Preparation method of ganoderma lucidum spore glswa-1 polysaccharide tetradecasaccharide and substructure and application of enhancing insulin protein activity

Abstract

The present application belongs to the technical field of biological medicine, and relates to a new use of polysaccharide, in particular to a preparation method of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide and its substructure and application of the Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide in enhancing insulin protein activity. The present application discloses structures and applications of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide 1 and its substructures pentasaccharide 2, pentasaccharide 3 and tetrasaccharide 4, and discloses the use of a series of polysaccharides in preparing Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide. Activity research shows that the Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide has obvious blood sugar lowering activity, and it is determined that the substructure pentasaccharide 2 with the insulin protein activity enhancing effect is an active center site, and further research shows that the essence of the activity of the substructure is to stabilize the three-dimensional structure of insulin to maintain the content of insulin, thereby playing a key role in reducing blood sugar. The disclosure fills the gap of Ganoderma lucidum polysaccharide research and application, and indicates a new path for the research and development of drugs and health care products with hypoglycemic effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a new use of polysaccharide, in particular to a preparation method of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide and substructure and application of the polysaccharide tetradecasaccharide and substructure in enhancing insulin protein activity. BACKGROUND

[0002] As one of the four molecules necessary for life activities, polysaccharide plays an important role in the process of life activities of organisms. Synthesis and preparation of complex oligosaccharides and conjugates with determined structure as molecular tools to meet the needs of structural, biochemical and biological research, and to carry out research and development of sugar drugs, have become an important task of sugar chemistry and glyco-biochemistry research. Chemical synthesis is a reliable means to obtain sugar compounds with determined structure and uniformity. However, unlike nucleotides and proteins which can be programmed to complete automatic solid-phase synthesis, the synthesis of sugar compounds is more difficult, and its development is more backward. The problem of regioselectivity and stereoselectivity is the most important challenge in the synthesis of sugar compounds, which also leads to a long time-consuming and long-step process for the synthesis of sugar compounds. At present, there are still few reports on the synthesis of long, branched and complex sugar compounds.

[0003] Ganoderma lucidum belongs to Ganodermataceae and Ganoderma. Ganoderma contains various bioactive components, including triterpenes, polysaccharides, nucleotides, sterols and the like. Recent studies have shown that polysaccharides extracted from the fruiting bodies, mycelium and spores of Ganoderma have high biological activity. These polysaccharides have many pharmacological activities, such as antioxidant, antitumor and immunomodulatory and other functions. Although more than 200 polysaccharides have been isolated from Ganoderma, in many cases, the in vitro and in vivo biological effects of these polysaccharides are still unclear due to the lack of structural data. Ganoderma spores are extremely small, fog-like brown oval spores released at the mature Ganoderma cap. These spores contain the entire genetic material and biological material of Ganoderma. They are considered to have greater medicinal value than the fruiting bodies of Ganoderma. However, due to the difficulty of collection, the study of polysaccharides in Ganoderma spores is less.

[0004] In 2017, Chinese scientists Yang Jinsong and Tang Qingjiu et al. first reported the complete structure of GLSWA-1 polysaccharide tetradecasaccharide fragment from Ganoderma spores. GLSWA-1 glucan has the following structural characteristics: (1) a long chain structure with 10 main glucose, containing 4 β-(1→3) glycosidic bonds, 4 β-(1→6) glycosidic bonds and 1 β-(1→4) glycosidic bond; (2) 3 branch structures connected to the main chain by β-(1→4) glycosidic bond and β-(1→6) glycosidic bond. The structure is as follows:

[0005]

[0006] However, although the biological activity of Ganoderma lucidum extracts represented by Ganoderma lucidum polysaccharides has been studied in the prior art, the separate biological activity of water-soluble glucan GLSWA-1 isolated from the spore powder of Ganoderma lucidum and its action mechanism have not been reported.

[0007] In addition, there is no literature report on the total synthesis of the fourteen-sugar fragment of Ganoderma spore GLSWA-1 polysaccharide and its substructure. SUMMARY

[0008] Therefore, the first object of the present application is to solve the technical problems existing in the prior art by efficiently synthesizing the target structure and its three substructures based on the orthogonal one-pot glycosylation reaction strategy of glycosyl N-phenyltrifluoroacetimidate, glycosyl o-alkynylbenzoate and glycosyl o-(1-phenylalkenyl)benzoate, thereby avoiding the problems of aglycone transfer, leaving group interference and strong thiol odor existing in the orthogonal one-pot glycosylation reaction strategy based on thioglycoside. Figure 1 The structure of the target oligosaccharide is shown in the following formula:

[0009] It is worth noting that the structural characteristics of the fourteen-sugar fragment of Ganoderma spore GLSWA-1 polysaccharide are as follows: from the perspective of sugar composition, GLSWA-1 polysaccharide is a typical glucan compound composed of D-glucose; from the perspective of structure, the main chain of GLSWA-1 is formed by the connection of ten glucose in turn, containing two monosaccharide branches and one disaccharide branch; from the perspective of connection mode, the main chain structure of GLSWA-1 is connected by 4 beta-(1→3) glycosidic bonds, 1 beta-(1→4) glycosidic bond and 4 beta-(1→6) glycosidic bonds from the non-reducing end to the reducing end; the three branch structures are all connected to the O-6 position of the fifth D-glucose (monosaccharide branch structure), the O-6 position of the sixth D-glucose (disaccharide branch structure) and the O-4 position of the seventh D-glucose (monosaccharide branch structure) from the non-reducing end to the reducing end.

[0010] In order to achieve the above object, the present application adopts the following technical solutions:

[0011] A preparation method of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide, first, an orthogonal one-pot glycosylation reaction of sugar-based ortho(1-phenylalkenyl)benzoate PVB, sugar-based N-phenyltrifluoroacetimidate PTFAI and sugar-based ortho-alkynylbenzoate ABz is used to construct sugar-based PTFAI tetrasaccharide donor 5 and penta-saccharide PVB double functional group acceptor 6 and a reducing end penta-saccharide acceptor 7; then the fragments of sugar-based PTFAI tetrasaccharide donor 5, penta-saccharide PVB double functional group acceptor 6 and reducing end penta-saccharide acceptor 7 are used to prepare Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide 1 through 【4+5+5】 orthogonal one-pot glycosylation reaction and functional group transformation; wherein the structures of sugar-based PTFAI tetrasaccharide donor 5, penta-saccharide PVB double functional group acceptor 6 and reducing end penta-saccharide acceptor 7 are shown in the reaction pathway:

[0012]

[0013] The structural research of fungal polysaccharides is of great significance for understanding the mechanism and structure-activity relationship of fungal polysaccharides and screening new drug targets. The synthesis of long-chain and multi-branched complex carbohydrates has always been a difficulty in chemical synthesis of polysaccharides, and Ganoderma lucidum spore polysaccharide GLSWA-1 is a typical representative, which contains multiple branches and different connection modes. If the traditional sequential glycosylation connection is used, it will increase the workload and cause serious waste of raw materials and cost. The method of orthogonal one-pot glycosylation reaction has the following advantages: 1) the glycosylation process does not require any functional group protection, does not require purification of intermediates, and can effectively improve the synthesis speed and reduce chemical waste; 2) the high-efficiency modular orthogonal one-pot glycosylation method not only solves the problem of difficult synthesis of polysaccharide splicing and low efficiency, but also provides multiple natural product templates for subsequent activity research, which provides convenient conditions for the study of the structure-activity relationship of polysaccharides.

[0014] The reaction steps of the 【4+5+5】 orthogonal one-pot glycosylation reaction include:

[0015] The 【4+5】 glycosylation reaction of the tetrasaccharide N-phenyltrifluoroacetimidate donor and the penta-saccharide ortho(1-phenylalkenyl)benzoate double functional group acceptor generates a non-sugar intermediate, which then further undergoes a 【9+5】 glycosylation reaction with the reducing end penta-saccharide acceptor to prepare the fully protected Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide.

[0016] The reaction steps of the functional group transformation include: sodium methoxide is used to remove all benzoyl groups of the target substrate, and then palladium / carbon (Pd / C) is used to remove the remaining benzyl protecting groups.

[0017] It is worth noting that the orthogonal one-pot glycosylation reaction strategy based on glycosyl ortho-(1-phenylalkenyl) benzoate disclosed in the application effectively avoids the problems of aglycone transfer, leaving group interference and strong thiol odor existing in the orthogonal one-pot glycosylation reaction strategy based on thioglycoside, and the orthogonality of ortho-(1-phenylalkenyl) benzoate, N-phenyl trifluoroacetate and ortho-alkynyl benzoate and the difference in reactivity of ortho-(1-phenylalkenyl) benzoate and thioglycoside can be utilized to realize four-step one-pot glycosylation for efficient synthesis of the acceptor pentasaccharide at the reducing end.

[0018] In addition, the preparation method disclosed in the application has the following characteristics: on the one hand, it is constructed by orthogonal one-pot glycosylation reaction of multiple 1,2-trans glycosidic bonds; on the other hand, the target tetradecasaccharide is synthesized by convergent modular 【4+5+5】 orthogonal one-pot method. The preparation method provides a very economical and green chemical synthesis method for quickly obtaining GLSWA-I tetradecasaccharide and its three substructures, and effectively makes up for the problem of scarcity of natural ganoderma spore polysaccharide.

[0019] The second object of the application is to provide an application of the ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide obtained by the preparation method, i.e., the application of the ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide in preparing a preparation with hypoglycemic efficacy.

[0020] The preparation of the application is prepared from the ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide and a pharmaceutically acceptable excipient. The preparation is a solid preparation or a liquid preparation.

[0021] It is worth noting that the hypoglycemic efficacy is to stabilize the three-dimensional structure of insulin to maintain the content of insulin.

[0022] The activity study shows that the ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide has the effect of enhancing the activity of insulin protein, and can improve the thermal stability of the tertiary structure of insulin.

[0023] The third object of the application is to provide a pentasaccharide with the effect of enhancing the activity of insulin protein, and a derivative structure of the pentasaccharide 2 is an intermediate reducing end pentasaccharide acceptor 7 for preparing the ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide, and the structure of the pentasaccharide 2 is as follows:

[0024]

[0025] Further, the application also provides an application of the pentasaccharide with the effect of enhancing the activity of insulin protein, i.e., the application of the pentasaccharide 2 in preparing a preparation and a compound with hypoglycemic efficacy.

[0026] Further, the hypoglycemic efficacy is to stabilize the three-dimensional structure of insulin to maintain the content of insulin.

[0027] The activity study shows that the pentasaccharide 2 has the effect of enhancing the activity of insulin protein and can improve the thermal stability of the tertiary structure of insulin.

[0028] It is worth noting that the preparation of the present application is prepared from the pentasaccharide 2 with the effect of enhancing the activity of insulin protein and the pharmaceutically acceptable adjuvant. The preparation is a solid preparation or a liquid preparation.

[0029] Further, the application in the preparation of the compound with the hypoglycemic effect includes the preparation of the Ganoderma spore GLSWA-1 polysaccharide fourteen saccharide 1 by the orthogonal one-pot glycosylation reaction and the functional group transformation using the derivative of the pentasaccharide 2.

[0030] In order to technically complete the disclosure, the present application also provides a preparation method of the biologically active pentasaccharide as described above. However, it should be clear that this disclosure does not limit the technical solutions contained in the present application. The pentasaccharide 2 and its derivatives obtained by the prior art without creative work or only by conventional technical means belong to the protection scope of the present application.

[0031] A preparation method of a biologically active pentasaccharide, including preparing the pentasaccharide 2 by the orthogonal one-pot glycosylation method and the deprotection method, first constructing the substructure pentasaccharide 20 by the four-step one-pot glycosylation reaction of the orthogonality of N-phenyl trifluoroacetate, o-alkynyl benzoate and o-(1-phenylalkenyl) benzoate and the poor reactivity of o-(1-phenylalkenyl) benzoate and 4-phenylthiol, and then preparing the biologically active pentasaccharide 2 by the functional group deprotection transformation.

[0032] The specific synthesis method is as follows:

[0033] Under the condition of argon protection, the glycosyl PTFAI donor 8, the ABz acceptor 14 and the newly activated The molecular sieve was dissolved in dry dichloromethane, stirred at room temperature for 15 min, then cooled to 0℃, and trifluoromethanesulfonic acid (HOTf) was added. After reaction at 0℃ for 2 h, the PVB acceptor 15 and the newly prepared PPh3AuOTf dichloromethane solution were added in turn, and the reaction was carried out at room temperature for 4 h. After monitoring the reaction completion by thin layer chromatography, the temperature was lowered to -30℃, and the SToL acceptor 16, N-iodosuccinimide NIS and trifluoromethanesulfonic acid HOTf were added in turn. After reaction at -30℃ for 4 h, the reaction completion was monitored by thin layer chromatography, the temperature was raised to 0℃, the Linker acceptor 17, N-iodosuccinimide NIS and trifluoromethanesulfonic acid HOTf were added in turn, and the reaction was carried out at 0℃ for 3 h. The reaction was quenched with triethylamine, filtered with diatomite, the filtrate was concentrated to remove the solvent by a rotary evaporator, and the product was purified by silica gel column chromatography to obtain the pentasaccharide 20.

