Synthesis method and application of ganoderma brownii galactomannan oligosaccharide and polysaccharide

Abstract

The application discloses a synthesis method and application of Ganoderma annularis galactomannan oligosaccharide and polysaccharide, and belongs to the field of sugar chemistry and medicinal chemistry. Five tetrasaccharide building blocks are used as synthesis subunits, the construction efficiency of Ganoderma annularis galactose 1,2-cis glycosidic bond is improved through remote participation of benzoyl at the 4th position of galactose, and through [4+4], [8+8] and [16+8] glycosylation strategies, Ganoderma annularis octasaccharide, hexadecasaccharide and twenty-four saccharide are stereoselectively constructed; the obtained oligosaccharide and polysaccharide are deprotected to obtain six Ganoderma annularis galactomannan oligosaccharides and polysaccharides with anti-inflammatory activity, which have definite structures and single components. Through cell immunity experiments, it is found that the above oligosaccharide and polysaccharide can inhibit the inflammatory reaction of LPS-induced mouse macrophage RAW 264.7, has potential application in anti-inflammatory drugs, and is also a good antigen of Ganoderma annularis sugar vaccine, and can be developed into a sugar vaccine or a sugar drug.

Description

Technical Field

[0001] This invention relates to a method for synthesizing galactomannan oligosaccharides and polysaccharides from Antrodia camphorata and their application as immunomodulators, belonging to the fields of glycochemistry and medicinal chemistry. Background Technology

[0002] In recent years, medicinal fungi have received widespread attention due to their potent therapeutic activities, particularly as immunomodulatory agents. *Antrodia camphorata* (… Antrodia cinnamomea As a widely studied medicinal fungus, *Antrodia camphorata* has been used for centuries to treat food poisoning and enhance liver function. In recent decades, it has been incorporated into Asian folk medicine, showing significant effectiveness in treating certain inflammatory diseases, cancer, hypertension, and hepatitis. Based on its medicinal properties, the total market value of *Antrodia camphorata* products (including fruiting bodies and health foods) is estimated to exceed US$100 million annually. Int. J. Biol. Sci. 2018, 14 (1378-1388). These therapeutic effects have encouraged many research groups to conduct in-depth biological and phytochemical studies on Antrodia camphorata, thereby isolating different classes of pharmacologically active secondary metabolites.

[0003] Hundreds of small molecules from *Antrodia camphorata* with different biological activities have been identified as active ingredients. Among them, the most important pharmacologically active components include triterpenes, steroids, benzoquinone derivatives, and polysaccharides. Pharmacology & Therapeutics 2013, 139 ,124–156). Some crude polysaccharide components also possess certain biological activities, including immunomodulatory, antitumor, antioxidant, and anti-angiogenic activities. The chemical structure of *Antrodia camphorata* polysaccharide has been determined to be repeating galactomannan (PS), exhibiting phagocytic activity. In 2017, Wu Shenghua's research group ( Org. Lett. 2017, 19 A polysaccharide composed of octasaccharide repeating fragments was isolated from Antrodia camphorata polysaccharide (3486−3489), with the main chain being {→6)-α-D-Man. p -(1→2)- α -D-Man p -(1→2)-[α-D-Man p -(1→3)- α -D-Man p -(1→2)-α-D-Man p -(1→6)- α -D-Gal p -(1→6)- α -D-Man p -(1→6)- α -D-Gal p -(1→} nIts composition is 3 / 4 mannose and 1 / 4 galactose; the mannose residues (Man) and galactose residues (Gal) communicate with each other via... α -1,2、 α -1,3 and α -1,6 glycosidic bonds are linked together, and its structure is shown in the following formula.

[0004]

[0005] In glycochemistry, the 1,2-trans glycosidic bond of galactose is relatively easy to construct, mainly relying on the interaction of the neighboring group of the acyl group at the 2-position of the glycosyl group; however, the construction of the 1,2-cis glycosidic bond is more difficult. The 1,2-cis glycosidic bond of galactose exists in Antrodia camphorata polysaccharides, but due to the steric hindrance effect of the C2 axial bond of galactose, the synthesis of the 1,2-cis glycosidic bond of Antrodia camphorata galactose is very challenging. Currently, only the research group of Professor Guofeng Gu (Chinese Chemical Letters 2024, 35, 109089) has reported on the synthesis of Antrodia camphorata oligosaccharides. Their synthetic strategy uses disaccharide building blocks as basic building units, constructing three tetrasaccharide building blocks, two hexasaccharide building blocks, and one octasaccharide building block through [2+2], [2+2+2], and [2+2+2+2] synthetic strategies. The authors also found through cell experiments that oligosaccharides 1a–1f have a significant stimulatory effect on Raw264.7 macrophages.

[0006] However, the construction efficiency of 1,2-cis glycosidic bonds in Antrodia camphorata oligosaccharides is not high, making it difficult to prepare large quantities of Antrodia camphorata polysaccharides. Further synthesis of Antrodia camphorata polysaccharides also faces certain limitations. Therefore, there is an urgent need for a direct and stereo-controlled method to construct galactose 1,2-cis glycosidic bonds in order to efficiently synthesize Antrodia camphorata galactomannan polysaccharides. Summary of the Invention

[0007] To address the aforementioned problems, the first objective of this invention is to provide a method for synthesizing *Antrodia camphorata* oligosaccharides and polysaccharides by efficiently constructing 1,2-cis-glycosidic bonds in *Antrodia camphorata* galactose.

