-OH基团,所述糖链包含一个甘露糖或由两个以上甘露糖串联构成,所述连接基团含有S或O。本发明的方法可一步合成所需要的多糖化合物,不仅可以节约大量的人力和时间,使目的化合物的大量制备能够顺利、简洁、快速和高效地进行,而且提高了产物的合成产率,节省大量的合成原料,使生产成本大大降低,同时也有利于环保,进而突破氨基酸糖基化的研究和应用壁垒。(The invention discloses an amino acid derivative and a preparation method thereof. Wherein the amino acid derivative is obtained by linking an amino acid containing an-SH and/or-OH group to a sugar chain containing one mannose or consisting of two or more mannose in tandem via a linking group containing S or O. The method can synthesize the required polysaccharide compound in one step, not only can save a large amount of manpower and time, but also can smoothly, concisely, quickly and efficiently prepare a large amount of target compounds, improve the synthesis yield of products, save a large amount of synthesis raw materials, greatly reduce the production cost, and simultaneously is beneficial to environmental protection, thereby breaking through the research and application barriers of amino acid glycosylation.)

1. An amino acid derivative obtained by linking an amino acid and a sugar chain through a linking group, wherein the amino acid contains an-SH and/or-OH group, the sugar chain contains one mannose or is composed of two or more mannose in series, and the linking group contains S or O.

2. The amino acid derivative according to claim 1, wherein the amino acid has a structure represented by the following formula (I):

wherein A is S or O, R is H or C₁-C₃An alkyl group.

3. The amino acid derivative according to claim 1, wherein said sugar chain consists of 2 to 10 mannose in tandem.

4. Amino acid derivative according to claim 1, characterized in that it has a structure of the levorotatory and/or dextrorotatory isomer.

5. A method for preparing an amino acid derivative, comprising the steps of:

(1) dissolving a mannose compound II with hydroxyl groups protected and one of the hydroxyl groups substituted by bromine in chloroform, and reacting with 2,6-lutidine to obtain a compound III containing 1, 2-orthoester;

(2) adding an initiator to react the compound III to obtain a derivative consisting of a plurality of mannose;

(3) replacing the methyl group in the product of step (2) with acetyl group to obtain acetylated derivative;

(4) reacting the product of step (3) with an amino acid, to give a first amino acid derivative, wherein the amino acid comprises-SH and/or-OH groups and the amino group is protected.

6. The method of producing an amino acid derivative according to claim 5, further comprising (5) a step of reacting a hydroxyl group-protected mannose compound I with an amino acid to obtain a second amino acid derivative, wherein the amino acid comprises an-SH and/or-OH group and an amino group is protected.

7. The process for producing an amino acid derivative according to claim 6, wherein in the step (5), the hydroxyl group-protected compound I is reacted with the Fmoc-protected amino acid in boron trifluoride diethyl etherate to obtain the compound VI in one step, the reaction solvent is anhydrous acetonitrile, and the reaction temperature is room temperature.

8. The method for producing an amino acid derivative according to claim 5, wherein the sugar chain in the first amino acid derivative contains two or more mannose, and the second amino acid derivative contains one mannose.

9. The method for preparing amino acid derivatives according to claim 5, wherein in the step (2), the compound III is subjected to trimethylsilyl trifluoromethanesulfonate-promoted reaction to obtain a compound IV, a compound VII and a compound X in one step, wherein the reaction solvent is dichloromethane, the reaction temperature is-30 ℃ to 0 ℃, and the compound IV is two mannose tandem compounds, the compound VII is three mannose tandem compounds, and the compound X is four mannose tandem compounds.

10. The method for producing an amino acid derivative according to claim 5, wherein in step (4), the product of step (3) is reacted with the Fmoc-protected amino acid in boron trifluoride diethyl etherate to obtain the first amino acid derivative in one step, the reaction solvent is anhydrous acetonitrile, and the reaction temperature is room temperature.

Technical Field

The invention relates to the technical field of glycosylated protein organic synthesis, in particular to an amino acid derivative and a preparation method thereof, and especially relates to a preparation method of mannose glycosylated amino acid and enantiomers, half enantiomers and analogues containing thio-glycoside bonds thereof.

Background

The amino acids in the side chains of proteins, mainly serine, threonine and aspartate, are covalently linked to sugar molecules, which form glycosylated proteins, called glycoproteins for short. Glycosylation is a very important post-translational modification of proteins. The glycosylation modification can not only make different labels on the protein, but also limit the change of the local conformation of the protein, and increase the stability of the protein, the specificity of the combination of the protein and the diversity of functions. Sugars attached to proteins are also often used as specific ligands for exogenous receptors, utilized by various viruses, bacteria, and parasites; the sugar connected on the protein can also be used as a specific ligand of an endogenous receptor and participates in mediated clearance, turnover and intracellular trafficking; in addition, the sugar linked to the protein plays an important role in fertilization.

To better understand the specific roles of the sugars on proteins and thus to better utilize glycoproteins or sugar analogs for disease therapy and diagnosis, it is necessary to prepare glycoproteins with high purity and homogeneous composition by organic synthesis. These synthetic glycoproteins may contain natural sugar and amino acid structures, as well as their enantiomeric or partial enantiomeric structures, or other non-natural structures that are greatly altered. These non-native structures are of great value in the study and application of glycosylated proteins. However, these studies and applications have been very slow to advance due to the difficulty in obtaining large quantities of glycosylated amino acids necessary to prepare these molecules.

Glycosylated amino acids are largely divided into two groups, the first being O-linked glycosylated amino acids formed by the attachment of a sugar through the O atom of the hydroxyl group of the serine and threonine side chains, depending on the amino acid to which the sugar is attached. The other is an N-linked glycosylated amino acid formed by the attachment of the N atom of the amide group of the aspartic acid side chain to a sugar.