[0034] The pentasaccharide substrate 20 is dissolved in dry tetrahydrofuran under the protection of argon, hydrogen fluoride pyridine is slowly added, reaction is carried out at room temperature for 40 h, the reaction is quenched with triethylamine, the reaction solution is diluted with dichloromethane, sequentially washed with saturated sodium bicarbonate aqueous solution, saturated brine, the organic phase is dried with anhydrous sodium sulfate, filtered, the filtrate is concentrated under reduced pressure, and column chromatography on silica gel is carried out to obtain the pentasaccharide acceptor 7;

[0035] The pentasaccharide acceptor 7 is dissolved in methanol / dichloromethane under the protection of argon, sodium methoxide is added to adjust the pH to 12, reaction is carried out at room temperature for 24 h, thin layer chromatography is used to monitor the reaction, 4M hydrochloric acid is used to adjust the pH to 7, filtration is carried out, the filtrate is concentrated under reduced pressure by using a rotary evaporator, and column chromatography on Sephadex TM LH-20 gel is carried out to obtain an intermediate, the intermediate and palladium hydroxide / carbon (Pd(OH)2 / C) are dissolved in tetrahydrofuran / methanol / water / acetic acid, hydrogen is introduced into the reaction system, reaction is carried out at room temperature for 18 h, the reaction solution is filtered by using a 25mm polyether sulfone microporous filter membrane, the filtrate is concentrated by using a rotary evaporator, and column chromatography on Sephadex TM LH-20 gel is carried out to obtain the fully deprotected pentasaccharide substructure 2;

[0036] The reaction path is as follows:

[0037]

[0038] It is worth noting that in the process of synthesizing the pentasaccharide substructure 20, not only the orthogonality of ortho-(1-phenylalkenyl) benzoate, N-phenyl trifluoroacetate and ortho-alkynyl benzoate is utilized, but also the activity difference between ortho-(1-phenylalkenyl) benzoate and STol is utilized, and four-step one-pot glycosylation is further realized, so that the synthesis of a pentasaccharide based on a natural product as a template by utilizing four-step one-pot glycosylation is rarely reported.

[0039] Further, the application further discloses a pentasaccharide with biological activity, a derivative structure of the pentasaccharide 3 is an intermediate pentasaccharide ortho-(1-phenylalkenyl) benzoate bifunctional acceptor 6 for preparing Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide, and a structure of the pentasaccharide 3 is as follows:

[0040]

[0041] Further, the application discloses application of the pentasaccharide 3 in preparation of the Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1.

[0042] In addition, the application further discloses a tetrasaccharide with biological activity, a derivative structure of the tetrasaccharide 4 is an intermediate glycosyl PTFAI tetrasaccharide donor 5 for preparing Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide, and a structure of the tetrasaccharide 4 is as follows:

[0043]

[0044] Further, the application discloses application of the tetrasaccharide 4 in preparation of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide 1.

[0045] Compared with the prior art, the application discloses structures and applications of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide 1, pentasaccharide 2, pentasaccharide 3 and tetrasaccharide 4 and a series of derivative products thereof, and discloses a new use of the series of polysaccharides in preparation of Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide with a blood sugar lowering effect. Active research shows that the Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide has obvious blood sugar lowering activity. After in-depth research and comparison of the blood sugar lowering activity of the three substructures and the complete structure, the substructure pentasaccharide 2 with the insulin protein activity enhancing effect is successfully found as an active center site. Further research shows that the essence of the activity of the substructure is to stabilize the three-dimensional structure of insulin to maintain the content of insulin and then play a key role in reducing blood sugar. The disclosure fills the gap of Ganoderma lucidum polysaccharide research and application, and indicates a new path for research and development of drugs and health care products with a blood sugar lowering effect.

[0046] In addition, synthesis of complex carbohydrates with long chains and multiple branches has always been a difficulty in chemical synthesis of polysaccharides. The application provides a high-efficiency modular orthogonal one-pot glycosylation strategy for the total synthesis of the GLSWA-1 polysaccharide tetradecasaccharide fragment with a long chain structure and a branched structure. First, the orthogonal one-pot glycosylation of sugar-based ortho (1-phenylalkenyl) benzoic acid ester, sugar-based N-phenyltrifluoroacetic acid acetylimidate and sugar-based ortho alkyne benzoic acid ester is used to synthesize three polysaccharide substructures, so as to simplify chemical synthesis of oligosaccharides. Second, the three substructures are efficiently spliced through 【4+5+5】 orthogonal one-pot glycosylation to obtain the Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide fragment, so as to avoid complexity and uneconomicalness caused by synthesis of long chains and branched structures. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, brief introduction will be given to the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only belong to the embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of the provided drawings

[0048] Figure 1 Structure of the target oligosaccharide Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide.

[0049] Figure 2 Experimental results obtained in experiment 2 of the present application.

[0050] Figure 3 Experimental results obtained in experiment 3 of the present application. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0052] Herein the word "embodiment" as "illustrative" of any embodiment described does not necessarily mean that the described embodiment is preferred or better than other embodiments. In the performance index test of the embodiments of the present application, unless otherwise specified, the conventional test method in the art is adopted. It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the disclosure of the present application.

[0053] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present application belongs; as the test methods and technical means not specially noted in the present application are all the experimental methods and technical means commonly used by those skilled in the art.

[0054] In order to better illustrate the content of the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that without some specific details, the present application can also be implemented. In the embodiments, some methods, means, instruments, equipment and the like which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0055] The technical features disclosed in the embodiments of the present application can be combined in any way without conflict, and the technical solutions obtained belong to the disclosure of the embodiments of the present application.

[0056] The structure of the target oligosaccharide of the present application is shown as Figure 1 .

[0057] The specific Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide fragment and its substructure full synthesis preparation method is:

[0058]

[0059] Preparation of substrate 9: A known compound S1 (4.87 g, 9.96 mmol) was dissolved in pyridine (20 mL). After cooling to 0 °C, B2Cl (3.5 mL, 29.89 mmol) and DMAP (1.22 g, 9.96 mmol) were slowly added to the system. The reaction mixture was then heated to 70 °C in an oil bath and stirred for 24 h. After the reaction was complete as monitored by thin-layer chromatography, the reaction solution was diluted with ethyl acetate and washed successively with 4 M HCl, saturated sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1 to 10:1) to give an intermediate (5.91 g), 100% yield. Under argon protection, this intermediate (3 g, 5.06 mmol) was dissolved in dry dichloromethane (50.61 mL) and freshly activated... Molecular sieves were used. After stirring at room temperature for 15 min, the mixture was cooled to 0 °C, and trimethylsilyltrifluoromethanesulfonic acid (TMSOTf) (0.46 mL, 2.53 mmol) and BH3 (20.24 mL, 20.24 mmol) were added dropwise to the reaction system. The mixture was stirred at 0 °C for 12 h. After the reaction was completed by thin-layer chromatography, the reaction was quenched with Et3N / MeOH = 10 / 1. The mixture was filtered through diatomaceous earth, and the reaction solution was extracted with ethyl acetate and washed successively with saturated sodium bicarbonate aqueous solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to give a white solid (2.93 g), yield 97%. The above solid (3.9 g, 6.56 mmol) was dissolved in dry tetrahydrofuran (32.78 mL). Under argon protection at 0 °C, 60% (314.68 mg, 13.11 mmol) of NaH was added. After stirring for 15 min, BnBr (1.68 g, 9.83 mmol) and TBAI (1.21 g, 3.28 mmol) were added sequentially. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete as monitored by thin-layer chromatography, the reaction was quenched with water. The reaction solution was diluted with dichloromethane and washed sequentially with saturated sodium bicarbonate aqueous solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 25:1 to 7:1) to give a white solid (4.49 g), 100% yield. The above solid (4.49 g, 6.56 mmol) was dissolved in tetrahydrofuran (21.85 mL), and pyridine hydrogen fluoride (70%, 5.32 mL) was slowly added dropwise. The mixture was stirred at room temperature for 24 h. After the reaction was complete as monitored by thin-layer chromatography, triethylamine was slowly added to quench the reaction. The reaction solution was diluted with dichloromethane and washed successively with saturated sodium bicarbonate aqueous solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to give white foam S2 (3.67 g), yield 98%.

[0060] Under argon, intermediate S2 (3.67 g, 6.43 mmol) and LevOH (1.12 g, 9.65 mmol) were dissolved in DCM (64 mL) and EDCI (2.22 g, 11.58 mmol), DMAP (785.64 mg, 6.43 mmol) and DIPEA (3.19 mL, 19.29 mmol) were added sequentially. The reaction was stirred at room temperature overnight and monitored by TLC. The reaction was concentrated under reduced pressure using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, 5:1 to 3:1) to give a white solid (4.3 g) in 100% yield. The white solid (4.3 g, 6.43 mmol) was dissolved in acetone / H2O (128 mL / 32 mL) and cooled to 0 °C. TCCA (1.49 g, 6.43 mmol) was added and the reaction was maintained at 0 °C for 2 h. The reaction was monitored by TLC and quenched with saturated NaHC03solution. The reaction was diluted with ethyl acetate and the organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1 to 2:1 to dichloromethane:methanol = 20:1) to give a white foam (2.77 g) in 77% yield. The foam (1.6 g, 2.84 mmol) and PhN=C(Cl)CF3(619.85 mg, 2.99 mmol) were dissolved in acetone and anhydrous potassium carbonate (589.55 mg, 4.27 mol) was added. The reaction was stirred at room temperature overnight. TLC showed that the reaction was complete and the solid was removed by filtration. The filtrate was concentrated under reduced pressure using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether / ethyl acetate, 8:1 with 1% Et3N) to give yellow syrup S3 (2.09 g) in 100% yield. Compound S3 was not further characterized and used directly in the subsequent glycosylation reaction.

[0061] Under argon, PTFAI donor S3 (850.0 mg, 1.16 mmol), ABzOH acceptor (323.5 mg, 1.74 mmol), and freshly activated Molecular sieves (1.2 g) were dissolved in dry dichloromethane (11 mL). After stirring at room temperature for 15 min, the temperature was lowered to -15 °C and HOTf (20.71 μL, 0.23 mmol) was added slowly. After 1 h at -15 °C, the reaction was quenched with triethylamine (0.5 mL), filtered over celite and the filtrate was concentrated over a rotary evaporator to remove the solvent and purified over a silica gel column (petroleum ether: ethyl acetate = 4: 1) to give a yellow foam (757.3 mg) in 85% yield. The above foam (343.7 mg, 0.47 mmol) was dissolved in pyridine / acetic acid (3 mL / 2 mL) under argon and hydrazine hydrate (NH2NH2-H2O) (0.069 mL, 1.41 mmol) was added dropwise. After stirring at room temperature overnight, the reaction was monitored by thin layer chromatography and after completion, the reaction was quenched by the addition of acetone and diluted with ethyl acetate. The organic phase was washed successively with 1 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and saturated aqueous brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated over a rotary evaporator under reduced pressure and purified over a silica gel column (petroleum ether: ethyl acetate = 5: 1) to give 9 as a white foam (236.6 mg) in 80% yield.

[0062] Data for 9: [a] D 24 = +7.54 (c 0.38 CHCI3); 1 H NMR (400 MHz, Chloroform-d) δ 7.87 (d, J = 7.6 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.30 (d, J = 7.7 Hz, 1H), 7.27 - 7.10 (m, 14H), 7.07 (t, J = 7.6 Hz, 1H), 5.97 (d, J = 8.2 Hz, 1H), 5.30 (t, J = 8.8 Hz, 1H), 4.74 (d, J = 11.2 Hz, 1H), 4.54 (d, J = 11.9 Hz, 2H), 4.41 (d, J = 12.1 Hz, 1H), 3.93 (t, J = 9.0 Hz, 1H), 3.73 (t, J = 9.2 Hz, 1H), 3.70 (d, J = 2.7 Hz, 2H), 3.63 (dt, J = 9.8, 2.8 Hz, 1H), 3.02 (s, 1H), 1.37 (m, 1H, CH-ABz), 0.77 - 0.70 (m, 4H, 2 x CH2-ABz). 13C NMR(101MHz,Chloroform-d)δ166.25,163.75,138.11,137.89,134.28,133.36,132.24,130.88,129.84,129.67,129.21,128.49,128.40,128.37,12 8.01,127.94,127.91,127.69,127.00,125.58,100.28,92.51,77.66,75. 90,75.82,74.82,74.40,73.95,73.54,68.22,8.96,8.93.HRMS(ESI)calcd forC 39 H 36 O8[M+Na] + 655.2302, found 655.2304.