[0008] Another object of the present invention is to provide the application of the antrodia camphorata oligosaccharides and polysaccharides synthesized by the above method as immunomodulators, for the preparation of anti-inflammatory drugs and for the development of sugar vaccines and sugar drugs.

[0009] This invention uses five tetrasaccharide building blocks 7-11 as raw materials, and utilizes the long-range participation of the benzoyl group at the 4-position of galactose to obtain four octasaccharides, one hexadecase, and one ticosaccharide through [4+4], [8+8], and [16+8] glycosylation strategies and stereoselective α-D-galactosylation reactions. Finally, deprotection is performed to obtain the target oligosaccharides and polysaccharides 1-6.

[0010] .

[0011] The reaction mechanism for constructing the 1,2-cis-glycosidic bond of Antrodia camphorata galactose using the long-range participation of the benzoyl group at the 4-position of galactose in this invention is shown in the following equation:

[0012] .

[0013] During glycosylation, compound a leaves an anodic leaving group under the catalysis of an accelerator, generating an anodic oxonium ion intermediate b. After oxonium ion rearrangement, intermediate c is obtained, followed by ester recombination to obtain e. Through a ring-closing reaction with the 4-benzoyl group, intermediate d with the 4-acyl group is obtained. This intermediate then shields the sugar ring from above, resulting in product d where the acceptor attacks from below the sugar ring, thus yielding the 1,2-cis-glycosylated galactose product.

[0014] To achieve the above objectives, the specific technical solution provided by the present invention is as follows:

[0015] A method for synthesizing galactomannan oligosaccharides and polysaccharides from Antrodia camphorata, characterized by comprising the following steps:

[0016] Step 1: Synthesis of glycosyl donor 13 and glycosyl acceptors 14 and 15:

[0017]

[0018]

[0019] The protected tetrasaccharide compound 7 was hydrolyzed to remove the anopropylthioglycoside at the anomeric position, and then reacted with N-phenyltrifluoroimine chloride (PTFA-Cl) to obtain tetrasaccharide donor 13.

[0020] The protected tetrasaccharide compound 8 was hydrolyzed to remove the anopropylthioglycoside at the anomeric position, yielding compound 14. The protected tetrasaccharide compound 8 was then directly glycosylated with an azidehexanol acceptor under catalytic conditions to yield acceptor 15 containing a linker arm. This step utilizes the long-range involvement of the benzoyl group at the 4-position of galactose to directly construct an α-D-galactosidic bond with high stereoselectivity. The hydrolysis reagent was dibromohydantoin (DBDMH); the catalytic agent was a Lewis acid or a protic acid, selected from one or more of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and N-iodosuccinimide (NIS).

[0021] Step Two: Synthesis of octasaccharides 16, 17, 18, 19, 20 and hexadecaccharides 23 and twentiethaccharides 25:

[0022]

[0023]

[0024]

[0025] Glycosyl donors 7, 13, or 22 and glycosyl acceptors 8, 9, 10, 11, 14, 21, or 24 are dissolved in a solvent, and molecular sieves and promoters are added to carry out the reaction, yielding octasaccharides 16, 17, 18, 19, and 20, and hexadecanoates 23 and tetradecanoates 25, respectively. This step utilizes the long-range participation of the benzoyl group at the 4-position of galactose to directly construct α-D-galactosidic bonds with high stereoselectivity.

[0026] The accelerator is a Lewis acid or a protic acid, selected from one or more of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and N-iodosuccinimide (NIS).

[0027] Step 3: Remove Protective Groups

[0028] Octasaccharides 16, 17, 18, and 19, and hexadecasaccharides 23 and 25, containing connecting arms, were first desilicified by hydrofluoric acid, then deesterified by NaH, and finally debenzyl and 2-naphthylmethyl groups were removed by Pd-catalyzed hydrogenation to obtain octadecasaccharides 3, 4, 5, and 6, and hexadecasaccharides 2 and 1, with fully exposed hydroxyl groups.

[0029]

[0030] .

[0031] This invention also provides the application of the oligosaccharides and polysaccharides of Antrodia camphorata synthesized by the above method in anti-inflammatory drugs, laying the foundation for the development of sugar vaccines and sugar drugs in the future.

[0032] The beneficial effects of this invention are:

[0033] This invention provides a highly efficient method for synthesizing *Antrodia camphorata* galactomannan oligosaccharides and polysaccharides, achieving stereospecific direct construction of 1,2-cis-α-D-glycosidic bonds. Furthermore, based on this method, the octa-, hexadecyl, and twentidecyl sugars of *Antrodia camphorata* galactomannan oligosaccharides containing linker arms were chemically synthesized, and their activity in inhibiting lipopolysaccharide-induced inflammatory responses in mouse RAW 264.7 macrophages was subsequently tested. The results showed that the *Antrodia camphorata* oligosaccharides and polysaccharides synthesized in this invention can inhibit LPS-induced inflammatory responses in mouse RAW 264.7 macrophages, significantly suppressing the secretion of IL-6 and TNF-α in RAW 264.7 cells, exhibiting excellent immunomodulatory effects, with the octa-saccharide 4 showing the strongest inhibitory effect. The *Antrodia camphorata* oligosaccharides and polysaccharides synthesized in this invention can be used to prepare anti-inflammatory drugs and developed into glycopeptides and glycopeptide vaccines. Attached Figure Description

[0034] Figure 1This is a diagram showing the inhibitory effects of different Antrodia camphorata galactomannan oligosaccharides and polysaccharides on lipopolysaccharide-induced IL-6 release.