For example, an O-glycosylated threonine (residue) having a sugar moiety in eukaryotic proteoglycans was prepared by the above-described O-glycosylation in Sariya Talat et al, using a synthetic method in which a specific compound is linked to an amino acid and then debenzylated (S.Talat, et al. glycocony J.2011,28,537-555.). As another example, in the document D.Varon, et al.Aust Jchem,2002,55,161-165, mannose trisaccharide is obtained by a method of linking mannose disaccharide and mannose monosaccharide to obtain mannose trisaccharide, and then a series of operations are performed to obtain mannose trisaccharide-glycosylated amino acid. The above published documents are hereby incorporated by reference in their entirety.

To date, there have been few reports of worldwide optimization of glycosylated amino acid synthesis, particularly with respect to enantiomers, hemi-enantiomers, and analogs containing thioglycosidic linkages. In a few reports, the synthesis steps are relatively more, the complexity is relatively high, and the yield is low. Meanwhile, the synthesized product mainly takes glycosylated serine and threonine containing mannose monosaccharide and disaccharide with relatively simple structures as well as synthesis of enantiomer, hemienantiomer and analog containing thioglycosidic bond. Therefore, there is still a need to develop a simple, fast, efficient and low-cost preparation method to break through the research and application barriers of protein glycosylation.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention provides an amino acid derivative and a preparation method thereof. The present inventors have found through extensive studies that a desired polysaccharide compound can be obtained by one-step synthesis of a specific compound, and the desired product can be obtained by one-step ligation without protecting the amino acid side chain and the carboxyl group. In addition, the compound obtained by the preparation method has high yield and purity, and is basically free of impurities. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided an amino acid derivative obtained by linking an amino acid and a sugar chain through a linking group, wherein the amino acid comprises an-SH and/or-OH group, the sugar chain comprises one mannose or consists of two or more mannose chains connected in series, and the linking group contains S or O.

According to the amino acid derivative of the present invention, preferably, the amino acid has a structure represented by the following formula (I):

wherein A is S or O, R is H or C₁-C₃An alkyl group.

According to the amino acid derivative of the present invention, preferably, the sugar chain is composed of 2 to 10 mannose in tandem.

According to the amino acid derivatives of the present invention, preferably, the amino acid derivatives have a levorotatory and/or dextrorotatory isomer structure.

In a second aspect of the present invention, there is provided a method for producing an amino acid derivative, comprising at least the steps of:

(1) dissolving a mannose compound II with hydroxyl groups protected and one of the hydroxyl groups substituted by bromine in chloroform, and reacting with 2,6-lutidine to obtain a compound III containing 1, 2-orthoester;

(2) adding an initiator to react the compound III to obtain a derivative consisting of a plurality of mannose;

(3) replacing the methyl group in the product of step (2) with acetyl group to obtain acetylated derivative;

(4) reacting the product of step (3) with an amino acid, to give a first amino acid derivative, wherein the amino acid comprises-SH and/or-OH groups and the amino group is protected.

The method for producing an amino acid derivative according to the second aspect of the present invention preferably further comprises (5) a step of reacting a hydroxyl group-protected mannose compound I with an amino acid, which contains a-SH and/or-OH group and whose amino group is protected, to obtain a second amino acid derivative.

According to the method for preparing an amino acid derivative according to the second aspect of the present invention, preferably, in step (5), the hydroxyl protected compound I is reacted with the Fmoc protected amino acid in boron trifluoride diethyl etherate solution to obtain compound VI in one step, the reaction solvent is anhydrous acetonitrile, and the reaction temperature is room temperature.

According to the method for producing an amino acid derivative according to the second aspect of the present invention, preferably, the sugar chain in the first amino acid derivative contains two or more mannose, and the second amino acid derivative contains one mannose.

According to the preparation method of the amino acid derivative of the second aspect of the present invention, preferably, in the step (2), the compound III is subjected to trimethylsilyl trifluoromethanesulfonate-promoted reaction to obtain the compound IV, the compound VII and the compound X in one step, wherein the reaction solvent is dichloromethane, and the reaction temperature is-30 ℃ to 0 ℃, wherein the compound IV is two mannose tandem compounds, the compound VII is three mannose tandem compounds, and the compound X is four mannose tandem compounds.

According to the method for preparing an amino acid derivative of the second aspect of the present invention, preferably, in step (4), the product of step (3) is reacted with the Fmoc-protected amino acid in boron trifluoride diethyl etherate solution to obtain the first amino acid derivative in one step, the reaction solvent is anhydrous acetonitrile, and the reaction temperature is room temperature.

In a third aspect of the present invention, there is provided the use of an amino acid derivative according to the first aspect of the present invention or an amino acid derivative obtained by the preparation method according to the second aspect in a glycosylated protein or glycopeptide.

In a fourth aspect of the present invention, there is provided a use of the amino acid derivative according to the first aspect of the present invention or the amino acid derivative obtained by the preparation method according to the second aspect in a drug containing a glycosylated protein or glycopeptide.

The synthetic route and the method not only can save a large amount of manpower and time, lead the mass preparation of the mannose glycosylation amino acid and the enantiomer, the semi-enantiomer and the analog containing the thio-glycosidic bond to be carried out smoothly, concisely, quickly and efficiently, but also can improve the synthetic yield of the product, save a large amount of synthetic raw materials, reduce the production cost, lead the target compound product to have higher product cost performance, and be beneficial to environmental protection. The high product cost performance is more beneficial for scientific research workers to use the products as starting raw materials, and further breaks through the research and application barriers of protein glycosylation.