[0063]

[0064] Preparation of substrate 10: Under argon protection, PTFAI donor S3 (1 g, 1.36 mmol), PVBOH acceptor (458.46 mg, 2.04 mmol), and freshly activated... Molecular sieve (1.5 g) was dissolved in dry dichloromethane (14 mL). After stirring at room temperature for 15 minutes, the mixture was cooled to -15 °C, and HOTf (21.76 μL, 0.27 mmol) was slowly added. After reacting at -15 °C for 1 h, the mixture was quenched with triethylamine (0.5 mL), filtered through diatomaceous earth, and the filtrate was concentrated by rotary evaporation to remove the solvent. The filtrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give yellow foam (847 mg), with a yield of 81%. Under argon protection, the foam (847 mg, 1.1 mmol) was dissolved in pyridine / acetic acid (1.32 mL / 0.88 mL), and hydrazine hydrate (NH₂NH₂·H₂O) (0.16 mL, 3.3 mmol) was added dropwise. The mixture was stirred overnight at room temperature. After the reaction was monitored by thin-layer chromatography, acetone was added to quench the reaction. After dilution with ethyl acetate, the mixture was washed successively with 1M hydrochloric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator. The filtrate was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 10 white foam (514.1 mg), with a yield of 70%.

[0065] Data for 10: [α] D 24 = -11.43 (c 0.22CHCl3); 1H NMR (400 MHz, Chloroform-d) δ 7.93 (dd, J = 15.6, 7.8 Hz, 3H), 7.49 (t, J = 7.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.36 - 7.33 (m, 4H), 7.33 - 7.30 (m, 3H), 7.30 - 7.26 (m, 4H), 7.26 - 7.22 (m, 3H), 7.22 - 7.17 (m, 3H), 7.17 - 7.12 (m, 3H), 5.84 (d, J = 8.2 Hz, 1H), 5.59 (s, 1H, H-PVB), 5.28 (t, J = 8.8 Hz, 1H), 5.03 (s, 1H, H-PVB), 4.81 (d, J = 11.2 Hz, 1H), 4.67 - 4.54 (m, 2H), 4.48 (d, J = 12.1 Hz, 1H), 3.90 (t, J = 9.0 Hz, 1H), 3.77 - 3.67 (m, 3H), 3.58 (d, J = 9.6 Hz, 1H), 3.09. (s, 1H). 13 C NMR (101 MHz, Chloroform-d) δ 165.96, 164.79, 148.83, 143.65, 140.20, 138.06, 137.92, 133.27, 132.22, 131.23, 130.49, 129.82, 129.21, 128.92, 128.40, 128.31, 128.04, 127.91, 127.87, 127.80, 127.62, 127.53, 127.38, 126.45, 114.02, 92.35, 77.51, 75.78, 75.72, 74.68, 73.67, 73.45, 68.12. HRMS (ESI) calcd for C 42 H 38 O8[M+NH4] + 688.2905, found 688.2917.

[0066]

[0067] Preparation of substrate 11:

[0068] Compound S2 was prepared according to the procedure described in Example 1.1, using compound S1 as starting material. b(1.2 g, 2.13 mmol) was dissolved in DMF (11.0 mL), imidazole (290.41 mg, 4.27 mmol) was added, and the mixture was cooled to 0 °C, and then TdSCl (0.84 mL, 4.27 mmol) was slowly added. After the mixture was stirred at 0 °C for 15 min, it was slowly warmed to room temperature and allowed to react overnight. After the reaction was completed as monitored by TLC, the reaction was quenched by the addition of water and diluted with ethyl acetate. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 6: 1) to give a white solid (1.56 g) in 100% yield. The solid (1.56 g, 2.21 mmol) was dissolved in pyridine / acetic acid (13 mL / 8 mL) under argon, and hydrazine hydrate (NH2NH2-H2O) (0.33 mL, 6.64 mmol) was added dropwise. After stirring at room temperature overnight, the reaction was completed as monitored by TLC, and the reaction was quenched by the addition of acetone. After dilution with ethyl acetate, the mixture was washed sequentially with 1 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 6: 1) to give 11 as a white foam (1.2 g) in 89% yield.

[0069] Data for substrate 11: [a] D 25 = -23.08 (c 0.18 CHCl3); 1 H NMR (400 MHz, Chloroform-d) δ 7.93 (d, 2H), 7.44 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.7 Hz, 2H), 7.28 - 7.10 (m, 10H), 4.92 (t, J = 9.6, 7.5 Hz, 1H), 4.71 (d, J = 11.1 Hz, 1H), 4.68 (d, J = 7.28 Hz, 1H), 4.57 (d, J = 11.1 Hz, 1H), 4.54 - 4.45 (m, 2H), 3.76 (t, J = 9.1 Hz, 1H), 3.66 - 3.59 (m, 2H), 3.53 (t, J = 9.2 Hz, 1H), 3.43 - 3.37 (m, 1H), 2.42 (s, 1H), 0.64 - 0.60 (m, 12H, 3 x CH3-TDS), 0.06 (s, 3H, CH3-TDS), 0.03 (s, 3H, CH3-TDS). 13C NMR (101 MHz, Chloroform-d) δ 166.52, 138.45, 138.37, 133.26, 129.97, 128.64, 128.49, 128.42, 128.21, 128.03, 127.78, 127.70, 95.84, 78.65, 76.19, 75.21, 74.87, 73.67, 69.19, 34.04, 24.85, 20.08, 19.97, 18.57, 18.52. HRMS (ESI) calcd for C 35 H 46 O7Si[M+NH4] + 624.3351, found 624.3351.

[0070]

[0071] Preparation of substrate 14: Known compound S4 (4.62 g, 5.66 mmol) was dissolved in acetone / H2O (112 mL / 29 mL) under argon protection, cooled to 0 °C, and TCCA (1.31 g, 5.66 mmol) was added. The reaction was maintained at 0 °C for 2 h, TLC monitoring showed the reaction was completed, quenched with saturated NaHC03solution, the reaction was diluted with ethyl acetate and extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5: 1) to give white solid (3.56 g) with 89% yield. The above solid (300 mg, 0.42 mmol) and PhN=C(Cl)CF3(91.98 mg, 0.44 mmol) were dissolved in acetone, and anhydrous potassium carbonate (87.48 mg, 0.6337 mol) was added. The reaction was stirred at room temperature overnight, TLC showed the reaction was completed, the solid was removed by filtration, and the filtrate was concentrated under reduced pressure using a rotary evaporator, and purified by silica gel column chromatography (petroleum ether / ethyl acetate, 8: 1, containing 1% Et3N) to give yellow syrup S5 (372.2 mg) with 100% yield. Compound S5 was not further characterized and was used directly for the subsequent glycosylation reaction.

[0072] PTFAI donor S5 (380 mg, 0.43 mmol), ABzOH acceptor (120 mg, 0.646 mmol), and freshly activated 4 A molecular sieves (100 mg) were dissolved in anhydrous pyridine (2 mL) under argon protection. The reaction was stirred at room temperature for 24 h, TLC monitoring showed the reaction was completed, the solid was removed by filtration, and the filtrate was concentrated under reduced pressure using a rotary evaporator, and purified by silica gel column chromatography (petroleum ether / ethyl acetate, 8: 1, containing 1% Et3N) to give white solid S6 (370 mg) with 100% yield. Compound S6 was not further characterized and was used directly for the subsequent glycosylation reaction. Molecular sieves (500 mg) were dissolved in dry dichloromethane (4 mL). After stirring at room temperature for 15 min, the solution was cooled to -15 °C and HOTf (6.88 μL, 0.086 mmol) was added slowly. After 1 h at -15 °C, the reaction was quenched with triethylamine (0.5 mL), filtered over celite and the filtrate was concentrated by rotary evaporation to remove the solvent. Purification by column chromatography on silica gel (petroleum ether: ethyl acetate = 8:1 to 6:1) gave a yellow foam (285.7 mg, single beta configuration) in 75% yield. The foam (285.7 mg, 0.325 mmol) was dissolved in pyridine / acetic acid (1.2 mL / 0.8 mL) under argon and hydrazine hydrate (NH2NH2-H2O) (0.047 mL, 0.974 mmol) was added dropwise. After stirring at room temperature overnight, the reaction was monitored by thin layer chromatography and then quenched by the addition of acetone. After dilution with ethyl acetate, the mixture was washed sequentially with 4 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and saturated aqueous brine. The organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated by rotary evaporation under reduced pressure. Purification by column chromatography on silica gel (petroleum ether: ethyl acetate = 8:1) gave 14 as a white foam (172.5 mg) in 68% yield.

[0073] Data for substrate 14: [a] D 26 = +15.6 (c 0.180 CHCI3); 1 H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 7.7 Hz, 2H), 7.94 (d, J = 7.9 Hz, 1H), 7.72 (m, 4H), 7.53 (t, J = 7.4 Hz, 1H), 7.45 - 7.34 (m, 10H), 7.26 - 7.24 (m, 3H), 7.22 - 7.16 (m, 3H), 6.08 (d, J = 8.2 Hz, 1H), 5.57 (t, J = 8.8 Hz, 1H), 4.82 (s, 2H), 4.09 (t, J = 9.0 Hz, 1H), 4.03 - 3.98 (m, 2H), 3.87 (t, J = 9.1 Hz, 1H), 3.72 (dt, J = 9.3, 4.4 Hz, 1H), 3.00 (s, 1H), 1.53 - 1.46 (m, 1H, CH-ABz) 1.11 (s, 9H, 3 x CH3-TBDPS), 0.85 (d, J = 6.7 Hz, 4H, 2 x CH2-ABz). 13C NMR (101 MHz, Chloroform-d) δ 165.32, 163.69, 137.99, 135.75, 135.69, 134.35, 133.30, 132.95, 132.82, 132.32, 130.97, 129.97, 129.93, 129.84, 129.71, 129.58, 128.47, 128.46, 128.10, 127.90, 127.87, 127.85, 127.12, 125.73, 100.36, 92.68, 82.31, 75.69, 74.77, 74.44, 72.44, 72.08, 64.25, 26.95, 19.33, 9.00, 8.97. HRMS (ESI) calcd for C 48 H 48 O8 Si[M + Na] + 803.3011, found 803.3007.

[0074]

[0075] Preparation of substrate 18: Known compound S6 (1.47 g, 1.67 mmol) was dissolved in dry tetrahydrofuran (3.28 mL) under argon protection, hydrogen fluoride pyridine (70%, 1.33 mL) was added slowly. The reaction was stirred at room temperature for seven hours, then quenched with triethylamine. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution, saturated brine solution successively, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2.5:1) to give the intermediate (937.1 mg) in 87% yield.

[0076] Data of substrate 18: [a] D 26 = +45.28 (c 0.64, CHCI3). 1H NMR (400 MHz, Chloroform-d) δ 7.86 (d, J = 7.3 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 7.4 Hz, 2H), 7.25 (d, J = 4.9 Hz, 5H), 7.22 - 7.16 (m, 1H), 7.12 (t, J = 7.4 Hz, 1H), 6.01 (d, J = 8.2 Hz, 1H), 5.49 (t, J = 9.4 Hz, 1H), 5.36 (t, J = 9.7 Hz, 1H), 4.67 - 4.57 (m, 2H), 3.83 (t, J = 9.5 Hz, 2H), 3.73 - 3.67 (m, 1H), 3.67 - 3.61 (m, 1H), 2.40 (m, J = 6.5, 5.8 Hz, 2H, CH2-Lev), 2.28 (m, J = 6.5, 5.8 Hz, 2H, CH2-Lev), 1.88 (s, 3H, CH3-Lev), 1.39 (m, 1H, CH-ABz), 0.77 (d, J = 8.0 Hz, 4H, 2 x CH2-ABz). 13 C NMR (101 MHz, Chloroform-d) δ 205.75, 171.76, 165.29, 163.34, 137.60, 134.30, 133.34, 132.37, 130.72, 129.80, 129.31, 128.98, 128.44, 128.38, 128.10, 127.95, 127.03, 125.66, 100.47, 92.38, 76.40, 74.92, 74.74, 74.66, 74.24, 71.32, 60.98, 37.63, 29.60, 29.41, 27.90, 8.91, 8.89, 0.63. HRMS (ESI) calcd for C 37 H 36 O 10 [M+Na] + 663.2201, found 663.2198.

[0077]

[0078] Preparation of substrate 19: PTFAI donor S5 (372 g, 0.42 mmol), PVBOH acceptor (141.9 mg, 0.633 mmol), and freshly activated Molecular sieves (550 mg) were dissolved in dry dichloromethane (4 mL). After stirring at room temperature for 15 min, HOTf (6.74 μL, 0.084 mmol) was added slowly at -15 °C. After 1 h at -15 °C, the reaction was quenched with triethylamine (0.5 mL), filtered over celite, and the filtrate was concentrated to remove the solvent using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 8: 1) to give a yellow foam (357 mg, a: b = 1: 6) in 92% yield. The above foam (357 mg, 0.39 mmol) was dissolved in pyridine / acetic acid (1.2 mL / 0.8 mL) under argon protection, and hydrazine hydrate (NH2NH2-H2O) (0.057 mL, 1.17 mmol) was added dropwise. After stirring at room temperature overnight, the reaction was monitored by thin layer chromatography, and after completion, the reaction was quenched by adding acetone, diluted with ethyl acetate, and washed successively with 4 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated aqueous brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator and purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 8: 1) to give 10 as a white foam (265.8 mg) in 83% yield.