[0035] In the figure, data are expressed as mean ± standard deviation (n = 3);* P < 0.05;** P < 0.01; *** P <0.001; **** P < 0.0001 compared to the LPS group, unpaired t-test.

[0036] Figure 2 This diagram illustrates the inhibitory effects of different Antrodia camphorata galactomannan oligosaccharides and polysaccharides on lipopolysaccharide-induced TNF-α release.

[0037] In the figure, data are expressed as mean ± standard deviation (n = 3);* P < 0.05;** P < 0.01; *** P <0.001; **** P < 0.0001 compared to the LPS group, unpaired t-test.

[0038] Figure 3 This is a Western blot analysis of p38, p-p38, p65, and p-p65 in RAW264.7 cell lysates after treatment with octasaccharide compound 4. Detailed Implementation

[0039] The present invention will be described in detail below with reference to specific embodiments to provide a better understanding of the invention. However, those skilled in the art will readily understand that the description of the specific embodiments is for illustrative purposes only and should not, and will not, limit the scope of protection claimed in the claims.

[0040] The solvents and reagents used in the following specific examples were all purchased from reagent manufacturers and require no special treatment or purification before use. The following five tetrasaccharide compounds were all purchased from reagent manufacturers on a custom basis, and the hydroxyl groups were protected. Bz, Bn, TBS, TBDPS, Nap, Lev, and Azmb represent benzoyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, 2-naphthylmethyl, acetylpropionyl, and azidobenzoyl, respectively.

[0041]

[0042] The dried DMF, DCM, and THF solvents were mainly obtained using a Braun solvent purification and drying system. All reaction solutions were monitored using thin-layer chromatography plates (HSGF254) purchased from the Yantai Chemical Industry Research Institute. Generally, observation was performed first under UV light, followed by color development with a colorimetric reagent (carbohydrates were typically developed using a 5% sulfuric acid methanol solution). Column chromatography separation was performed using 300-400 mesh silica gel purchased from the Yantai Chemical Industry Research Institute. The developing solvent was two or more mixed solvents selected from DCM, MeOH, PE, and EA.

[0043] 1 H NMR, 13 All C NMR measurements were performed using an Agilent DD2 500 MHz or a Bruker AVANCE NEO 400 MHz NMR spectrometer; the deuterated solvent used was CDCl3 or D2O; the solvent peaks in the spectra were labeled as follows: TMS: d H = 0.00 ppm, CDCl3: d H = 7.26 ppm, d C = 77.0 ppm. High-resolution mass spectrometry (HR-MS) was performed using a Thermo LTQ Orbitrap XL mass spectrometer. Optical rotation was determined using a JASCO P-1010 digital polarimeter. The UV lamp was a Shanghai Jiapeng Technology Co., Ltd. ZF-6 model; the rotary evaporator was an EYELA, BUCHI, R-114: DTC-21 model; and the stirrer was a DHJF-8002 model.

[0044] Example 1:

[0045] The synthesis of compounds 13, 14 and 15 is shown in the following formula:

[0046]

[0047]

[0048] Under argon protection, tetrasaccharide compound 7 (885 mg, 0.43 mmol, 1 equiv) was dissolved in acetone / H2O = 10:1 (10 mL), and dibromohydantoin (DBDMH) (246 mg, 0.86 mmol, 2 equiv) was added for hydrolysis. After 2 min of reaction, TLC was used to monitor the reaction of the starting material until it was complete. Na2S2O3 and NaHCO3 were added to quench the reaction. After extraction of the reaction solution, the organic phase was concentrated and dried. The crude product 12 was directly used for the next reaction.

[0049] Under argon protection, the crude product obtained in the previous step was dissolved in acetone (10 mL), and K2CO3 (102 mg, 0.65 mmol, 1.5 equiv) and N-phenyltrifluoroacetylimine chloride (PTFA-Cl) (95) were added. m After reacting at room temperature for 10 h (L, 0.56 mmol, 1.0 equiv), the resulting tetrasaccharide donor 13 was concentrated and used directly in the next reaction.

[0050] Under argon protection, tetrasaccharide compound 8 (750 mg, 0.39 mmol, 1 equiv) was dissolved in acetone / H2O = 10:1 (40 mL), and dibromohydantoin (DBDMH) (225 mg, 0.79 mmol, 2 equiv) was added for hydrolysis. After 2 min of reaction, TLC was used to monitor the reaction of the starting material until it was complete. Na2S2O3 and NaHCO3 were added to quench the reaction. After extraction of the reaction solution, the organic phase was concentrated and dried to obtain tetrasaccharide acceptor 14 with exposed anodic hydroxyl groups. After concentration, it was directly used for the next reaction.