Drawings

FIG. 1 is a scheme for the synthesis of the object compounds of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Any intermediate products obtained during the synthesis of the present invention and the product of the desired compound can be determined by known means by those skilled in the art, including, but not limited to, High Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), or gas chromatography-mass spectrometry (GC-MS). And further by, for example¹H、¹³C and various two-dimensional Nuclear Magnetic Resonance (NMR) technologies to characterize the molecular structure of any compound in the preparation process.

The term "amino acid" is meant to include both naturally occurring and synthetic amino acids, as well as amino acid analogs. Naturally occurring amino acids refer to those amino acids encoded by the genetic code, as well as modified amino acids, for example, modified amino acids including, but not limited to, hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Synthetic amino acids refer to amino acids having the basic amino acid chemical structure, i.e., hydrogen-binding alpha carbon, carboxyl group, amino group, and R group, obtained by artificial synthesis. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, e.g., homoserine, and the like. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. As used herein, "amino acid" refers to the D-and/or L-isomer of an amino acid.

The term "enantiomer", sometimes also referred to as enantiomer, refers to stereoisomers that are not superimposable but physical or mirror images of each other, which have optical rotation, one of which is levorotatory and one of which is dextrorotatory, and thus enantiomers herein are also referred to as optical isomers. In this case, the sign (+) denotes the right-handed rotation and the sign (-) denotes the left-handed rotation.

The term "hemienantiomer" refers to a molecule in which only the sugar portion is the enantiomer of the natural structure or only the amino acid portion is the enantiomer of the natural structure.

The term "analog containing a thioglycosidic linkage" refers to a molecule in which the oxygen atom of the sugar attached to the amino acid is replaced with a sulfur atom.

The term "group" refers to any portion of a compound.

Unless otherwise indicated, configurations, element symbols, dashes, solid and dashed wedge lines, and the like in the structural formulae shown herein have the definitions customary in the art.

[ amino acid derivatives ]

The "amino acid derivative" (also referred to herein as "glycosylated amino acid" in some cases) of the present invention is obtained by linking an amino acid comprising an-SH and/or-OH group and a sugar chain comprising one mannose or consisting of two or more mannose in tandem via a linking group containing S or O.

Herein, for the purpose of illustrating the method, the amino acid derivatives are classified into a first amino acid derivative and a second amino acid derivative, wherein the first amino acid derivative refers to a compound containing one mannose residue, and the second amino acid derivative refers to a derivative containing two or more mannose residues. Wherein the linking means between two or more mannose residues is not particularly limited. In certain embodiments, two or more mannose are linked in a tandem fashion, thereby forming a linear structure, and are linked to an amino acid through an S-or O-containing linking group. In certain embodiments, two or more mannose residues form a branched structure and are linked to an amino acid by a linking group comprising one S or O.

Preferably, the amino acid has the structure shown in the following formula (I):

wherein A is S or O, R is H or C₁-C₃An alkyl group. Also preferably, R is CH₃。

The sugar chain of the present invention is preferably composed of 2 to 10 mannose chains in tandem. More preferably, the mannose contains 2 to 9 mannose residues in tandem. For example, it consists of 2, 3, 4, 5, 6, 7, 8 or 9 mannose in tandem.

[ production method ]

The method for producing an amino acid derivative of the present invention includes at least the following steps. In the following methods, the mannose raw material used includes, but is not limited to, D- (+) -mannose and L- (-) -mannose, and the amino acid raw material used includes, but is not limited to, Fmoc-L- (-) -serine, Fmoc-D- (+) -serine, Fmoc-L- (-) -threonine and Fmoc-D- (+) -threonine.

In the step (1), a mannose compound II in which the hydroxyl groups are protected and one of the hydroxyl groups is substituted with bromine is dissolved in chloroform and reacted with 2,6-lutidine to obtain a 1, 2-orthoester-containing compound III.

Wherein, the mannose compound II can be obtained by reacting mannose with acetic anhydride under the condition suitable for reaction and in the presence of sodium bicarbonate to obtain the compound I with the full acetyl protection, and then reacting with, for example, an acetic acid solution of hydrobromic acid at room temperature to obtain the mannose compound II containing bromine. The conditions suitable for the reaction here refer to anhydrous sodium acetate as a catalyst, and the reaction conditions are 100-120 ℃ oil bath. Preferably an oil bath at the temperature of 101-119 ℃. It is also preferably an oil bath at a temperature of 105 ℃ and 112 ℃. The concentration of hydrobromic acid acetic acid is 20-40%. Preferably 22 to 38%, and still more preferably 25 to 36%.

The reaction with 2,6-lutidine at room temperature is preferably carried out with an organic solution of 2,6-lutidine, including but not limited to methanol, ethanol, diethyl ether, etc.

In the step (2), an initiator is added to cause a reaction between the compounds III to obtain a derivative composed of a plurality of mannose. Illustratively, compound III can obtain compound IV, compound VII and/or compound X which can be separated in one step under the trimethylsilyl trifluoromethanesulfonate-promoted reaction. The reaction solvent here is dichloromethane and the reaction temperature is from-30 ℃ to 0 ℃. Further preferably, the reaction temperature is from-20 ℃ to-5 ℃. Also preferably, the reaction temperature is from-15 ℃ to-5 ℃. By "isolatable" is meant that the methods of isolation of the above compounds are known to those skilled in the art.

In certain embodiments, a single mannose protected by a hydroxyl group may be reacted directly with an amino acid to give a second amino acid derivative. The amino acids here contain-SH and/or-OH groups and the amino groups are protected. Preferably, compound I is reacted with Fmoc protected amino acid in boron trifluoride etherate in one step to obtain mannosylated amino acid, such as compound VI, in anhydrous acetonitrile as a reaction solvent at room temperature.