[0079] Data for substrate 19: [a] D 26 = -7.15 (c 0.12 CHCI3); 1 H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J = 7.3 Hz, 2H), 8.02 - 7.83 (m, 3H), 7.69 (d, J = 7.4 Hz, 4H), 7.59 (t, J = 7.5 Hz, 1H), 7.50 - 7.37 (m, 9H), 7.27 (d, J = 9.0 Hz, 2H), 7.19 (d, J = 3.0 Hz, 5H), 7.16 - 7.12 (m, 2H), 7.06 (t, J = 7.3 Hz, 2H), 7.01 (d, J = 7.1 Hz, 1H), 5.79 (d, J = 8.2 Hz, 1H), 5.56 (s, 1H, H-PVB), 5.41 (t, 1H), 5.02 (s, 1H, H-PVB), 4.77 (q, 2H), 4.01 (t, J = 8.8 Hz, 1H), 3.90 - 3.82 (m, 2H), 3.73 (t, J = 9.1 Hz, 1H), 3.58 - 3.52 (m, 1H), 2.99 (s, 1H), 1.09 (s, 9H, 3xCH3-TBDPS). 13C NMR (101 MHz, Chloroform-d) δ 165.14, 164.82, 149.01, 143.85, 140.40, 137.99, 135.73, 135.67, 133.25, 132.87, 132.79, 132.35, 131.41, 130.58, 130.01, 129.98, 129.91, 129.66, 129.07, 128.44, 128.41, 128.09, 127.94, 127.89, 127.81, 127.71, 127.42, 126.54, 114.15, 92.51, 82.21, 75.28, 74.70, 72.40, 72.21, 64.45, 26.94, 19.32, 14.21. HRMS (ESI) calcd for C 51 H 50 O8Si[M+Na] + 841.3167, found 841.3173.

[0080]

[0081] One-pot synthesis of tetrasaccharide 24: under argon protection, glycosyl PTFAI donor 8 (80 mg, 0.11 mmol) and ABz acceptor 9 (63.4 mg, 0.1 mmol), and freshly activated Molecular sieves (250 mg) were dissolved in dry dichloromethane (1.5 mL). After stirring at room temperature for 15 min, HOTf (0.16 mL, 0.02 M) was added at -15 °C. After 1.5 h at -15 °C, TLC monitoring showed the reaction was complete, and PVB acceptor 10 (53.77 mg, 0.08 mmol) was added, followed by freshly prepared PPh3AuOTf in dichloromethane (0.25 mL, 0.02 M). After 12 h at room temperature, TLC monitoring showed the reaction was complete, and the reaction was quenched with triethylamine (Et3N) (0.5 mL) at -30 °C, followed by filtration over celite. The filtrate was concentrated by rotary evaporation to remove the solvent, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 9: 1) to give tetrasaccharide product 24 (104 mg) in 64% one-pot yield.

[0082] Data of tetrasaccharide 24: [a] D 20 = +15.07 (c 0.3, CHCl3); 1H NMR (600 MHz, Chloroform-d) δ 7.87 (d, J = 7.8 Hz, 2H), 7.77 (d, J = 7.7 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 7.7 Hz, 3H), 7.60 - 7.53 (m, 3H), 7.48 (t, J = 7.7 Hz, 2H), 7.42 - 7.30 (m, 28H), 7.29 (s, 10H), 7.25 - 7.18 (m, 10H), 7.16 - 7.09 (m, 3H), 7.02 (d, J = 7.4 Hz, 2H), 5.23 (t, J = 8.8 Hz, 1H), 5.10 (t, J = 8.6 Hz, 1H), 5.05 (d, J = 11.0 Hz, 1H), 4.99 (m, 4H), 4.79 (d, J = 10.7 Hz, 1H), 4.74 (d, J = 7.7 Hz, 1H), 4.64 - 4.55 (m, 7H), 4.51 (d, J = 11.7 Hz, 5H), 4.43 (d, J = 10.9 Hz, 1H), 4.41 (d, J = 12.3 Hz, 1H), 4.37 (t, J = 13.6 Hz, 3H,), 4.14 (t, J = 8.9 Hz, 1H), 3.93 (m, 2H), 3.77 (t, J = 10.2 Hz, 3H), 3.71 (d, J = 11.2 Hz, 2H), 3.69 - 3.65 (m, 1H), 3.65 - 3.57 (m, 2H), 3.57 - 3.50 (m, 2H), 3.49 - 3.40 (m, 5H), 3.40 - 3.35 (m, 1H), 3.34 - 3.28 (m, 1H), 1.49-1.38 (m, 1H, H-TDS), 0.74 - 0.62 (m, 12H, 4 x CH3-TDS), 0.12 (s, 3H, CH3-TDS), 0.00 (s, 3H, CH3-TDS). 13C NMR (151 MHz, Chloroform-d) δ 168.98, 168.04, 167.79, 167.71, 142.42, 142.18, 142.08, 142.04, 141.99, 141.97, 141.41, 141.14, 136.97, 136.80, 136.59, 136.52, 134.57, 133.60, 133.54, 133.51, 133.24, 133.08, 133.00, 132.10, 132.01, 131.98, 131.96, 131.91, 131.79, 131.76, 131.71, 131.69, 131.56, 131.47, 131.20, 131.13, 131.10, 131.06, 131.04, 131.01, 130.96, 130.88, 103.92, 103.78, 99.24, 86.52, 83.63, 83.20, 82.79, 81.58, 79.93, 79.75, 79.70, 79.13, 79.02, 78.93, 78.89, 78.80, 78.72, 78.46, 78.31, 78.27, 77.64, 77.49, 77.31, 77.03, 76.94, 76.86, 73.33, 72.92, 72.85, 72.35, 37.24, 33.33, 28.17, 23.30, 23.24, 21.94, 21.90, 4.67, 1.65, 0.00. HRMS (ESI) calcd for C 123 H 130 O 25 Si[M+NH4] + 2052.9009, found 2052.9008.

[0083]

[0084] Preparation of tetrasaccharide 25: The glycosyl PTFAI donor 5 (122 mg, 0.059 mmol) and Linker acceptor (29.0 mg, 0.089 mmol), and freshly activated Molecular sieves (200 mg) were dissolved in dry dichloromethane (1.0 mL). After stirring at room temperature for 15 min, HOTf (0.14 mL, 0.018 M) was added at 0 °C. After 2 h at 0 °C, the reaction was monitored by TLC and quenched with triethylamine (Et3N) (0.5 mL), filtered over celite, and the filtrate was concentrated by rotary evaporation to remove the solvent and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4: 1) to give tetrasaccharide product 25 (122.3 mg) in 61% yield.

[0085] Data for tetrasaccharide 25: [a] D 24 = +10.06 (c 0.19, CHCI3). 1 H NMR (600 MHz, Chloroform-d) δ 7.77 (t, J = 8.4 Hz, 2H), 7.70 (d, J = 7.7 Hz, 2H), 7.63 (t, J = 7.6 Hz, 4H), 7.54 - 7.47 (m, 2H), 7.42 (t, J = 7.5 Hz, 3H), 7.35 - 7.13 (m, 53H), 7.10 - 7.03 (m, 4H), 6.96 (d, J = 7.3 Hz, 2H), 5.17 (t, 1H), 5.12 (s, 2H), 5.04 (t, J = 8.5 Hz, 1H), 4.98 (t, J = 11.0 Hz, 2H), 4.95 - 4.91 (m, 2H), 4.73 (d, J = 10.7 Hz, 1H), 4.67 (d, J = 7.8 Hz, 1H), 4.57 - 4.48 (m, 6H), 4.46 (s, 2H), 4.42 (m, 2H), 4.39 (d, J = 10.1 Hz, 1H), 4.36 (d, J = 5.1 Hz, 2H), 4.34 (s, 1H), 4.29 (t, J = 11.8 Hz, 3H), 4.09 (t, J = 8.6 Hz, 1H), 3.88 (m, 2H), 3.74 - 3.63 (m, 6H), 3.62 - 3.56 (m, 2H), 3.53 (t, J = 9.0 Hz, 1H), 3.50 - 3.35 (m, 7H), 3.35 - 3.30 (m, 1H), 3.26 - 3.22 (m, 1H), 2.87 - 2.81 (m, 1H), 1.32-1.17 (m, 4H, 2xCH2-Linker), 1.00 - 0.84 (m, 2H, CH2-Linker). 13C NMR (151 MHz, Chloroform-d) δ 165.39, 164.44, 164.28, 164.17, 156.64, 156.05, 138.66, 138.45, 138.37, 138.34, 138.22, 138.05, 137.97, 137.79, 137.53, 136.91, 136.83, 133.20, 132.96, 129.95, 129.92, 129.74, 129.69, 129.67, 129.65, 129.62, 129.47, 128.52, 128.51, 128.44, 128.41, 128.36, 128.33, 128.20, 128.18, 128.13, 128.00, 127.97, 127.91, 127.89, 127.83, 127.78, 127.75, 127.72, 127.63, 127.60, 127.58, 127.54, 127.49, 127.47, 127.39, 127.37, 127.28, 127.20, 127.14, 127.12, 100.74, 100.51, 100.39, 100.21, 82.92, 80.15, 79.93, 79.23, 77.99, 76.31, 76.11, 75.45, 75.34, 75.30, 75.21, 75.14, 75.11, 74.88, 74.84, 74.77, 74.09, 73.93, 73.72, 73.42, 73.39, 73.35, 69.62, 69.37, 69.30, 69.13, 68.77, 67.07, 50.48, 50.17, 47.08, 46.08, 46.01, 28.93, 27.66, 27.26, 23.02. HRMS (ESI) calcd for C 135 H 135 NO 27 [M+Na] + 2224.9114, found2224.9116.

[0086]

[0087] Preparation of tetrasaccharide 4:

[0088] The tetrasaccharide substrate 25 (118 mg, 0.054 mmol) was dissolved in methanol / dichloromethane (4 mL, v / v 1 : 1) under argon protection, sodium methoxide was added to adjust the pH to 12. The reaction system was placed in a 45 °C oil bath for 48 h, thin layer chromatography was used to monitor the reaction, the pH was adjusted to 7 with 4 M hydrochloric acid, filtered, the filtrate was concentrated under reduced pressure with a rotary evaporator, and purified by silica gel column chromatography (dichloromethane:methanol = 25:1) followed by Sephadex™ LH-20 gel column chromatography (MeOH-DCM = 1:1) to obtain the intermediate (95 mg) with a yield of 99%. The above intermediate (95 mg, 0.053 mmol) and palladium on carbon (Pd / C) (692.5 mg, 10%) were dissolved in ethyl acetate / methanol / water / acetic acid (5 mL / 10 mL / 1 mL / 0.25 mL). Hydrogen was introduced into the reaction system, and the reaction was carried out at room temperature for 36 h. The reaction solution was filtered with a 25 mm diameter polyether sulfone microporous filter, the filtrate was concentrated with a rotary evaporator, and purified by Sephadex™ LH-20 gel column chromatography (MeOH-H2O = 1:1) to obtain colorless transparent solid 4 (33.4 mg) with a yield of 84%.

[0089] Data of tetrasaccharide 4: [a] D 25 = -23.79 (c 0.16, H2O). 1 H NMR (400 MHz, Deuterium Oxide) δ 4.77 (s, 1H), 4.74 (m, 2H), 4.48 (d, J = 8.0 Hz, 1H), 3.91 (d, J = 12.8 Hz, 5H, CH-Linker), 3.81 - 3.64 (m, 9H, CH2-Linker), 3.58 - 3.43 (m, 12H, CH2-Linker), 3.43 - 3.31 (m, 3H), 3.00 (t, J = 7.5 Hz, 2H, CH2-Linker), 1.72 - 1.61 (m, 4H, 2 x CH2-Linker), 1.50 - 1.41 (m, 2H, CH2-Linker). 13 C NMR (151 MHz, Deuterium Oxide) δ 102.76, 102.50, 102.46, 101.88, 84.35, 84.15, 83.96, 75.96, 75.59, 75.57, 75.52, 75.49, 73.39, 73.24, 73.19, 72.86, 70.05, 69.52, 68.11, 68.06, 68.02, 60.65, 60.62, 39.31, 28.11, 26.35, 22.04. HRMS (ESI) calcd for C 29 H53 NO 21 [M+H] + 752.3183, found 752.3188.

[0090]

[0091] One-pot synthesis of trisaccharide 21: Under argon protection, glycosyl PTFAI donor 8 (170 mg, 0.234 mmol) and ABz acceptor 18 (125 mg, 0.195 mmol), along with newly activated... Molecular sieve (500 mg) was dissolved in dry dichloromethane (3 mL). After stirring at room temperature for 15 minutes, the mixture was cooled to 0 °C, and HOTf (0.47 mL, 0.059 M) was added. The reaction was carried out at 0 °C for 1.5 hours. After the reaction was completed as monitored by thin-layer chromatography, PVB receptor 19 (160 mg, 0.195 mmol) and freshly prepared PPh3AuOTf dichloromethane solution (0.25 mL, 0.039 M) were added sequentially. The reaction was carried out at room temperature for 3 hours. After the reaction was completed as monitored by thin-layer chromatography, the reaction was quenched with triethylamine (Et3N) (0.5 mL), filtered through diatomaceous earth, and the filtrate was concentrated by rotary evaporation to remove the solvent. The filtrate was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 3:1) to obtain trisaccharide product 21 (283.3 mg), with a one-pot yield of 80%.