[0051] Under argon protection, tetrasaccharide compound 8 (800 mg, 0.42 mmol, 1 equiv) and azidehexanol receptor (58.4 mg, 0.408 mmol, 1.2 equiv) were dissolved in dry Et₂O (8.4 mL, 0.05 M). Activated 4 ÅMS (800 mg) was added and stirred for 10 min. The mixture was cooled to -40 ºC, and NIS (236 mg, 1.05 mmol, 2.5 equiv) was added and stirred for 2 min, followed by the addition of TMSOTf (7.6 mg). m L, 42 m The reaction was carried out at -40 ºC for 2 h (mol, 0.1 equiv). TLC monitoring showed that the acceptor reaction was complete. The insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain a colorless paste compound 15. = +90.78 ( c = 0.05, CHCl3); 1 H NMR (400 MHz, Chloroform- d ) d 8.07 (d, J = 7.7 Hz, 2H), 7.87 (d, J = 7.8 Hz, 2H), 7.81 (d, J= 7.8 Hz,2H), 7.76 (s, 1H), 7.68 (m, 3H), 7.59 (t, J = 7.5 Hz, 1H), 7.47 (m, 6H),7.39–7.10 (m, 46H), 7.03 (t, J = 7.4 Hz, 1H), 5.76 (s, 3H), 5.23 (s, 1H),5.18 (s, 1H), 4.96 (d, J = 3.5 Hz, 1H), 4.89–4.77 (m, 5H), 4.72 (d, J = 3.9Hz, 3H), 4.66 (s, 1H), 4.63 (s, 2H), 4.61–4.49 (m, 6H), 4.40 (s, 2H), 4.28–4.07 (m, 7H), 4.04 (s, 1H), 3.93 (d, J = 9.2 Hz, 5H), 3.86–3.64 (m, 9H), 3.60(s, 2H), 3.43 (m, 1H), 3.33 (dd, J = 9.9, 6.5 Hz, 1H), 3.19 (s, 2H), 2.70–2.50 (m, 4H), 2.06 (s, 3H), 1.76 (s, 1H), 1.66 (m, 2H), 1.56 (m, 2H), 1.39(m, 5H), 1.26 (s, 1H), 0.84 (s, 10H); 13 C NMR (101 MHz, Chloroform- d ) d206.4, 165.5, 138.3, 138.2, 133.2, 133.0, 129.98, 129.90, 129.7, 128.57, 128.51, 128.4, 128.39, 128.35, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 126.1, 125.9, 125.7, 100.3, 99.2, 99.1, 97.2, 80.0, 78.2, 75.7, 75.1, 75.0, 74.5, 73.9, 73.7, 73.4, 73.2, 73.1, 72.7, 72.5, 72.4, 72.2, 71.5, 70.46, 70.41, 69.8, 69.0, 68.3, 67.6, 65.5, 63.1, 62.5, 51.4, 37.9, 29.8, 29.3, 28.8, 27.8, 26.6, 26.0, 25.9, 18.3.

[0052] Example 2:

[0053] The synthesis of compound 16 is shown in the following formula:

[0054]

[0055] Under argon protection, N 13 (640 mg, 0.30 mmol, 1.2 equiv) of phenyltrifluoroacetylimine ester (PTFAI) donor compound 13 and acceptor compound 8 (530 mg, 0.27 mmol, 1.0 equiv) were dissolved in dry Et2O (13 mL), and activated 4 Å MS (1.30 g) was added. The mixture was stirred for 10 min, cooled to -40 ºC, and TMSOTf (10 g) was added. m The reaction was carried out at -40 ºC for 1 h (L, 0.05 mmol, 0.2 equiv). TLC monitoring showed that the receptor reaction was complete. The insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA = 6:1) to obtain a colorless paste compound 16 (α:β > 20:1, 766 mg, 0.19 mmol, 72%). = +683.8( c = 0.01, CHCl3); 1 H NMR (400 MHz, Chloroform- d ) d 8.19–8.05 (m, 7H), 7.97 (d, J = 7.7 Hz, 2H), 7.90 (d, J = 7.8Hz, 2H), 7.83 (d, J = 7.7 Hz, 2H), 7.76–7.52 (m, 16H), 7.51–7.15 (m, 84H),7.12 (t, J = 6.2 Hz, 9H), 5.88 (d, J = 3.4 Hz, 1H), 5.84 (d, J = 3.2 Hz, 1H),5.82–5.73 (m, 4H), 5.64 (d, J = 3.0 Hz, 1H), 5.41 (s, 1H), 5.35 (s, 1H), 5.30(d, J = 3.5 Hz, 1H), 5.26 (s, 1H), 5.16 (s, 1H), 5.04 (d, J = 3.5 Hz, 1H),5.02–4.93 (m, 3H), 4.93–4.81 (m, 6H), 4.79 (s, 2H), 4.77–4.54 (m, 22H), 4.50(d, J = 14.4 Hz, 1H), 4.48–4.36 (m, 6H), 4.35–4.22 (m, 6H), 4.22–3.95 (m,16H), 3.95–3.71 (m, 15H), 3.71–3.54 (m, 3H), 3.46 (m, 2H), 3.35 (d, J = 9.1Hz, 1H), 3.28 (t, J = 6.8 Hz, 2H), 2.84–2.57 (m, 4H), 2.16 (s, 3H), 1.80 (d, J = 4.2 Hz, 3H), 1.65 (m, 5H), 1.54–1.40 (m, 5H), 0.79 (s, 9H), 0.00 (s, 3H),-0.03 (s, 3H); 13 C NMR (101 MHz, Chloroform- d ) d206.4, 172.3, 165.4, 165.2, 138.5, 138.3, 138.1, 138.0, 137.99, 137.94, 135.8, 133.2, 133.1, 133.0, 132.7, 131.7, 130.2, 129.9, 129.8, 129.7, 129.5, 129.48, 129.43, 128.56, 128.50, 128.47, 128.41, 128.38, 128.32, 128.29, 128.21, 128.18, 128.15, 128.10, 128.0, 127.99, 127.96, 127.94, 127.91, 127.87, 127.84, 127.79, 127.73, 127.6, 127.57, 127.53, 127.49, 127.42, 127.3, 126.1, 125.78, 125.71, 125.5, 100.2, 99.8, 99.7, 99.6, 99.2, 98.5, 97.3, 97.2, 80.1, 78.1, 75.0, 74.8, 74.4, 74.1, 73.6, 73.3, 73.2, 73.0, 72.6, 72.5, 72.2, 72.1, 72.0, 71.5, 71.3, 70.6, 70.3, 69.0, 68.8, 68.6, 68.3, 67.4, 66.1, 63.0, 62.8, 53.1, 51.3, 37.9, 29.8, 29.2, 28.8, 27.8, 26.6, 25.8, 18.1, -5.0, -5.4.