In step (3), the methyl group in the product of step (2) is replaced with an acetyl group under conditions suitable for the reaction to give an acetylated derivative. The conditions suitable for the reaction here mean that the reaction is carried out at from-5 ℃ to 5 ℃ with an acid solution of acetic anhydride. Acids herein include, but are not limited to, hydrochloric acid, sulfuric acid, and the like. Preferably sulfuric acid. Further preferably 98% sulfuric acid. The temperature here is preferably from-2 ℃ to 5 ℃ and also preferably from-2 ℃ to 1 ℃.

In step (4), the product of step (3) is reacted with an amino acid, to give a first amino acid derivative, wherein the amino acid here comprises-SH and/or-OH groups and the amino group is protected. Preferably, the product of step (3) is reacted with Fmoc protected amino acid in boron trifluoride ether solution to obtain the first amino acid derivative in one step, the reaction solvent is anhydrous acetonitrile, and the reaction temperature is room temperature.

It should be noted that, although the production of the first amino acid derivative and the second amino acid derivative is separately described herein, it is easily understood by those skilled in the art that both the step for producing the first amino acid derivative and the step for producing the second amino acid derivative may be performed simultaneously or sequentially in the same reaction system, or may be performed simultaneously or sequentially in different reaction systems.

It will be appreciated by those skilled in the art that other steps or operations, such as purification or water washing to increase yield, may be included before or after steps (1) - (4) or between steps of the preparation process of the present invention, as long as the objects of the present invention are achieved, and that the process of the present invention may be further optimized and/or improved.

Example 1

This example is an exemplary preparation of an amino acid derivative, the synthetic process of which is shown in FIG. 1, comprising the following steps:

1. synthesis of Compound I

D- (+) -mannose (40g,222mmol) was used as a starting material, which was dissolved in acetic anhydride (400mL), and then anhydrous sodium acetate (24g) was added as a catalyst. The reaction was stirred in a 110 ℃ oil bath and the extent of reaction progress was monitored using TLC plates. After 2 hours, the reaction was quenched by addition of ice saturated sodium bicarbonate solution, and the organic phase was then extracted, dried and concentrated to give compound I in 98% yield.

2. Synthesis of Compound II

To compound I (86g,222mmol) was added 33% hydrobromic acid in acetic acid (520mL), stirred at room temperature and the extent of reaction progress was monitored using TLC plates. After 30 minutes, the reaction was diluted with dichloromethane (5L) and extracted. The combined organic phases were washed with ice water (about 200mL each time) until neutral; drying and concentrating the organic phase to obtain a crude product of the compound II without purification.

3. Synthesis of Compound III

A crude compound of compound II (88g,214mmol) was dissolved in chloroform (600mL), and a methanol solution of 2,6-Lutidine (v/v ═ 1/10,660mL) was slowly added to the solution, and the reaction was stirred at room temperature overnight. The reaction was monitored by TLC plate and after confirming completion of the reaction, dichloromethane was added to dilute the reaction, washed once with ice 3% sodium bicarbonate solution, the organic phase was dried and concentrated, petroleum ether was added to the concentrate, the precipitated white crystalline material was filtered, washed, dissolved in dichloromethane and purified by column chromatography to give compound III in about 64% yield.

4. Synthesis of Compound IV, Compound VII, Compound X

Under the protection of nitrogen, dissolving compound III (30g,82.845mmol) in anhydrous dichloromethane (1050mL), cooling the solution to-30 ℃, dropwise adding trimethylsilyl trifluoromethanesulfonate (45mL,248.536mmol) into the solution to trigger the reaction, continuing the reaction at-30 ℃ for 15min after the dropwise addition is finished, monitoring the reaction completion by using a TLC plate, diluting the reaction solution with dichloromethane (500mL), quenching with ice saturated sodium bicarbonate solution (500mL), extracting, drying and concentrating, and purifying the crude product by column chromatography to obtain compound IV with the yield of about 36%, compound VII with the yield of about 16%, compound X with the yield of about 8%.

5. Synthesis of Compound VI

Compound I (15g, 38.428mmol) and Fmoc-Ser-OH (15.1g,46.114mmol) or Fmoc-Thr-OH (15.6g,46.114mmol) were dissolved in anhydrous acetonitrile (460mL) under nitrogen, boron trifluoride diethyl etherate (14mL,115.284mmol) was slowly added dropwise to the solution, stirred at room temperature for 17h, after monitoring the completion of the reaction using TLC plates, acetonitrile was removed by rotary evaporation, ethyl acetate (500mL) was then added, the organic phase was extracted several times with water, dried, concentrated and the crude product was purified by column chromatography to give Compound VI. When R ═ H, the yield was about 29%; r is CH₃The yield was 27%.

6. Synthesis of Compound IX

Compound IV (10g,15.371mmol) was dissolved in acetic anhydride (150mL), the solution was then cooled to 0 deg.C, a mixture of 98% sulfuric acid (0.5mL) dissolved in acetic anhydride (10mL) was slowly added dropwise thereto, stirred at 0 deg.C for 1 hour, after completion of the reaction was monitored using a TLC plate, diluted with dichloromethane, followed by addition of ice saturated sodium bicarbonate solution with stirring to neutrality, extracted, dried, concentrated, and then purified by column chromatography to give Compound V (9.1g, 88%).