[0092] Data for trisaccharide 21: [α] D 26 = +20.9(c 0.16, CHCl3). 1H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 7.7 Hz, 2H), 7.89 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.76 - 7.63 (m, 7H), 7.55 (t, J = 7.4 Hz, 1H), 7.49 - 7.42 (m, 7H), 7.42 - 7.18 (m, 30H), 7.18 - 6.96 (m, 20H), 5.61 (d, J = 8.3 Hz, 1H), 5.47 (s, 1H, H-PVB), 5.42 (t, J = 8.9 Hz, 1H), 5.35 - 5.27 (m, 2H), 5.22 - 5.14 (m, 2H), 5.06 (d, J = 8.1 Hz, 1H), 4.95 (s, 1H, H-PVB), 4.81 - 4.61 (m, 5H), 4.61 - 4.44 (m, 5H), 4.43 - 4.33 (m, 2H), 4.31 - 4.21 (m, 2H), 3.83 - 3.73 (m, 6H), 3.69 (t, J = 9.6 Hz, 2H), 3.61 - 3.53 (m, 2H), 3.46 (t, J = 8.3 Hz, 2H), 3.13 (d, J = 9.6 Hz, 1H), 2.41 (t, J = 7.0 Hz, 2H, CH2-Lev), 2.23 (t, J = 7.0 Hz, 2H, CH2-Lev), 1.96 (s, 3H, CH3-Lev), 1.03 (s, 9H, 3 x CH3-TBDPS). 13C NMR (101 MHz, Chloroform-d) δ 205.72, 171.58, 165.03, 165.01, 164.79, 148.90, 143.85, 140.32, 138.86, 138.40, 138.15, 138.03, 137.98, 135.84, 135.44, 134.30, 134.16, 133.61, 133.28, 133.13, 133.01, 132.54, 132.23, 131.34, 130.58, 130.08, 130.01, 129.88, 129.85, 129.75, 129.72, 129.38, 129.22, 129.08, 128.61, 128.52, 128.50, 128.43, 128.36, 128.29, 128.27, 128.14, 128.12, 128.06, 127.98, 127.96, 127.79, 127.73, 127.70, 127.63, 127.59, 127.40, 127.16, 126.54, 114.15, 101.20, 100.19, 92.57, 82.84, 80.09, 78.23, 77.36, 76.17, 76.07, 75.94, 75.48, 75.06, 74.91, 74.84, 74.41, 74.33, 74.09, 73.65, 72.60, 71.86, 69.05, 67.29, 61.10, 37.91, 29.57, 28.08, 26.97, 19.52. HRMS (ESI) calcd for C 110 H 108 O 22 Si[M+Na] + 1831.6994, found 1831.7002.

[0093]

[0094] Preparation of tri-saccharide bifunctional acceptor 13: The tri-saccharide substrate 21 (360 mg, 0.199 mmol) was dissolved in dry tetrahydrofuran (3 mL) under argon protection, hydrogen fluoride pyridine (70%, 0.179 mL) was added slowly. The reaction was stirred at room temperature for 36 h, then quenched with triethylamine. The reaction solution was diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution, saturated brine solution successively, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to give tri-saccharide bifunctional acceptor 13 (256.4 mg) with a yield of 82%.

[0095] Data for trisaccharide difunctional acceptor 13: [a] D 20 = -11.72 (c 0.43, CHCI3). 1 H NMR (600 MHz, Chloroform-d) δ 8.11 - 8.07 (m, 2H), 7.99 - 7.96 (m, 2H), 7.79 - 7.77 (m, 2H), 7.77 - 7.75 (m, 1H), 7.60 - 7.57 (m, 1H), 7.56 - 7.52 (m, 1H), 7.50 - 7.47 (m, 1H), 7.47 - 7.44 (m, 2H), 7.44 - 7.42 (m, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.35 - 7.32 (m, 2H), 7.29 (dd, J = 5.0, 1.6 Hz, 8H), 7.28 - 7.22 (m, 7H), 7.22 - 7.15 (m, 7H), 7.15 - 7.08 (m, 12H), 7.03 - 6.99 (m, 1H), 6.97 (dd, J = 8.1, 6.4 Hz, 2H), 5.61 - 5.58 (m, 2H, H-1, H-PVB), 5.46 (t, J = 9.4, 8.3 Hz, 1H), 5.34 (t, J = 9.8, 8.5 Hz, 1H), 5.27 (t, J = 9.7, 8.0 Hz, 1H), 5.15 (t, J = 9.8, 8.0 Hz, 1H), 5.07 (s, 1H, H-PVB), 4.98 (t, J = 11.9 Hz, 2H), 4.85 - 4.82 (m, 3H), 4.76 (d, J = 8.1 Hz, 1H), 4.67 (d, J = 12.5 Hz, 1H), 4.56 (d, J = 12.2 Hz, 1H), 4.51 (d, J = 11.0 Hz, 1H), 4.48 (d, J = 12.3 Hz, 1H), 4.45 - 4.39 (m, 2H), 4.14 - 4.10 (m, 1H), 4.05 (t, J = 9.2 Hz, 1H), 3.95 (t, J = 9.4 Hz, 1H), 3.76 - 3.70 (m, 2H), 3.64 - 3.60 (m, 1H), 3.60 - 3.58 (m, 1H), 3.56 (d, J = 9.9, 8.4 Hz, 1H), 3.53 (d, J = 10.9, 2.1 Hz, 1H), 3.50 (dd, J = 11.0, 4.5 Hz, 1H), 3.47 - 3.43 (m, 1H), 3.41 - 3.36 (m, 1H), 3.27 - 3.22 (m, 1H), 3.19 - 3.15 (m, 1H), 2.50 - 2.38 (m, 2H, CH2-Lev), 2.34 - 2.24 (m, 2H, CH2-Lev), 1.97 (s, 3H, CH3-Lev). 13C NMR(151MHz,Chloroform-d)δ205.90,171.82,165.37,165.26,164.94,164.4 5,149.18,144.01,140.64,138.69,138.55,138.42,137.63,133.56,133.18, 133.06, 132.73, 131.74, 130.44, 130.40, 130.08, 129.98, 129.87, 129.58, 129.25, 128.70, 128.67, 128.52, 128.47, 128.42, 128.40, 128.38, 128.32, 128.2 4,128.11,128.09,127.92,127.87,127.80,127.73,127.61,127.56,127.43,127.39,126.58,114.28,100.72,100.15,92.56,82.94,80.37,78.20,76.36, 75.92,75.68,75.37,75.06,75.02,74.88,74.70,74.57,74.54,73.55,72.73 ,71.96,68.83,66.57,60.10,37.85,29.83,29.65,28.01.HRMS(ESI)calcdfor C 94 H 90 O 22 [M+Na] + 1593.5816, found 1593.5817.

[0096]

[0097] One-pot synthesis of pentasaccharide 22: Under argon protection, glycosyl PTFAI donor 8 (160 mg, 0.22 mmol) and ABz acceptor 12 (116.24 mg, 0.184 mmol), along with newly activated... Molecular sieve (700 mg) was dissolved in dry dichloromethane (2.2 mL). After stirring at room temperature for 15 min, it was cooled to 0 °C and HOTf (0.3 mL, 0.037 M) was added. After 1 h at 0 °C, TLC monitoring indicated the reaction was complete and PVB acceptor 13 (260 mg, 0.165 mmol), freshly prepared PPh3AuOTf in dichloromethane (0.3 mL, 0.037 M) was added sequentially. The reaction was allowed to warm to room temperature and stirred for 4 h. TLC monitoring indicated the reaction was complete and the reaction was quenched with triethylamine (Et3N) (0.5 mL). The mixture was filtered through celite and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4: 1) to give pentasaccharide 22 (401.7 mg) in 95% one-pot yield.

[0098] Data for pentasaccharide 22: [a] D 21 = +15.39 (c 0.33, CHCI3). 1H NMR (600 MHz, Chloroform-d) δ 8.14 (t, J = 7.6 Hz, 4H), 8.09 (d, J = 7.8 Hz, 2H), 7.97 (d, J = 7.8 Hz, 2H), 7.92 - 7.88 (m, 3H), 7.72 (t, J = 7.4 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 8.0 Hz, 3H), 7.55 - 7.51 (m, 3H), 7.51 - 7.36 (m, 38H), 7.35 - 7.23 (m, 23H), 7.23 - 7.10 (m, 15H), 5.72 (d, J = 7.8 Hz, 1H), 5.62 (s, 1H, H-PVB), 5.48 (t, J = 8.0 Hz, 2H), 5.38 (t, J = 8.8 Hz, 1H), 5.33 (t, J = 9.5 Hz, 1H), 5.28 (t, J = 8.6 Hz, 1H), 5.17 (t, 1H), 5.10 (d, J = 12.0 Hz, 1H), 5.07 (s, 1H, H-PVB), 5.02 (d, J = 12.7 Hz, 1H), 4.98 (d, J = 11.1 Hz, 1H), 4.94 (d, J = 11.2 Hz, 1H), 4.90 (d, J = 11.0 Hz, 2H), 4.88 - 4.75 (m, 7H), 4.72 (d, J = 12.8 Hz, 1H), 4.70 (d, J = 7.9 Hz, 1H), 4.68 (d, J = 12.1 Hz, 1H), 4.62 (t, J = 11.5 Hz, 2H), 4.59 (s, 2H), 4.54 (d, J = 7.9 Hz, 1H), 4.45 (d, J = 11.7 Hz, 2H), 4.39 (d, J = 11.5 Hz, 1H), 4.22 (t, J = 9.1 Hz, 1H), 4.07 (d, J = 9.1 Hz, 2H), 3.99 (t, J = 9.4 Hz, 2H), 3.95 (d, J = 9.0 Hz, 1H), 3.91 (d, J = 10.7 Hz, 1H), 3.86 - 3.75 (m, 6H), 3.74 - 3.63 (m, 6H), 3.63 - 3.58 (m, 2H), 3.54 - 3.44 (m, 4H), 3.15 (d, J = 10.6, 4.1 Hz, 1H), 2.89 (d, 1H), 2.56 - 2.49 (m, 2H, CH2-Lev), 2.40 - 2.32 (m, 2H, CH2-Lev), 2.11 (s, 3H, CH3-Lev). 13C NMR (151 MHz, Chloroform-d) δ 205.82, 171.69, 165.23, 165.13, 165.00, 164.90, 164.87, 164.38, 148.98, 143.79, 140.41, 138.81, 138.80, 138.35, 138.31, 138.28, 138.24, 138.14, 138.01, 137.96, 133.69, 133.39, 133.11, 133.02, 132.98, 132.12, 131.24, 130.66, 130.03, 129.97, 129.94, 129.92, 129.84, 129.80, 129.75, 129.65, 129.34, 128.98, 128.72, 128.67, 128.59, 128.57, 128.49, 128.41, 128.34, 128.30, 128.27, 128.25, 128.06, 128.01, 127.95, 127.90, 127.88, 127.85, 127.71, 127.70, 127.62, 127.58, 127.52, 127.50, 127.41, 127.12, 127.04, 126.57, 114.08, 101.28, 100.79, 100.63, 100.07, 92.42, 82.88, 82.79, 80.58, 79.98, 78.26, 78.09, 77.57, 76.12, 75.58, 75.41, 75.22, 75.12, 75.11, 74.97, 74.90, 74.87, 74.78, 74.63, 74.27, 74.18, 74.02, 74.00, 73.62, 73.51, 73.45, 72.97, 72.85, 71.87, 68.84, 68.65, 67.68, 66.90, 66.53, 37.79, 29.78, 29.62, 27.96. MS (Maldi-TOF) calcd for C 155 H 148 O 34 [M+Na] + 2575.9744, found 2575.9745.

[0099]

[0100] Preparation of pentasaccharide 6: The pentasaccharide substrate 22 (164 mg, 0.064 mmol) was dissolved in dichloromethane (1.6 mL) under argon protection, and a mixture of hydrazine hydrate (NH2NH2-H2O) and pyridine acetic acid (1 M, 0.2 mL, 0.2 mmol, V hydrazinehydrate / V pyridine / V AcOH = 1 / 1 1.2 / 8). After stirring overnight at room temperature, thin layer chromatography was used to monitor the completion of the reaction. The reaction was quenched by the addition of acetone, and the reaction solution was concentrated using a rotary evaporator. After dilution with ethyl acetate, the solution was washed with 4 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator. Purification was performed using silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain pentasaccharide 6 (146 mg) as a white foam with a yield of 93%.