[0056] Example 3:

[0057] The synthesis of compound 17 is shown in the following formula:

[0058]

[0059] Under argon protection, the glucosinolate donor compound 7 (36 mg, 17.4 mg) was... m mol, 1.1 equiv) and receptor compound 9 (35 mg, 17.6 mol, 1.1 equiv) m 1 mol, 1.0 equiv) dissolved in dry DCM / Et2O ( v / v= 1 / 1, 2 mL), add activated 3 Å MS (1.30 g), stir for 10 min, cool to -40 ºC, add NIS (20 mg, 88.9 g). m mol, 5.0 equiv) / TMSOTf (2 m L, 13.0 m The reaction was carried out at -40 ºC for 1 h (mol, 0.5 equiv). TLC monitoring showed that the acceptor reaction was complete. The insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA = 6:1) to obtain a colorless paste compound 17 (69%). = +33.3( c = 0.10, CHCl3); 1 H NMR (400 MHz, CDCl3) d 8.18–8.03 (m, 8H), 7.96–7.88 (m, 4H), 7.83–7.75 (m, 3H), 7.72–7.08 (m, 100H), 5.90(d, J = 3.4 Hz, 1H), 5.87–5.77 (m, 4H), 5.75 (t, J = 2.5 Hz, 1H), 5.71 (dd, J = 3.0, 1.8 Hz, 1H), 5.65–5.61 (m, 1H), 5.37 (d, J = 1.8 Hz, 1H), 5.34 (d, J =1.9 Hz, 1H), 5.32 (d, J = 3.5 Hz, 1H), 5.28 (d, J = 1.9 Hz, 1H), 5.26 (d, J =3.5 Hz, 1H), 5.05–5.00 (m, 2H), 4.99–4.94 (m, 2H), 4.92–4.87 (m, 2H), 4.86–4.83 (m, 2H), 4.84–4.77 (m, 6H), 4.76–4.65 (m, 12H), 4.65–4.57 (m, 6H), 4.53(dd, J= 11.2, 2.2 Hz, 2H), 4.47–4.35 (m, 6H), 4.28–4.07 (m, 16H), 4.06–3.93(m, 4H), 3.92–3.72 (m, 14H), 3.65–3.55 (m, 2H), 3.55–3.44 (m, 2H), 3.34 (t, J = 9.2 Hz, 1H), 3.29 (t, J = 6.9 Hz, 2H), 1.68–1.59 (m, 4H), 1.47–1.40 (m,4H), 0.79 (s, 9H), 0.00 (s, 3H), -0.03 (s, 3H); 13 C NMR (151 MHz, CDCl3) d166.2, 165.9, 165.7, 165.50, 165.49, 165.46, 165.4, 165.4, 165.3, 139.0, 138.8, 138.7, 138.6, 138.5, 138.4, 138.2, 138.1, 138.0, 137.9, 137.7, 135.7, 133.3, 133.2, 133.1, 133.0, 132.9, 132.8, 132.7, 131.8, 131.6, 130.4, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.1, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 126.7, 126.2, 125.8, 125.7, 100.1, 99.9, 99.8, 99.4, 98.6, 98.3, 98.0, 97.3, 80.2, 79.6, 78.7, 78.5, 78.4, 78.2, 75.5, 75.4, 75.3, 75.2, 75.1, 74.9, 74.5, 74.4, 74.3, 74.2, 74.1, 74.0, 73.6, 73.4, 73.3, 73.1, 72.8, 72.3, 72.2, 72.1, 72.0, 71.9, 71.8, 71.7, 71.6, 71.4, 70.7, 70.4, 70.3, 69.9, 69.4, 69.1, 69.0, 68.7, 67.8, 67.6, 67.5, 67.2, 66.2, 65.3, 62.9, 62.5, 53.2, 53.1, 51.5, 29.8, 29.4, 28.8, 26.6, 25.9, 18.2, -5.0, -5.4.

[0060] Example 4:

[0061] The synthesis of compound 18 is shown in the following formula:

[0062]