Under nitrogen protection and at room temperature, compound V (12g,17.684mmol) and Fmoc-Ser-OH (8.683g,26.526mmol) or Fmoc-Thr-OH (9.05g,26.526mmol) were dissolved in anhydrous acetonitrile (400mL), boron trifluoride ether (6.547mL,53.051mmol) was slowly added dropwise to the solution, and the reaction was monitored using TLC plates. After completion of the reaction, the reaction system was diluted with ethyl acetate (400mL), then washed with water, and the organic phase was dried and concentrated to obtain a crude product. After purification of the crude product by column chromatography, compound IX was obtained in about 39% yield when R ═ H; when R ═ CH3, the yield was 44%.

7. Synthesis of Compound XII

Compound VII (5g,5.33mmol) was dissolved in acetic anhydride (60mL), and after the solution was cooled to 0 deg.C, a mixture of 98% sulfuric acid (0.2mL) dissolved in acetic anhydride (10mL) was slowly added dropwise to the solution. After stirring at 0 ℃ for 1 hour and monitoring completion of the reaction using TLC plates, the reaction was diluted with dichloromethane (300mL) and the diluted reaction was treated several times with a small amount of saturated solution of ice-added sodium bicarbonate, and after completion of the extraction, the organic phase was dried, concentrated and purified by column chromatography to give VIII (5.1g, 99%).

Compound VIII (5g,5.171mmol) and Fmoc-Ser-OH (2.539g,7.757mmol) or Fmoc-Thr-OH (2.65g,7.757mmol) were dissolved in dry acetonitrile (150mL) under nitrogen at room temperature, and boron trifluoride ether (2.2g,15.514mmol) was slowly added dropwise to the resulting solution and the reaction was monitored using TLC plates. After the reaction was completed, ethyl acetate (1000mL) was added, followed by washing with water, the organic phase was dried and concentrated, and the crude product was purified by column chromatography to obtain compound XII, which is about 38% yield when R ═ H; r is CH₃The yield was 48%.

8. Synthesis of Compound XIII

Compound X was dissolved in acetic anhydride, the resulting solution was cooled to 0 deg.C, a mixture of acetic anhydride and 98% sulfuric acid was slowly added dropwise to the solution, and the reaction was monitored using a TLC plate. After 1 hour, the reaction was diluted with dichloromethane and the organic phase was washed several times with a small amount of saturated sodium bicarbonate solution with ice. The organic phase was dried, concentrated and the crude product was purified by column chromatography to give XI.

Compounds XI and Fmoc-Ser-OH or Fmoc-Thr-OH were dissolved in anhydrous acetonitrile under nitrogen, and to the resulting solution boron trifluoride etherate was slowly added dropwise and the reaction was monitored using TLC plates. After the reaction is finished, adding ethyl acetate into the reaction system to dilute the reaction system, washing the diluted system with water, drying an organic phase, and concentrating to obtain a crude product. The crude product was purified by column chromatography to give compound XIII in about 45% yield when R ═ H; r is CH₃The yield was 50%.

9. Synthesis of Compound XXVII

Compound I (682mg,1.75mmol) and Fmoc-Cys-OH (900mg,2.62mmol) were dissolved in anhydrous acetonitrile (35mL) under nitrogen, and to the resulting solution was added boron trifluoride ether (0.81mL,5.25mmol) slowly dropwise, stirred under nitrogen for 28h, and the reaction was monitored using TLC plates. After the reaction is finished, adding ethyl acetate into the reaction system to dilute the reaction system, washing the diluted system with water, drying an organic phase, and concentrating to obtain a crude product. The crude product was purified by column chromatography to afford compound XXVII in 34% yield.

10. Synthesis of Compound XXVIII

Compound V (678mg,1.0mmol) and Fmoc-Cys-OH (514mg,1.5mmol) were dissolved in anhydrous acetonitrile (20mL) under nitrogen, and to the resulting solution was added boron trifluoride ether (0.46mL,3.0mmol) slowly dropwise, stirred under nitrogen for 19h, and the reaction was monitored using TLC plates. After the reaction is finished, adding ethyl acetate into the reaction system to dilute the reaction system, washing the diluted system with water, drying an organic phase, and concentrating to obtain a crude product. The crude product was purified by column chromatography to afford compound XXVIII in 43% yield.

Example 2

This example is a characterization of the molecular structure prepared in example 1, and the experimental results were determined by nmr spectroscopy, specifically as follows:

a compound I:¹H NMR(500MHz,Chloroform-d)δ6.08(d,J＝2.0Hz,1H),5.36–5.32(m,2H),5.26(t,J＝2.3Hz,1H),4.29(td,J＝12.7,5.1Hz,1H),4.19–4.00(m,2H),2.22(s,3H,OAc),2.17(d,J＝4.0Hz,3H,OAc),2.09(s,3H,OAc),2.05(s,3H,OAc),2.00(s,3H,OAc).ESI-MS:Calc.for C₁₆H₂₂O₁₁:390.1162.Found:429.08[M+K]⁺,413.11[M+Na]⁺compound II:¹H NMR(500MHz,Chloroform-d)δ6.29(d,J＝1.6Hz,1H),5.72(dd,J＝10.2,3.4Hz,1H),5.45(dd,J＝3.4,1.7Hz,1H),5.37(t,J＝10.2Hz,1H),4.33(dd,J＝12.5,5.0Hz,1H),4.22(ddd,J＝10.3,5.1,2.2Hz,1H),4.14(dd,J＝12.5,2.3Hz,1H),2.17(s,3H,OAc),2.10(s,3H,OAc),2.07(s,3H,OAc),2.01(s,3H,OAc).