[0101] Data for pentasaccharide 6: [a] D 25 = +16.00 (c 0.19 CHCI3); 1H NMR (600 MHz, Chloroform-d) δ 7.97 (d, 2H), 7.86 (dd, J = 13.6, 7.8 Hz, 7H), 7.70 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.40 - 7.33 (m, 7H), 7.33 - 7.18 (m, 48H), 7.17 - 7.05 (m, 26H), 7.04 - 6.89 (m, 13H), 5.52 (d, J = 8.0 Hz, 1H), 5.43 (s, 1H, H-PVB), 5.34 (t, J = 8.6 Hz, 1H), 5.29 (m, 2H), 5.21 (t, J = 9.6, 8.0 Hz, 1H), 5.12 (t, J = 8.5 Hz, 1H), 4.93 - 4.84 (m, 5H, H-PVB), 4.80 (dd, J = 11.1, 5.2 Hz, 2H), 4.77 - 4.70 (m, 3H), 4.70 - 4.64 (m, 4H), 4.64 - 4.57 (m, 5H), 4.55 - 4.48 (m, 5H), 4.47 - 4.39 (m, 6H), 4.25 (d, J = 12.1 Hz, 1H), 4.17 (d, J = 7.9 Hz, 1H), 4.10 (t, J = 9.0 Hz, 1H), 4.01 (m, 1H), 3.88 (t, J = 9.2 Hz, 1H), 3.84 (t, J = 9.0 Hz, 1H), 3.79 (t, J = 9.3 Hz, 1H), 3.72 (m, 1H), 3.67 - 3.54 (m, 11H), 3.54 - 3.43 (m, 5H), 3.40 (dd, J = 10.8, 3.0 Hz, 2H), 3.37 - 3.35 (m, 1H), 3.35 - 3.30 (m, 2H), 3.28 - 3.22 (m, 2H), 2.93 (d, J = 9.4, 2.8 Hz, 1H), 2.23 (d, J = 4.6 Hz, 1H). 13C NMR (151 MHz, Chloroform-d) δ 166.38, 165.35, 165.18, 165.10, 164.99, 164.44, 149.05, 143.97, 140.54, 138.89, 138.79, 138.60, 138.46, 138.44, 138.34, 138.29, 138.06, 133.78, 133.54, 133.11, 133.10, 133.01, 132.23, 131.39, 130.57, 130.18, 130.13, 130.07, 130.01, 129.98, 129.94, 129.91, 129.84, 129.79, 129.37, 129.13, 128.82, 128.79, 128.75, 128.63, 128.59, 128.56, 128.53, 128.50, 128.48, 128.39, 128.36, 128.32, 128.20, 128.19, 128.16, 128.13, 128.09, 128.08, 127.99, 127.96, 127.94, 127.85, 127.80, 127.77, 127.74, 127.71, 127.68, 127.65, 127.63, 127.54, 127.27, 127.24, 126.74, 114.28, 101.26, 100.85, 100.46, 100.27, 92.67, 83.03, 82.98, 80.60, 80.29, 78.54, 78.38, 78.26, 76.29, 75.87, 75.55, 75.51, 75.36, 75.32, 75.28, 75.11, 75.09, 75.05, 74.82, 74.78, 74.76, 74.46, 74.40, 74.38, 73.74, 73.62, 73.57, 73.09, 72.04, 68.94, 68.79, 67.92, 66.99, 66.74, 29.93. MS (Maldi-TOF) calcd for C 150 H 142 O 32 [M+Na] + 2477.9376, found 2477.9374.

[0102]

[0103] One-pot synthesis of pentasaccharide 23: under argon, glycosyl PTFAI donor 8 (40 mg, 0.055 mmol) and ABz acceptor 12 (29 mg, 0.046 mmol), and freshly activated PPh3AuOTf in dichloromethane (0.25 mL, 0.009 M) were added sequentially to molecular sieves (250 mg) in dry dichloromethane (2 mL). After stirring at room temperature for 15 min, HOTf (0.73 mL, 0.009 M) was added at 0 °C. After 2 h at 0 °C, TLC monitoring indicated completion of the reaction, and PVB acceptor 13 (65 mg, 0.041 mmol) was added, followed by the freshly prepared PPh3AuOTf in dichloromethane (0.25 mL, 0.009 M). The reaction was stirred at room temperature for 4 h, after which TLC monitoring indicated completion of the reaction, and the reaction was quenched with triethylamine (Et3N) (0.5 mL) at 0 °C. The mixture was filtered through celite, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3.5: 1) to give pentasaccharide 23 (90.2 mg) in 72% one-pot yield.

[0104] Data for pentasaccharide 23: [a] D 26 = +8.28 (c 0.24, CHCl3). 1 ​H NMR (600 MHz, Chloroform-d) δ 8.04 (d, J = 7.8 Hz, 2H), 7.95 (t, J = 8.0 Hz, 7H), 7.81 (d, J = 7.8 Hz, 2H), 7.52 - 7.25 (m, 65H), 7.25 - 7.13 (m, 22H), 7.13 - 7.05 (m, 7H), 7.05 - 6.98 (m, 6H), 5.37 (t, J = 8.9 Hz, 1H), 5.29 (t, J = 9.1 Hz, 2H), 5.21 - 5.12 (m, 6H), 5.04 (d, J = 12.1 Hz, 2H), 4.89 (dd, J = 16.2, 11.4 Hz, 3H), 4.87 - 4.71 (m, 8H), 4.65 (t, J = 23.4, 11.4 Hz, 4H), 4.57 (t, 4H), 4.52 - 4.47 (m, 6H), 4.46 - 4.41 (m, 2H), 4.37 - 4.29 (m, 3H), 4.23 (m, 1H), 4.22-4.10 (m, 1H), 4.08 - 3.99 (m, 3H), 3.94 (d, J = 9.9 Hz, 2H), 3.90 - 3.82 (m, 2H), 3.74 (d, J = 9.2 Hz, 2H), 3.72 - 3.66 (m, 3H), 3.66 - 3.58 (m, 6H), 3.58 - 3.49 (m, 5H), 3.41 - 3.34 (m, 2H), 3.33 - 3.24 (m, 2H), 3.15 (t, J = 9.1 Hz, 1H), 2.98 - 2.73 (m, 4H), 2.51 - 2.42 (m, 2H, CH2-Lev), 2.40 - 2.27 (m, 2H, CH2-Lev), 1.98 (s, 3H, CH3-Lev), 1.26 - 1.01 (m, 4H, 2 x CH2-Linker), 1.00 - 0.76 (m, 4H, CH2-Linker), 0.14 (s, 4H). 13C NMR (151 MHz, Chloroform-d) δ 205.76, 171.69, 165.32, 165.27, 165.05, 164.88, 164.77, 156.64, 156.03, 138.65, 138.59, 138.55, 138.34, 138.32, 138.29, 138.23, 138.18, 138.10, 138.00, 137.93, 137.89, 137.66, 136.93, 133.77, 133.45, 133.03, 132.93, 132.80, 130.07, 130.04, 129.99, 129.93, 129.82, 129.77, 129.73, 129.70, 129.60, 128.92, 128.88, 128.80, 128.71, 128.62, 128.51, 128.49, 128.42, 128.39, 128.36, 128.33, 128.31, 128.20, 128.10, 128.08, 128.01, 127.98, 127.94, 127.92, 127.86, 127.82, 127.80, 127.77, 127.74, 127.71, 127.69, 127.62, 127.55, 127.52, 127.45, 127.28, 127.18, 127.16, 127.10, 101.51, 100.61, 100.51, 100.07, 82.88, 82.81, 80.84, 80.25, 78.48, 78.24, 78.12, 76.20, 76.11, 75.53, 75.39, 75.24, 74.96, 74.85, 74.62, 74.53, 74.34, 74.24, 73.88, 73.61, 73.47, 73.43, 73.29, 72.88, 72.68, 68.91, 68.79, 68.75, 68.27, 67.72, 67.05, 66.43, 50.54, 50.22, 47.17, 46.44, 46.15, 37.75, 29.76, 29.72, 29.57, 28.84, 28.71, 27.94, 27.66, 27.33, 23.02, 22.89, 1.10. HRMS (ESI) calcd for C 160 H 161 NO 35 [M+Na] + 2679.0741, found 2679.0734.

[0105]

[0106] Preparation of pentasaccharide 3: The pentasaccharide substrate 23 (118 mg, 0.046 mmol) was dissolved in methanol / dichloromethane (4 mL, v / v 1 : 1) under argon protection, and sodium methoxide was added to adjust the pH to 12. The reaction system was placed in an oil bath at 45 °C for 48 h, and thin layer chromatography was used to monitor the reaction. The pH was adjusted to 7 with 4 M hydrochloric acid, and the mixture was filtered and concentrated under reduced pressure using a rotary evaporator. Purification was performed using silica gel column chromatography (dichloromethane:methanol = 20: 1) followed by Sephadex™ LH-20 gel column chromatography (MeOH-DCM = 1: 1) to obtain the intermediate (82.4 mg) in a yield of 88%. The intermediate (82.4 mg, 0.04 mmol) and palladium on carbon (Pd / C) (574.2 mg, 10%) were dissolved in ethyl acetate / methanol / water / acetic acid (7 mL / 10 mL / 1 mL / 0.25 mL). Hydrogen was bubbled into the reaction system, and the reaction was allowed to proceed at room temperature for 36 h. The reaction solution was filtered using a 25 mm diameter polyether sulfone microporous filter, and the filtrate was concentrated using a rotary evaporator. Purification was performed using Sephadex™ LH-20 gel column chromatography (MeOH-H2O = 1: 1) to obtain colorless transparent solid 3 (24 mg) in a yield of 65%.

[0107] Data for pentasaccharide 3: [a] D 23 = -13.91 (c 0.09, H2O). 1H NMR (600 MHz, Deuterium Oxide) δ 4.55 (d, J = 7.9 Hz, 1H), 4.54 (d, J = 4.2 Hz, 1H), 4.52 (d, J = 4.2 Hz, 1H), 4.50 (d, J = 8.0 Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.26 (d, J = 11.2 Hz, 1H), 4.19 (d, J = 11.7, 2.2 Hz, 1H), 3.97 (d, J = 12.4, 2.2 Hz, 1H), 3.94 - 3.87 (m, 5H, CH-Linker), 3.87 - 3.84 (m, 1H), 3.81 (dd, J = 12.3, 5.0 Hz, 1H), 3.76 (d, J = 9.2 Hz, 1H, 3.74 - 3.69 (m, 4H), 3.69 - 3.66 (m, 1H, CH-Linker), 3.66 - 3.60 (m, 5H), 3.60 - 3.57 (m, 1H), 3.52 - 3.42 (m, 7H), 3.41 (d, J = 9.4 Hz, 1H), 3.40 - 3.37 (m, 1H), 3.37 - 3.33 (m, 1H), 3.33 - 3.27 (m, 4H), 2.99 (t, J = 7.5 Hz, 2H, CH2-Linker), 1.70 - 1.63 (m, 4H, 2 x CH2-Linker), 1.47 - 1.40 (m, 2H, CH2-Linker). 13 C NMR (151 MHz, D2O) δ 102.64, 102.54, 102.37, 102.04, 78.59, 78.41, 75.93, 75.82, 75.57, 75.43, 75.31, 74.71, 74.63, 74.35, 74.15, 73.36, 73.08, 72.97, 72.79, 72.71, 70.14, 69.52, 69.41, 69.38, 68.52, 67.33, 60.62, 60.49, 59.90, 39.30, 28.10, 26.35, 23.20, 22.02, 19.99. HRMS (ESI) calcd for C 35 H 63 NO 26 [M+H] + 914.3711, found 914.3714.

[0108]

[0109] One-pot synthesis of pentasaccharide 20: Under argon protection, glycosyl PTFAI donor 8 (80 mg, 0.11 mmol) and ABz acceptor 14 (79 mg, 0.10 mmol), along with newly activated... Molecular sieve (350 mg) was dissolved in dry dichloromethane (2 mL). After stirring at room temperature for 15 minutes, the mixture was cooled to 0 °C, and TMOTf (0.33 mL, 0.018 M) was added. The reaction was carried out at 0 °C for 2 hours. After the reaction was completed as monitored by thin-layer chromatography, PVB receptor 15 (62 mg, 0.092 mmol) and freshly prepared PPh3AuOTf dichloromethane solution (0.3 mL, 0.018 M) were added sequentially. The reaction was carried out at room temperature for 2 hours. After the reaction was completed as monitored by thin-layer chromatography, the mixture was cooled to -30 °C, and STol receptor 16 (55 mg, 0.092 mmol), NIS (21 mg, 0.096 mmol), and TMOTf (0.33 mL, 0.018 M) were added sequentially. The reaction was carried out at -30℃ for 3 hours. After the reaction was monitored by thin-layer chromatography, the temperature was raised to 0℃, and receptor 17 (61 mg, 0.079 mmol), NIS (26.5 mg, 0.118 mmol), and TMOTf (0.2 mL, 0.009 M) were added sequentially. The reaction was carried out at 0℃ for 3 hours, and the reaction was quenched with triethylamine (Et3N) (0.5 mL). The mixture was filtered through diatomaceous earth, and the filtrate was concentrated by rotary evaporation to remove the solvent. The filtrate was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 6:1 to 3:1) to give pentasaccharide product 20 (130.6 mg), with a one-pot yield of 59%.