[0063] Under argon protection, donor compound 7 (194 mg, 94.9 g / L) was... mmol (1.05 equiv) and receptor compound 10 (177 mg, 92.7 mol, 1.05 equiv) m Dissolve 1 mol (1.0 equiv) in dry DCM (5 mL), add activated 4 Å MS (1.1 g), stir for 10 min, cool to -40 ºC, and add NIS (63 mg, 0.27 mmol, 2.7 equiv) / TMSOTf (13 mol, 1.0 equiv) to TMS. m L,90 m The reaction was carried out at -40 ºC for 1 h (mol, 1.0 equiv). TLC monitoring showed that the acceptor reaction was complete. Insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA = 3:1) to give a colorless paste compound 18 (302 mg, 76.5 mol, 1.0 equiv). m mol, 82%). = +31.0( c = 0.10, CHCl3); 1 H NMR (400 MHz, CDCl3) d 8.19–8.07 (m, 8H), 8.02 (d, J = 8.4, 2H), 7.95–7.87 (m, 7H), 7.83–7.74 (m, 3H), 7.69–7.53 (m, 9H), 7.49–7.07 (m, 92H), 6.00–5.97 (m, 1H), 5.93 (d, J = 3.4 Hz, 1H), 5.89 (d, J = 3.4 Hz, 1H), 5.86–5.83 (m, 1H), 5.83–5.80 (m, 1H), 5.77 (d, J = 2.1 Hz, 1H), 5.70–5.62 (m, 2H), 5.46 (d, J = 1.8 Hz, 1H), 5.37 (d, J =1.9 Hz, 1H), 5.30 (d, J = 1.9 Hz, 1H), 5.30–5.25 (m, 2H), 5.08–4.53 (m, 33H), 4.50–4.38 (m, 4H), 4.34–4.02 (m, 19H), 3.98–3.53 (m, 19H), 3.49–3.45 (m, 1H),3.36 (t, J= 9.0 Hz, 1H), 3.20 (t, J = 7.0 Hz, 2H)., 1.57–1.45 (m, 4H), 1.32–1.24 (m, 4H), 0.82 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H); 13 C NMR (101 MHz, CDCl3) d 165.9, 165.8, 165.6, 165.6, 165.5, 165.3, 139.0, 138.6, 138.5, 138.3, 138.2, 138.1, 138.0, 137.8, 135.9, 133.3, 133.1, 133.0, 132.9, 131.7, 130.3, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.3, 126.3, 125.9, 125.7, 99.9, 99.7, 99.6, 99.0, 98.63, 98.62, 98.3, 98.2, 80.1, 79.6, 78.3, 75.3, 75.0, 74.9, 74.5, 74.4, 74.1, 73.6, 73.4, 73.0, 72.5, 72.4, 72.0, 71.8, 71.7, 71.6, 71.3, 70.7, 70.1, 69.5, 69.1, 68.7, 67.6, 67.4, 67.1, 53.1, 51.3, 29.5, 28.7, 26.5, 25.9, 25.7, 18.2, -5.0, -5.4.

[0064] Example 5:

[0065] The synthesis of compound 19 is shown in the following formula:

[0066]

[0067] Under argon protection, donor compound 7 (197 mg, 96.4 mg) was... m mol (1.0 equiv) and receptor compound 11 (201 mg, 95.2 mol, 1.0 equiv) and receptor compound 11 (201 mg, 95.2 equiv). mDissolve 1 mol (1.0 equiv) in dry DCM (6 mL), add activated 4 Å MS (700 mg), stir for 10 min, cool to -40 ºC, add NIS (60 mg, 0.26 mmol, 2.5 equiv) / TMSOTf (10 mol, 1.0 equiv) m L, 62 m The reaction was carried out at -40 ºC for 1 h. TLC monitoring showed that the acceptor reaction was complete. Insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA:DCM = 3:1:1) to give a colorless slurry compound 19 (288 mg, 70.6 mol, 0.6 equiv). m mol, 74%). = +44.4 ( c = 0.10, CHCl3); 1 H NMR (400 MHz, CDCl3) d 8.19–8.13(m, 4H), 8.13–8.08 (m, 3H), 7.97–7.90 (m, 4H), 7.90 – 7.86 (m, 2H), 7.75–7.60(m, 12H), 7.60–7.49 (m, 6H), 7.49–7.09 (m, 90H), 7.07–6.92 (m, 5H), 6.06 (d, J = 3.4 Hz, 1H), 5.93–5.87 (m, 2H), 5.87–5.80 (m, 2H), 5.78 (t, J = 2.4 Hz, 1H), 5.66 (t, J = 2.5 Hz, 1H), 5.36 (d, J = 9.1 Hz, 2H), 5.34–5.30 (m, 2H), 5.28 (d, J = 2.0 Hz, 1H), 5.24 (d, J = 3.5 Hz, 1H), 5.07 (d, J = 1.6 Hz, 1H),5.05–4.97 (m, 3H), 4.93–4.69 (m, 18H), 4.69–4.55 (m, 9H), 4.52 (d, J = 8.8Hz, 1H), 4.49–4.38 (m, 7H), 4.36 (d, J= 9.4 Hz, 1H), 4.33–4.03 (m, 19H),3.97–3.78 (m, 12H), 3.75–3.61 (m, 6H), 3.41–3.28 (m, 2H), 3.23 (t, J = 6.9Hz, 2H), 1.60–1.41 (m, 4H), 1.36–1.22 (m, 4H), 1.05 (s, 9H), 0.82 (s, 9H),0.03 (s, 3H), -0.00 (s, 3H); 13 C NMR (101 MHz, CDCl3) d165.8, 165.7, 165.46, 165.45, 165.37, 165.36, 165.30, 165.28, 138.9, 138.7, 138.5, 138.4, 138.3, 138.2, 138.1, 138.0, 137.9, 137.8, 137.7, 135.7, 135.5, 133.2, 133.1, 133.0, 132.9, 132.8, 132.7, 131.7, 130.3, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6,129.5, 129.4, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8,127.7, 127.6, 127.5, 127.4, 127.3, 126.1, 125.7, 125.5, 101.4, 99.84, 99.83,99.5, 99.0, 98.7, 98.3, 97.6, 79.9, 79.8, 79.6, 78.3, 78.2, 78.0, 77.3, 76.0,75.6, 75.3, 75.2, 75.0, 74.9, 74.5, 74.2, 73.7, 73.4, 73.3, 73.2, 73.1, 73.0, 72.3, 72.1, 71.9, 71.6, 71.5, 70.7, 70.4, 70.2, 69.7, 69.6, 69.2, 69.1, 68.7, 67.5, 67.4, 67.2, 62.8, 62.5, 61.5, 53.1, 51.3, 29.7, 29.3, 28.7, 26.7, 26.5, 25.8, 25.7, 19.0, 18.1, -5.0, -5.5.