compound III:¹H NMR(500MHz,Chloroform-d)δ5.49(d,J＝2.6Hz,1H),5.29(t,J＝9.7Hz,1H),5.14(dd,J＝9.9,4.0Hz,1H),4.61(dd,J＝4.1,2.6Hz,1H),4.23(dd,J＝12.2,5.0Hz,1H),4.14(dd,J＝12.1,2.7Hz,1H),3.68(ddd,J＝9.5,4.9,2.7Hz,1H),3.27(s,3H,OMe),2.12(s,3H,OAc),2.07(s,3H,OAc),2.05(s,3H,OAc),1.74(s,3H,OMe).ESI-MS:Calc.for C₁₅H₂₂O₁₀:362.1213.Found:385.11[M+Na]⁺.

compound IV:¹HNMR(500MHz,Chloroform-d)δ5.37(dd,J_3c,4c＝10.1,J_2c,3c＝3.2Hz,1H,H-3c),5.33–5.21(m,6H,H-2c,H-3a,H-3b,H-4a,H-4b,H-4c),5.09(d,J_1b,2b＝2.0Hz,1H,H-1b),4.93(d,J_1c,2c＝1.8Hz,1H,H-1c),4.85(d,J_1a,2a＝2.0Hz,1H,H-1a),4.22–4.06(9H,m,H-2b,H-5b,H-5c,H-6a,H-6'a,H-6b,H-6'b,H-6c,H-6'c),4.04-3.94(dd,1H,H-2a),3.93-3.86(ddd,J_4a,5a＝9.6Hz,J_5a,6a＝4.5Hz,J_5a,6'a＝2.4Hz,1H,H-5a),3.40(s,3H,OMe).ESI-MS:Calc.for C₂₇H₃₈O₁₈:650.2058.Found:689.17[M+K]⁺,673.20[M+Na]⁺,651.21[M+H]⁺.

compound VII:¹H NMR(500MHz,Chloroform-d)δ5.38(dd,J＝10.0,3.4Hz,1H),5.35–5.23(m,6H),5.10(d,J＝2.1Hz,1H),4.94(d,J＝1.9Hz,1H),4.85(d,J＝2.1Hz,1H),4.27–4.07(m,7H),4.02(d,J＝2.4Hz,1H),3.90(ddd,J＝7.3,4.5,2.3Hz,1H),3.41(s,2H),2.15(s,3H,OAc),2.13(s,3H,OAc),2.12(s,3H,OAc),2.08(s,3H,OAc),2.06(s,3H,OAc),2.05(s,3H,OAc),2.03(d,J＝1.1Hz,6H,OAc*2),2.02(s,3H,OAc),2.00(s,3H,OAc),1.64(s,3H,OMe).ESI-MS:Calc.for C₃₉H₅₄O₂₆:938.2903.Found:977.25[M+K]⁺,961.28[M+Na]⁺,939.30[M+H]⁺.

compound V:¹H NMR(500MHz,Chloroform-d)δ6.23(d,J＝2.2Hz,1H),5.46–5.37(m,2H),5.31–5.22(m,3H),4.94(d,J＝1.9Hz,1H),4.27–4.09(m,5H),4.06–3.97(m,2H),2.14(s,6H,OAc*2),2.13(s,3H,OAc),2.09(s,3H,OAc),2.08(s,3H,OAc),2.03(s,6H,OAc*2),2.00(s,3H,OAc).ESI-MS:Calc.for C₂₈H₃₈O₁₉:678.2007.Found:717.16[M+K]⁺,701.19[M+Na]⁺.

compound VIII:¹H NMR(500MHz,Chloroform-d)δ6.23(d,J＝2.4Hz,1H),5.37(d,J＝9.9Hz,1H),5.34–5.28(m,3H),5.28–5.24(m,2H),5.12(d,J＝2.2Hz,1H),4.93(d,J＝1.9Hz,1H),4.27–4.16(m,3H),4.12(dtd,J＝9.6,5.3,4.7,2.7Hz,5H),4.06(t,J＝2.8Hz,1H),4.01(ddd,J＝9.8,4.1,2.4Hz,1H),2.14(d,J＝1.5Hz,6H),2.12(s,3H),2.11(s,3H),2.08(s,3H),2.07(s,3H),2.04(s,3H),2.04–2.02(m,9H),1.99(s,3H),1.72(s,2H).ESI-MS:Calc.for C₄₀H₅₄O₂₇:966.2853.Found:1005.25[M+K]⁺,989.27[M+Na]⁺,967.29[M+H]⁺.

compound X:¹H NMR(500MHz,Chloroform-d)δ5.44–5.22(m,10H),5.13(d,J＝2.2Hz,1H),5.07(d,J＝2.4Hz,1H),4.96(d,J＝1.9Hz,1H),4.86(d,J＝2.2Hz,1H),4.27–4.01(m,13H),3.90(ddd,J＝9.7,4.5,2.4Hz,1H),2.15(s,3H),2.12(s,3H),2.11(s,3H),2.11(s,3H),2.08(d,J＝0.9Hz,6H),2.06(s,6H),2.03(s,3H),2.03(s,3H),2.02(s,3H),2.01(s,3H),2.00(s,3H),1.68(s,3H).ESI-MS:Calc.for C₅₁H₇₀O₃₄:1226.3749.Found:1265.34[M+K]⁺,1249.36[M+Na]⁺,1227.38[M+H]⁺.