[0110] Data for pentasaccharides 20: [α] D 20 = +8.93 (c 0.43, CHCl3). 1H NMR(600MHz,Chloroform-d)δ8.00(d,2H),7.91(t,J=7.5Hz,2H),7.87(d,J=7.7Hz,3H),7.82(d,J=7.8Hz,2H),7.77–7.72(m,8H),7.70(d,J=7.9Hz,5H),7.51–7.33(m,15H),7.33–7.16(m,52H),7.16–7.04(m,29H),7.04–7.00(m,7H),6.98–6.91(m,5H),5.65(t,J=9.6Hz,1H),5.37(t,1H),5.33(t,J=8.8Hz,1H),5.23–5.19(m,1H),5.16(t,J=9.8Hz,1H),5.14–5.08(m,4H),5.06(t,1H),5.03–4.98(m,2H),4.82–4.71(m,4H),4.64(d,J=11.1Hz,1H),4.61(d,J=10.9Hz,1H),4.58(d,J=7.6Hz,2H),4.55(d,J=20.1Hz,2H),4.52–4.47(m,3H),4.47–4.43(m,4H),4.41(d,J=8.1Hz,2H),4.36(d,J=9.1Hz,1H),4.33(d,J=11.4Hz,5H),4.27(d,J=8.0Hz,2H),4.25–4.21(m,1H),4.02(d,J=10.4Hz,1H),3.97(d,J=11.0Hz,1H),3.88(t,J=9.4Hz,1H),3.85–3.75(m,4H),3.74–3.65(m,6H),3.65–3.56(m,4H),3.49–3.43(m,2H),3.42–3.30(m,5H),3.27(t,J=9.0Hz,2H),3.07(d,J=24.5Hz,1H),2.93(d,J=9.5,2.2Hz,1H),2.81(t,J=7.5Hz,1H),1.32-1.18(m,4H,2×CH2-Linker),1.01(s,12H,3×CH3-TBDPS,CH2-Linker). 13C NMR (151 MHz, Chloroform-d) δ 165.82, 165.21, 165.19, 165.12, 164.97, 164.95, 156.72, 156.12, 138.76, 138.40, 138.15, 138.10, 138.06, 138.01, 137.98, 137.92, 137.79, 135.99, 135.58, 134.07, 133.38, 133.21, 133.18, 133.10, 132.36, 130.12, 130.05, 130.01, 129.94, 129.90, 129.87, 129.80, 129.77, 129.72, 129.55, 129.40, 128.98, 128.95, 128.58, 128.55, 128.52, 128.46, 128.41, 128.39, 128.38, 128.35, 128.33, 128.26, 128.22, 128.04, 128.02, 127.97, 127.94, 127.90, 127.82, 127.79, 127.72, 127.66, 127.64, 127.57, 127.54, 127.47, 127.21, 101.38, 101.09, 100.91, 100.20, 83.10, 82.71, 82.67, 80.03, 78.29, 77.73, 77.71, 75.72, 75.46, 75.39, 75.28, 75.21, 74.80, 74.76, 74.70, 74.66, 74.44, 74.37, 73.97, 73.83, 73.72, 73.51, 73.21, 73.06, 71.79, 69.70, 69.42, 69.28, 68.64, 67.90, 67.55, 67.48, 67.13, 61.19, 50.61, 50.29, 47.22, 46.24, 29.80, 29.03, 27.88, 27.45, 27.02, 23.21, 19.48. HRMS (ESI) calcd for C 171 H 169 NO 35 Si[M+NH4] + 2842.1583, found 2842.1576.

[0111]

[0112] Preparation of pentasaccharide acceptor 7: The pentasaccharide substrate 20 (1.1 g, 0.39 mmol) was dissolved in dry tetrahydrofuran (1.3 mL) under argon protection, hydrogen fluoride pyridine (70%, 1.41 mL) was added slowly. The reaction was stirred at room temperature for 40 hours, then quenched with triethylamine. The reaction solution was diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution, saturated brine solution successively, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1 to 1: 1) to give pentasaccharide acceptor 7 (950.5 mg) with a yield of 94%.

[0113] Data of pentasaccharide acceptor 7: [a] D 25 = -5.00 (c 0.30 CHCI3). 1H NMR(600MHz,Chloroform-d)δ8.11(d,J=7.9Hz,2H),8.03(d,J=7.9Hz,2H),7.97(t,J=6.9Hz,2H),7.94–7.87(m,7H),7.81(d,J=7.9Hz,2H),7.61(t,J=7.5Hz,1H),7.57–7.53(m,1H),7.52–7.46(m,6H),7.45–7.40(m,5H),7.40–7.36(m,5H),7.36–7.25(m,31H),7.23(d,J=7.5Hz,6H),7.22–7.17(m,9H),7.17–7.10(m,18H),7.08(d,J=8.0Hz,4H),7.07–7.03(m,4H),7.03–6.97(m,4H),5.79(t,J=9.7Hz,1H),5.47(t,J=8.8Hz,1H),5.42(t,J=8.4Hz,1H),5.33–5.27(m,2H),5.17(m,4H),5.08–5.02(m,2H),4.89(d,J=8.1Hz,1H),4.85(d,J=11.0Hz,1H),4.80(d,J=11.1Hz,1H),4.75(d,J=12.1Hz,1H),4.70(d,J=9.1Hz,2H),4.66(d,J=16.6Hz,2H),4.64–4.57(m,4H),4.55(d,J=11.3Hz,1H),4.50-4.43(m,5H),4.40(m,5H),4.32(m,3H),4.12(t,J=9.1Hz,1H),4.02(d,J=10.9Hz,1H),3.93(m,3H),3.89(d,J=8.5Hz,2H),3.82(m,2H),3.77–3.70(m,5H),3.68–3.58(m,7H),3.55(dd,J=11.0,4.9Hz,1H),3.49(q,J=8.8Hz,2H),3.34(dt,J=12.7,6.3Hz,2H),3.20–3.11(m,2H),1.43-1.24(m,6H,2×CH2-Linker),1.13-0.97(m,2H,CH2-Linker). 13C NMR (151 MHz, Chloroform-d) δ 165.82, 165.67, 165.16, 165.10, 164.99, 164.97, 156.69, 156.09, 138.71, 138.27, 138.17, 138.11, 138.04, 137.91, 137.88, 137.86, 137.81, 137.00, 136.86, 133.54, 133.37, 133.25, 133.14, 133.08, 133.03, 130.06, 130.04, 130.02, 129.84, 129.80, 129.75, 129.74, 129.70, 129.32, 128.93, 128.91, 128.66, 128.56, 128.52, 128.50, 128.46, 128.39, 128.37, 128.35, 128.32, 128.28, 128.23, 128.12, 128.05, 128.02, 127.99, 127.96, 127.94, 127.90, 127.87, 127.82, 127.79, 127.72, 127.69, 127.67, 127.61, 127.59, 127.48, 127.33, 127.24, 127.19, 127.13, 101.05, 100.93, 100.90, 100.85, 82.83, 82.64, 82.45, 80.13, 78.17, 77.65, 77.23, 76.58, 75.30, 75.24, 75.10, 74.94, 74.77, 74.74, 74.64, 74.51, 74.44, 74.35, 73.82, 73.65, 73.58, 73.37, 73.10, 72.82, 71.86, 70.11, 69.38, 69.25, 68.65, 68.11, 67.92, 67.75, 67.13, 67.09, 60.68, 50.57, 50.26, 47.18, 46.20, 29.00, 27.83, 27.41, 23.19, 23.15. MS (Maldi-TOF) calcd for C 155 H 151 NO 35 [M+Na] + 2608.9959, found 2609.9951.

[0114]

[0115] Preparation of pentasaccharide 2: The pentasaccharide substrate 7 (300 mg, 0.116 mmol) was dissolved in methanol / dichloromethane (5 mL, v / v 1 : 1) under argon protection, sodium methoxide was added to adjust the pH to 12. The reaction was stirred at room temperature for 24 h, monitored by thin layer chromatography, the pH was adjusted to 7 with 4 M hydrochloric acid, filtered, the filtrate was concentrated under reduced pressure using a rotary evaporator and purified by Sephadex™ LH-20 gel column chromatography (MeOH-DCM = 1 : 1) to give the intermediate (214.5 mg) in 100% yield. The intermediate (214.5 mg, 0.116 mmol) and palladium hydroxide on carbon (Pd(OH)2 / C) (400 mg, 20%) were dissolved in tetrahydrofuran / methanol / water / acetic acid (8 mL / 5 mL / 5 mL / 0.5 mL). Hydrogen was bubbled through the reaction mixture and the reaction was stirred at room temperature for 18 h. The reaction was filtered using a 25 mm diameter polyethersulfone micro pore filter and the filtrate was concentrated using a rotary evaporator and purified by Sephadex™ LH-20 gel column chromatography (MeOH-H2O = 1 : 1) to give pentasaccharide 2 as a colourless transparent solid (101.6 mg) in 97% yield.

[0116] Data for pentasaccharide 2: [a] D 20 = -18.44 (c 0.36 D2O) 1 H NMR (600 MHz, Deuterium Oxide) δ 4.53 (d, J = 7.8 Hz, 1H), 4.52 - 4.50 (m, 2H), 4.49 (m, 1H), 4.46 (d, J = 8.0 Hz, 1H), 4.20 (d, J = 11.8 Hz, 3H), 3.98 (d, 1H), 3.94 - 3.88 (m, 2H, CH-Linker), 3.85 (t, J = 6.0 Hz, 2H), 3.81 (dd, J = 13.7, 6.0 Hz, 2H), 3.72 (dd, J = 12.4, 5.8 Hz, 1H), 3.70 - 3.66 (m, 1H, CH-Linker), 3.66 - 3.56 (m, 7H), 3.51 - 3.38 (m, 10H), 3.35 (t, J = 8.3 Hz, 1H), 3.33 - 3.28 (m, 3H), 3.25 (t, J = 8.4 Hz, 1H), 3.00 (t, J = 7.5 Hz, 2H, CH2-Linker), 1.67 (m, 4H, 2 x CH2-Linker), 1.45 (p, J = 7.7 Hz, 2H, CH2-Linker). 13C NMR (151 MHz, Deuterium Oxide) δ 105.49, 105.39, 105.20, 105.08, 104.72, 81.12, 78.47, 78.22, 78.07, 77.98, 77.38, 77.25, 76.72, 75.64, 75.57, 75.53, 75.51, 75.34, 72.68, 71.93, 71.88, 71.24, 71.10, 63.05, 62.48, 41.85, 30.69, 28.92, 25.75, 24.60. HRMS (ESI) calcd for C 35 H 63 NO 26 [M+H] + 914.3711, found 914.3715.

[0117]

[0118] One-pot synthesis of tetradecaose 26: under argon protection, glycosyl PTFAI tetraose donor 5 (50 mg, 0.024 mmol) and PVB pentaose bifunctional acceptor 6 (54 mg, 0.022 mmol), and freshly activated molecular sieves (160 mg) were dissolved in dry toluene (1 mL). After stirring at room temperature for 15 min, the mixture was cooled to -60 °C, and a mixture of toluene and HOTf (0.1 mL, 0.0044 M) was added. After 2.5 h at -60 °C, the reaction was monitored by TLC, and the mixture was warmed to room temperature. Pentaose acceptor 7 (45.28 mg, 0.0176 mmol) was added, and the mixture was cooled to -60 °C. NIS (14.85 mg, 0.0148 mmol) and a mixture of dichloromethane and HOTf (0.1 mL, 0.009 M) were added sequentially. After 12 h at -60 °C, the reaction was monitored by TLC, and the mixture was quenched with triethylamine (Et3N) (0.5 mL). The mixture was filtered over celite, and the filtrate was concentrated by rotary evaporation. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1 to 1.5:1) gave tetradecaose GLSWA-I (26) (80.5 mg) in 68% one-pot yield.