[0068] Example 6:

[0069] The synthesis of compounds 20 and 22 is shown in the following formula:

[0070]

[0071] Under argon protection, donor compound 13 (747 mg, 0.34 mmol, 1.2 equiv) and acceptor compound 14 (530 mg, 0.29 mmol, 1.0 equiv) were dissolved in dry Et2O (12 mL), activated 4 Å MS (1.5 g) was added, and the mixture was stirred for 10 min. The mixture was then cooled to -40 ºC, and TMSOTf (10 g / mL) was added. m L, 58 m The reaction was carried out at -40 ºC for 2 h. TLC monitoring showed that the acceptor reaction was complete. The insoluble solids were removed by filtration, and the filtrate was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain a colorless paste compound 20 (802 mg, 0.21 mmol, 72%). 1 H NMR (400 MHz, Chloroform- d ) d 8.14 (m, 8H), 8.02–7.85 (m, 5H), 7.83–6.96 (m, 103H), 5.85 (m, 5H), 5.67 (t, J = 2.5 Hz, 1H), 5.49–5.33 (m, 3H), 5.33–5.12 (m, 3H), 5.08–3.56 (m, 75H), 3.52–3.34 (m, 1H), 2.88–2.56 (m, 4H), 2.18 (s, 3H), 0.82(d, J = 2.7 Hz, 8H), 0.10–-0.07 (m, 6H); 13 C NMR (101 MHz, Chloroform- d ) d206.5, 172.4, 165.5, 139.0, 137.9, 133.2, 133.0, 132.8, 131.7, 129.96, 129.92, 129.8, 129.7, 129.5, 128.5, 128.4, 128.3, 128.2, 128.16, 128.10, 128.07, 128.03, 127.9, 127.88, 127.80, 127.73, 127.70, 127.6, 127.59, 127.55, 125.8, 73.5, 72.3, 72.0, 71.5, 69.0, 62.8, 62.4, 53.1, 37.9, 27.8, 25.8, 18.1, -4.9, -5.4.

[0072] Compound 20 was further reacted with PTFA-Cl under the same conditions as in Example 1 to obtain octasaccharide donor 22, which was directly used in the next reaction.

[0073] Example 7:

[0074] The synthesis of compound 23 is shown in the following formula:

[0075]

[0076] Under argon protection, compound 16 (200 mg, 0.05 mmol, 1.0 equiv) was dissolved in dry CH2Cl2 (5 mL), and pyridine / acetic acid (60 mL) was added. m L, 40 m L), hydrazine hydrate (10) m The reaction mixture was reacted at room temperature for 6 h. TLC monitoring showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA:DCM = 5:1:1) to obtain a colorless slurry compound 21 (184.5 mg, 48 L). m mol, 96%). 1 H NMR (400 MHz, CDCl3) d 8.22–8.06 (m, 7H), 8.00 (d, J = 7.8 Hz, 2H), 7.93 (d, J = 7.9 Hz, 2H), 7.86 (d, J= 7.8 Hz, 2H), 7.77–7.47 (m, 18H), 7.46–7.36 (m, 27H), 7.35–7.05 (m, 58H), 5.94 (d, J = 3.4 Hz,1H), 5.89–5.80 (m, 4H), 5.80–5.76 (m, 1H), 5.69–5.64 (m, 1H), 5.48 (s, 1H),5.38 (s, 1H), 5.35–5.26 (m, 2H), 5.16 (s, 1H), 5.08–4.54 (m, 34H), 4.50–4.40(m, 5H), 4.30–3.63 (m, 38H), 3.55–3.42 (m, 2H), 3.30 (t, J = 6.9 Hz, 2H),1.76–1.61 (m, 4H), 1.53–1.45 (m, 4H), 0.82 (s, 9H), 0.03 (s, 3H), -0.00 (s,3H); 13 C NMR (101 MHz, CDCl3) d166.0, 165.8, 165.6, 165.5, 165.4, 139.0,138.75, 138.74, 138.70, 138.6, 138.4, 138.2, 138.1, 138.07, 138.01, 137.9,136.1, 133.2, 133.1, 132.8, 131.8, 130.3, 130.2, 130.08, 130.0, 129.9, 129.8,129.5, 128.7, 128.6, 128.59, 128.53, 128.50, 128.4, 128.39, 128.31, 128.2,128.18, 128.14, 128.11, 128.08, 128.04, 127.96, 127.92, 127.8, 127.77,127.70, 127.6, 127.5, 127.4, 126.1, 125.84, 125.83, 125.81, 125.6, 100.2,100.0, 99.9, 99.7, 99.4, 98.7, 97.5, 80.4, 79.6, 78.42, 78.3, 78.1, 75.4,75.3, 75.2, 75.1, 75.0, 75.0, 74.5, 74.48, 74.43, 74.3, 74.2, 73.7, 73.5,73.38, 73.30, 73.1, 73.0, 72.7, 72.4, 72.4, 72.2, 72.1, 71.9, 71.8, 71.7,70.8, 70.5, 70.2, 70.0, 69.5, 69.3, 69.1, 68.5, 67.5, 67.4, 67.3, 66.1, 62.9,62.0, 53.2, 51.5, 29.4, 28.9, 26.7, 25.9, 18.2, -4.8, -5.3. MS (MALDI-TOF) m / z calcd for C 226 H 232 O 49 N6NaSi [M+Na] + 3864.5508, found 3864.73.