compounds VI to Ser:¹H NMR(500MHz,Chloroform-d)δ7.75(d,J＝7.5Hz,2H,H-Fmoc),7.61(t,J＝8.2Hz,2H,H-Fmoc),7.38(t,J＝7.3Hz,2H,H-Fmoc),7.29(t,J＝7.5Hz,2H,H-Fmoc),6.55(d,J＝8.3Hz,1H,NH),5.45(dd,J＝9.9,3.5Hz,1H,H-3),5.31–5.20(m,2H,H-2,H-4),4.86(d,J＝1.6Hz,1H,H-1),4.70(m,1H,H-α),4.37(m,2H,CH₂-Fmoc),4.23(m,2H,CH-Fmoc,H-6),4.16–4.00(m,4H,H-5,H-6,CH₂-β),2.15,2.05,2.01,1.96(4s,each 3H,OAc).ESI-MS:Calc.for C₃₂H₃₅NO₁₄:657.2058.Found:696.17[M+K]⁺,680.19[M+Na]⁺,658.21[M+H]⁺.

compound IX-Ser:¹H NMR(500MHz,Chloroform-d)δ7.75(d,J＝7.6Hz,2H,H-Fmoc),7.67-7.57(d,2H,H-Fmoc),7.38(t,J＝7.5Hz,2H,H-Fmoc),7.30(t,J＝7.5Hz,2H,H-Fmoc),6.19(d,J＝8.2Hz,1H,H-NH),5.38(dd,J＝10.0,3.4Hz,1H,H-3'),5.34-5.21(m,4H,H-3,H-4,H-4',H-2'),4.97(d,J＝2.5Hz,1H,H-1),4.92(d,J＝2.0Hz,1H,H-1'),4.60(d,J＝8.2Hz,1H,H-α),4.46-3.95(m,12H，CH₂-Fmoc,H-6,H-6,CH-Fmoc,H-6',H-6',H-5',CH2-β,H-2,H-5)2.13(s,3H,OAc),2.10(s,3H,OAc),2.10(s,3H,OAc),2.08(s,3H,OAc),2.04(s,3H,OAc),2.01(s,3H,OAc),1.98(s,3H,OAc).ESI-MS:Calc.for C₄₄H₅₁NO₂₂:954.2903.Found:984.25[M+K]⁺,968.28[M+Na]⁺,946.30[M+H]⁺.

compound XII-Ser:1H NMR (500MHz, Acetone-d)₆)δ7.86(d,J＝7.5Hz,2H,H-Fmoc),7.73(d,J＝7.4Hz,2H,H-Fmoc),7.47-7.39(t,J＝7.4Hz,2H,H-Fmoc),7.34(m,2H,H-Fmoc),7.11(d,J＝8.4Hz,1H,NH),5.41-5.23(m,8H,H-1,H-2”,H-3,H-3',H-3”,H-4,H-4',H-4”),5.18(d,J＝2.0Hz,1H,H-1'),5.15(d,J＝1.9Hz,1H,H-1”),4.59(m,1H,H-α),4.36(m,2H,CH₂-Fmoc),4.29-4.10(m,13H,H-2',H-5,H-5',H-5”,H-6,H-6',H-6”,CH₂-β,CH-Fmoc),4.08-4.03(m,1H,H-2),2.11,2.09,2.07,2.03,2.03,2.02,2.01,1.97,1.96(9s,30H,CH₃-Ac).ESI-MS:Calc.for C₅₆H₆₇NO₃₀:1233.3748.

Found:1272.34[M+K]⁺,1256.36[M+Na]⁺,1234.38[M+H]⁺.

Compound VI-Thr:¹H-NMR(500MHz,DMSO-d₆)δ7.90(d,J＝7.5Hz,2H,H-Fmoc),7.76(d,J＝6.9Hz,2H,H-Fmoc),7.40-7.46(m,2H,H-Fmoc),7.31-7.37(m,2H,H-Fmoc),5.29(dd,J＝9.9,3.6Hz,1H,H-3),5.05(dd,J＝3.9,1.8Hz,1H,H-2),5.04(t,J＝9.9,1H,H-4),4.97(d,J＝1.5Hz,1H,H-1),4.22-4.32(m,4H,CH-β,CH-Fmoc,CH₂-Fmoc)4.02-4.20(m,4H,H-5,H-6,H-6,H-α),2.08(s,3H,OAc),2.03(s,3H,OAc),2.01(s,3H,OAc),1.92(s,3H,OAc),1.24(d,J＝6.3Hz,3H,CH3-γ).ESI-MS:Calc.for C₃₃H₃₇NO₁₄:671.2214.Found:710.18[M+K]⁺,694.21[M+Na]⁺,672.23[M+H]⁺.

compound IX-Thr:¹H NMR(500MHz,Acetone-d₆)δ7.88(d,J＝7.6Hz,2H,H-Fmoc),7.74(t,2H,H-Fmoc),7.43(t,2H,H-Fmoc),7.35(m,2H,H-Fmoc),7.07(d,J＝9.5Hz,1H,NH),5.38(dd,J＝10.2,3.3Hz,1H,H-3),5.34–5.27(m,4H,H-2',H-3',H-4,H-4'),5.24(d,J＝1.6Hz,1H,H-1),5.02(d,J＝1.6Hz,1H,H-1'),4.53(m,1H,CH-β),4.47-4.33(m,3H,H-α,CH₂-Fmoc),4.27(t,J＝7.0Hz,1H,CH-Fmoc),4.24-4.06(m,7H,H-2,H-5,H-5',H-6,H-6'),2.12-1.97(21H,OAc),1.44(d,J＝6.4Hz,3H,CH₃).ESI-MS:Calc.for C₃₃H₃₇NO₁₄:959.3059.Found:998.27[M+K]⁺,982.30[M+Na]⁺,960.31[M+H]⁺.