[0119] Data for tetradecaose 26: [a] D 21 = +2.31 (c 0.32, CHCI3). 1H NMR (600 MHz, Chloroform-d) δ 8.09 - 7.74 (m, 14H), 7.72 (d, J = 7.9 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.65 - 7.52 (m, 6H), 7.52 - 7.44 (m, 6H), 7.44 - 7.37 (m, 5H), 7.37 - 7.03 (m, 133H), 7.03 - 6.91 (m, 20H), 6.91 - 6.86 (m, 2H), 6.86 - 6.81 (m, 3H), 5.25 - 5.11 (m, 6H), 5.11 - 5.03 (m, 1H), 5.01 - 4.97 (m, 2H), 4.91 (s, 1H), 4.90 - 4.82 (m, 3H), 4.82 - 4.76 (m, 3H), 4.73 (d, J = 10.5 Hz, 3H), 4.70 - 4.53 (m, 9H), 4.54 - 4.38 (m, 16H), 4.38 - 4.31 (m, 6H), 4.31 - 4.18 (m, 7H), 4.18 - 4.02 (m, 5H, CH-Linker), 3.98 - 3.91 (m, 3H), 3.91 - 3.79 (m, 5H), 3.75 (t, J = 8.9 Hz, 3H), 3.69 (t, J = 10.5 Hz, 6H), 3.65 - 3.47 (m, 16H, CH-Linker), 3.47 - 3.38 (m, 6H), 3.35 (t, J = 9.1 Hz, 3H), 3.27 (d, J = 9.5 Hz, 3H), 3.22 (dd, J = 10.3, 4.3 Hz, 2H), 3.17 (s, 3H), 3.13 - 3.04 (m, 3H), 3.02 - 2.91 (m, 2H), 2.89 - 2.80 (m, 2H, CH2-Linker), 0.95 (dt, J = 48.4, 7.3 Hz, 6H, 2 x CH2-Linker), 0.86 (d, J = 6.8 Hz, 2H, CH2-Linker). 13C NMR (151 MHz, Chloroform-d) δ 166.11, 165.62, 165.45, 165.34, 165.22, 165.13, 164.53, 164.43, 164.41, 164.02, 139.24, 138.91, 138.73, 138.55, 138.50, 138.43, 138.38, 138.34, 138.23, 138.10, 138.07, 137.95, 137.92, 137.84, 137.58, 133.95, 133.56, 133.38, 133.18, 133.02, 132.95, 131.06, 130.25, 130.18, 129.98, 129.85, 129.73, 129.69, 129.65, 129.62, 129.53, 129.45, 129.32, 128.97, 128.94, 128.90, 128.68, 128.60, 128.57, 128.51, 128.47, 128.45, 128.39, 128.36, 128.28, 128.24, 128.22, 128.20, 128.13, 128.10, 128.03, 128.01, 127.96, 127.91, 127.88, 127.84, 127.81, 127.73, 127.69, 127.64, 127.57, 127.55, 127.52, 127.50, 127.45, 127.40, 127.38, 127.25, 126.87, 125.15, 101.28, 100.99, 100.69, 100.63, 100.51, 100.31, 83.00, 82.62, 80.55, 80.04, 79.58, 79.30, 78.12, 78.07, 76.41, 76.33, 76.21, 76.16, 75.48, 75.37, 75.31, 75.29, 75.25, 75.05, 74.98, 74.91, 74.76, 74.60, 74.54, 74.32, 74.00, 73.93, 73.77, 73.50, 73.45, 73.37, 73.29, 72.91, 72.11, 69.45, 69.29, 68.91, 68.73, 67.89, 67.11, 65.71, 50.59, 50.30, 47.29, 46.35, 39.02, 32.33, 32.06, 30.69, 30.44, 30.30, 30.17, 29.83, 29.50, 29.11, 29.01, 27.92, 27.48, 26.88, 26.53, 26.03, 24.10, 23.57,23.28,23.10,22.83,19.32,14.27,13.87,11.23,1.16,0.14.MS(Maldi-TOF)calcd for C. 405 H 391 NO 89 [M+Na] + 6714.5993, found 6714.5997.

[0120]

[0121] Preparation of GLSWA-I(1): Under argon protection, tetradecanoic acid substrate 26 (156 mg, 0.023 mmol) was dissolved in methanol / dichloromethane (5 mL, v / v 2:3), and sodium methoxide was added to adjust the pH to 12. The reaction system was placed in an oil bath at 45 °C for 48 h. The reaction was monitored by thin-layer chromatography. The pH was adjusted to 7 with 4 M hydrochloric acid, filtered, and the filtrate was concentrated under reduced pressure by rotary evaporator. After purification by silica gel column chromatography (dichloromethane:methanol = 25:1), the intermediate (102.6 mg) was obtained by Sephadex™ LH-20 gel column chromatography (MeOH-DCM = 1:1), with a yield of 88%. The above intermediate (102.6 mg, 0.020 mmol) and palladium / carbon (Pd / C) (698 mg, 10%) were dissolved in ethyl acetate / methanol / water / acetic acid (12.5 mL / 5 mL / 1 mL / 0.25 mL). Hydrogen gas was introduced into the reaction system and the reaction was carried out at room temperature for 3 days. The reaction solution was filtered through a 25 mm diameter polyethersulfone microporous membrane, the filtrate was concentrated by rotary evaporation, and purified by Sephadex™ LH-20 gel column chromatography (MeOH-H2O = 1:1) to obtain a colorless transparent solid 1 (30 mg), with a yield of 62%.

[0122] Data from GLSWA-I(1): [α] D 24 = -17.7 (c 0.2, H2O). 1 H NMR(600MHz,DeuteriumOxide)δ4.61–4.48(m,

[0123] 7H), 4.46 (d, J = 8.0 Hz, 1H), 4.28 (d, J = 10.9 Hz, 1H), 4.24 - 4.16 (m, 4H), 3.99 - 3.96 (m, 1H), 3.96 - 3.86 (m, 12H, CH-Linker), 3.86 - 3.82 (m, 4H), 3.80 (d, J = 6.1 Hz, 1H), 3.79 - 3.75 (m, 4H), 3.75 - 3.69 (m, 10H), 3.69 - 3.66 (m, 2H), 3.66 - 3.63 (m, 4H, CH-Linker), 3.63 - 3.56 (m, 6H), 3.55 (d, J = 2.9 Hz, 1H), 3.55 - 3.52 (m, 3H), 3.52 - 3.43 (m, 21H), 3.41 (d, J = 6.6 Hz, 1H), 3.39 (q, J = 3.6, 2.9 Hz, 2H), 3.38 - 3.33 (m, 5H), 3.31 (td, J = 8.6, 3.6 Hz, 5H), 3.25 (t, J = 8.5 Hz, 1H), 3.00 (t, J = 7.0, 6.2 Hz, 2H, CH2-Linker), 1.70 - 1.63 (m, 4H, 2 x CH2-Linker), 1.47 - 1.41 (m, 2H, CH2-Linker). 13 C NMR (151 MHz, D20) δ 103.64, 103.60, 103.57, 103.50, 103.42, 103.35, 103.30, 103.21, 103.15, 103.09, 102.92, 84.88, 84.71, 84.18, 79.41, 79.21, 78.70, 76.69, 76.66, 76.57, 76.42, 76.31, 76.27, 76.23, 76.19, 76.15, 75.64, 75.58, 75.39, 75.15, 74.94, 74.79, 74.18, 74.13, 74.06, 73.98, 73.93, 73.83, 73.79, 73.76, 73.72, 73.67, 73.58, 73.43, 70.88, 70.26, 70.20, 70.13, 70.08, 69.45, 69.37, 69.28, 68.80, 68.76, 68.66, 68.26, 68.04, 61.37, 61.27, 60.69, 40.06, 28.89, 27.12, 22.80. MS (Maldi-TOF) calcd for C 89 H 153 NO 71 [M+Na] +2394.8290, found 2394.8284.

[0124] In order to further prove the beneficial effects of the present application and better understand the present application, the properties of the artificially synthesized bioactive pentasaccharide and the Ganoderma spore GLSWA-I polysaccharide tetradecasaccharide containing the structure thereof are further illustrated by the following experiments below, but it is not to be understood as a limitation of the present application, and the reactivity obtained by other experiments made by the person skilled in the art according to the above disclosure and the application made according to the above properties are also considered to fall within the protection scope of the present application.

[0125] Experiment 1: Structural comparison between the artificially synthesized Ganoderma spore GLSWA-I (1) polysaccharide tetradecasaccharide and the Ganoderma spore tetradecasaccharide polysaccharide GLSWA-1 of natural origin

[0126] Table 1. NMR data comparison between the artificially synthesized Ganoderma spore GLSWA-I (1) polysaccharide tetradecasaccharide and the Ganoderma spore tetradecasaccharide polysaccharide GLSWA-1 of natural origin (D2O).

[0127]

[0128] a The values denote the chemical shifts of H-6a and b denote the chemical shifts of H-6b.

[0129] Experiment 2: Evaluation of the effect of Ganoderma spore GLSWA-I (1) polysaccharide tetradecasaccharide and its substructures on the activity of insulin protein

[0130] 2.1 Experimental reagents:

[0131] Insulin protein detection kit, min6 cells, acarbose (commercially available), polysaccharide Ganoderma tetradecasaccharide GLSWA-1 (1) and its three substructure pentasaccharides 2, 3 and tetrasaccharide 4, pure water.

[0132] Among them, the structure of polysaccharide Ganoderma tetradecasaccharide GLSWA-1 (1) and its three substructure pentasaccharides 2, 3 and tetrasaccharide 4 are as follows:

[0133]

[0134] 2.2 Experimental method:

[0135] The test polysaccharide was mixed with min 6 cells in a 96-well plate (final concentration 100, 200, 400 μg / ml) and incubated for 12 h, with three repeated holes set respectively, and blank control holes without drugs and acarbose control group were set. The cell supernatant was taken, and the experiment was carried out according to the kit operation instruction. Within 15 min after termination of the reaction, the OD value was measured by an enzyme-labeled instrument, the detection wavelength was 450 nm, and the insulin protein activity was calculated.

[0136] 2.3 Experimental results

[0137] The experimental results are shown in Table 1 and Figure 1, and it can be seen that polysaccharide substructure pentasaccharide 2 has a certain insulin protein activity enhancing effect at a concentration of 200 μg / ml. Polysaccharide ganoderma fourteen saccharides GLSWA-1 (1) have strong insulin protein activity enhancing effect at concentrations of 100, 200 and 400 μg / ml. Figure 2

[0138] Experiment 3: Insulin structure stability test

[0139] 3.1 Reagents

[0140] Insulin, ganoderma fourteen saccharides GLSWA-1 (1) and its three substructure pentasaccharides 2, pentasaccharides 3 and tetrasaccharides 4, pure water, etc.

[0141] 3.2 Experimental method

[0142] The insulin sample was diluted to 1 mg / ml in ultrapure water, and divided into five parts, one of which was left as a blank control, and the other four were mixed with different polysaccharides. The fluorescence was excited at 330 and 350 nm, the program was warmed up from 35 to 95 ℃ (30 ℃ / min), then the chromogenic amino acid (Tyr) in insulin was detected, and the inflection point temperature (Ti) and initial fluorescence ratio of fluorescence were calculated.

[0143] 3.3 Experimental results

[0144] The experimental results are shown in Table 2 and Figure 2, and the results show that polysaccharides pentasaccharides 2, pentasaccharides 3 and tetrasaccharides 4 can all improve the thermal stability of the tertiary structure of insulin. Figure 3 Table 2

[0145]

[0146]

[0147] ​​The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A pentasaccharide 2 having an insulin protein activity enhancing effect, characterized by, The structure of the pentasaccharide 2 is: 。 2. Use of pentasaccharide 2 having an insulin protein activity enhancing effect according to claim 1, characterized in that, The application of the pentasaccharide 2 in the preparation of preparations and compounds with enhanced insulin protein activity or improved thermal stability of the tertiary structure of insulin.

3. Use according to claim 2, characterized in that, The application in the preparation of the compound with enhanced insulin protein activity or improved thermal stability of the tertiary structure of insulin includes the preparation of Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1 by orthogonal one-pot glycosylation reaction and functional group transformation using the derivative of the pentasaccharide 2, and the structure of the Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1 is: 。 4. A pentasaccharide 3 having biological activity, characterized in that, The structure of the pentasaccharide 3 is: 。 5. Use of a pentasaccharide having biological activity according to claim 4, characterized in that, The application of the pentasaccharide 3 in the preparation of Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1 and the preparation of preparations and compounds with improved thermal stability of the tertiary structure of insulin.

6. A method for preparing Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide, characterized in that, Firstly, the ortho-one (1-phenylalkenyl) benzoate PVB, the sugar-based N -phenyltrifluoroacetimidate PTFAI and the sugar-based ortho-alkynyl benzoate ABz were used to construct the sugar-based PTFAI tetrasaccharide donor 5, the derivatization structure of the pentasaccharide 3 as claimed in claim 4, the derivatization structure of the pentasaccharide 2 as claimed in claim 1, the reducing end pentasaccharide acceptor 7, and the bifunctional acceptor 6 of the pentasaccharide ortho-one (1-phenylalkenyl) benzoate; then the Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1 was prepared by using the sugar-based PTFAI tetrasaccharide donor 5, the bifunctional acceptor 6 of the pentasaccharide ortho-one (1-phenylalkenyl) benzoate, and the reducing end pentasaccharide acceptor 7 fragments through 【4+5+5】orthogonal one-pot glycosylation and functional group transformation; wherein the structures of the sugar-based PTFAI tetrasaccharide donor 5, the bifunctional acceptor 6 of the pentasaccharide ortho-one (1-phenylalkenyl) benzoate, and the reducing end pentasaccharide acceptor 7 are shown in the reaction pathway: 。 7. The use of the Ganoderma lucidum spore GLSWA-1 polysaccharide tetradecasaccharide prepared by the method of claim 6, characterized in that, The application of the Ganoderma spore GLSWA-1 polysaccharide tetradecasaccharide 1 in the preparation of preparations with enhanced insulin protein activity or improved thermal stability of the tertiary structure of insulin.

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