compound XII-Thr:¹H NMR(500MHz,Acetone-d₆)δ7.88(d,J＝7.6Hz,2H,H-Fmoc),7.73(t,J＝6.5Hz,2H,H-Fmoc),7.43(t,J＝7.6Hz,2H,H-Fmoc),7.36(t,J＝7.5Hz,2H,H-Fmoc),5.47–5.24(m,9H,H-1,H-1',H-2”,H-3,H-3',H-3”,H-4,H-4',H-4”),5.22(s,1H,NH),5.19(d,J＝1.2Hz,1H,H-1”),4.58–4.50(m,1H,H-β),4.46–4.07(m,15H,H-2,H-2',H-5,H-5',H-5”,H-6,H-6',H-6”,H-α,CH-Fmoc,CH₂-Fmoc),2.14–1.97(m,30H,OAc),1.43(d,J＝6.2Hz,3H,CH₃).ESI-MS:Calc.for C₅₇H₆₉NO₂₂:1247.39.Found:1270.63[M+Na]⁺.

compound XXVII:¹H-NMR(500MHz,CDCl₃):δ2.00,2.05,2.06,and2.15(4s,each 3H,4CH₃-Ac),3.18(dd,1H,J_α,β1＝2.1Hz,J_β1β2＝14.7Hz,H-β1),3.31(dd,1H,J_α,β2＝4.5Hz,H-β2),4.22(m,4H,Fmoc-CH,Fmoc-CHH,H-6a,and H-6b),4.32(m,1H,H-5),4.42(m,1H,Fmoc-CHH),4.79(m,1H,H-α),5.19(dd,1H,J_2,3＝2.6Hz,J_3,4＝9.8Hz,H-3),5.30(m,2H,H-4and H-1),5.35(bs,1H,H-2),6.10(d,1H,J_NH,α＝8.1Hz,NH),7.54(m,8H,Fmoc-ArH).High-resolution MS data of C₃₂H₃₅NO₁₃S(M,673.1829):[M+Na]⁺found 696.308；calcd 696.173.

compound XXVIII:¹H-NMR(400MHz,CDCl₃)δ7.76(d,J＝7.6Hz,2H,H-Fmoc),7.61(d,J＝7.2Hz,2H,H-Fmoc),7.40(t,J＝7.4Hz,2H,H-Fmoc),7.32(t,J＝7.4Hz,2H,H-Fmoc),6.05(d,J＝7.2Hz,1H,NH),5.48(s,1H,H-1),5.40(dd,3H,J＝10.0,2.7Hz,H-3’),5.31-5.36(m,2H,H-4,H-4’),5.17-5.23(m,2H,H-2’,H-3),4.92(d,J＝1.2Hz,1H,H-1’),4.64(s,1H,H-α),4.27-4.45(m,5H,H-5’,H-6,CH₂-Fmoc),4.22(t,1H,J＝7.0Hz,CH-Fmoc),4.13-4.17(m,4H,H-2,H-5,H-6’),3.24(dd,2H,J＝66.0,13.6Hz,CH₂-β),2.14(s,3H,CH₃-Ac),2.13(s,3H,CH₃-Ac),2.12(s,3H,CH₃-Ac),2.08(s,3H,CH₃-Ac),2.04(s,3H,CH₃-Ac),2.02(s,3H,CH₃-Ac),2.01(s,3H,CH₃-Ac).HRMS(ESI)Calcd.for C₄₄H₅₁NNaO₂₁S[M+Na]⁺requires 984.2567,Found:984.2556。

example 3

This example is the mannose glycosylated amino acid enantiomer (where R is H or CH)₃) Preparation example (3). The synthetic route was the same as that of example 1, except that Fmoc-Ser-OH/Fmoc-Thr-OH and D- (+) -Mannose were replaced with Fmoc-D-Ser-OH/Fmoc-Thr-OH and L- (-) -Mannose during the synthesis. The obtained compounds XIV, XV, XVI and XVII are shown below.

Example 4

This example is the mannose glycosylated amino acid half enantiomer (where R is H or CH)₃) Preparation example (3). The synthetic route was the same as that of example 1, except that Fmoc-D-Ser-OH/Fmoc-D-Thr-OH or L- (-) -Mannose was used in the synthesis instead of Fmoc-Ser-OH/Fmoc-Thr-OH or D- (+) -Mannose, and the obtained compounds XVIII, XIX, XX, XXI, XXII, XXIV, XXV and XXVI were as follows.

Example 5

The mannosylated amino acids of this example which are analogues containing a thioglycosidic bond are shown below. The synthetic route was the same as that of example 1, except that Fmoc-Cys-OH was used instead of Fmoc-Ser-OH/Fmoc-Thr-OH in the synthesis, and XXVII, XXVIII, XXIX and XXX were obtained as follows:

in the description of the above exemplary embodiment of the present invention, compound IV was obtained with a yield of about 36% using D- (+) -mannose, L- (-) -mannose, Fmoc-L- (-) -serine, Fmoc-D- (+) -serine, Fmoc-L- (-) -threonine, Fmoc-D- (+) -threonine, etc. as starting materials, respectively; compound VII, yield about 17%; compound X, in about 8% yield; compound VI, R ═ H, yield approximately 29%, R ═ CH₃The yield was 27%; compound IX, R ═ H, yield was about 39%, R ═ CH₃The yield was 44%; the yield was about 38% for compound XII, R ═ H, and R ═ CH₃The yield was 48%; compound XIII, R ═ H, yield approximately 45%, R ═ CH₃The yield is 50%; the yields of the mannose-glycosylated amino acids enantiomers and the hemienantiomers XIV-XVII, XVIII-XXI and XXIII-XXVI are similar to the yields of the corresponding mannose-glycosylated amino acids VI, IX, XII and XIII. The invention is superiorCompared with the prior art, the novel chemical preparation method has the advantages of mild reaction conditions, simple and convenient steps and high utilization rate of raw materials.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments without departing from the scope or spirit of the present invention. The scope of the invention should be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.