[0077] Under argon protection, N-phenyltrifluoroacetylimine ester (PTFAI) donor compound 22 (432 mg, 0.11 mmol, 1.1 equiv) and acceptor compound 21 (378 mg, 0.10 mmol, 1.0 equiv) were dissolved in dry Et2O (25.0 mL), and activated 4 Å MS (2.7 g) was added. The mixture was stirred for 30 min, cooled to -40 ºC, and TMSOTf (3.6 g) was added. m The reaction was carried out at -40 ºC for 2 h (L, 0.02 mmol, 0.2 equiv). TLC monitoring showed that the reaction of the starting material was complete. The insoluble solids were removed by filtration, the filtrate was washed with DCM, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA:DCM = 4:1:1) to give a colorless paste compound 23 ( α : β > 20:1, 497 mg, 0.065 mmol, 65%). 1 H NMR (400 MHz, Chloroform- d ) d 8.19–8.04 (m, 13H), 7.98 (d, J = 7.8 Hz, 2H), 7.89 (t, J = 7.5 Hz, 6H), 7.79(t, J = 6.7 Hz, 4H), 7.72–6.94 (m, 201H), 5.96–5.70 (m, 12H), 5.63 (t, J =4.6 Hz, 2H), 5.46 (s, 1H), 5.38–5.29 (m, 4H), 5.29–5.21 (m, 4H), 5.18 (s,1H), 5.13 (d, J = 3.5 Hz, 1H), 5.09–3.20 (m, 151H), 2.77–2.52 (m, 4H), 2.09(s, 3H), 1.77 (s, 2H), 1.68 (t, J = 6.9 Hz, 2H), 1.59 (m, 2H), 1.43 (d, J =6.2 Hz, 5H), 0.79 (s, 8H), 0.78 (s, 7H), -0.00 (s, 5H), -0.03 (s, 5H); 13 C NMR (126 MHz, Chloroform- d ) d206.5, 172.3,165.9, 165.55, 165.50, 165.4, 165.35,165.32, 138.8, 138.6, 138.5, 138.4, 138.3, 138.2, 138.14, 138.11, 138.,137.94, 137.8, 133.3, 133.19, 133.14, 133.0, 132.8, 132.76, 132.74, 131.7,130.3, 130.2, 130.0, 129.9, 129.88, 129.84, 129.81, 129.76, 129.74, 129.5,129.4, 128.7, 128.6, 128.5, 128.49, 128.46, 128.43, 128.40, 128.3, 128.29,128.24, 128.21, 128.20, 128.17, 128.12, 128.09, 128.03, 128.00, 127.96,127.93, 127.91, 127.87, 127.85, 127.81, 127.77, 127.74, 127.71, 127.68,127.60, 127.57, 127.52, 127.4, 127.39, 127.33, 127.31, 127.2, 126.1, 126.0,125.7, 125.67, 125.61, 125.5, 100.7, 99.8, 99.7, 99.3, 98.5, 97.3, 97.2,80.1, 79.6, 78.3, 78.2, 75.4, 75.2, 75.1, 75.0, 74.9, 74.3, 74.1, 74.0, 73.7,73.6, 73.4, 73.37, 73.31, 73.2, 73.0, 72.74, 72.70, 72.55, 72.52, 72.4,72.39, 72.30, 72.0, 71.9, 71.89, 71.80, 71.6, 71.4, 71.3, 71.0, 70.7, 70.4,70.29, 70.21, 70.0, 69.2, 69.0, 68.8, 68.6, 68.4, 67.5, 67.3, 67.2, 66.5,66.2, 65.3, 62.9, 62.8, 62.5, 53.1, 51.3, 37.9, 29.8, 29.3, 28.8, 27.8, 26.6, 25.89, 25.85, 18.19, 18.17, -4.94, -4.95, -5.4.

[0078] Example 8:

[0079] The synthesis of compound 25 is shown in the following formula:

[0080]

[0081] Under argon protection, compound 23 (21.8 mg, 2.9 mg) was... m Dissolve 1 mol (1.0 equiv) in dry CH2Cl2 (3 mL), add pyridine / acetic acid (20 mol, 1.0 equiv) m L, 30 m L), hydrazine hydrate (0.4 L) m The reaction mixture was reacted at room temperature for 6 h. TLC monitoring showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and then subjected to silica gel column chromatography (PE:EA:DCM = 4:1:1) to obtain a colorless slurry compound 24 (21.1 mg, 2.8 L). m mol, 95%). 1 H NMR (400 MHz, CDCl3) d 8.15–8.06 (m, 13H),8.01–7.95 (m, 2H), 7.94–7.84 (m, 6H), 7.83–7.98 (m, 4H), 7.70–7.32 (m, 95H),7.32–6.89 (m, 112H), 5.96–5.88 (m, 2H), 5.85–5.72 (m, 9H), 5.65–5.60 (m, 2H), 5.51 (s, 1H), 5.38–5.31 (m, 3H), 5.31–5.22 (m, 4H), 5.22–5.11 (m, 3H), 5.06–3.44 (m, 153H), 3.36–3.27 (m, 2H), 3.24 (t, J = 6.9 Hz, 2H), 1.66–1.55 (m,4H), 1.44–1.40 (m, 4H), 0.79 (s, 9H), 0.78 (s, 9H), 0.02, (s, 3H), 0.00 (s,3H), -0.03 (s, 3H), -0.04 (s, 3H); 13 C NMR (126 MHz, CDCl3) d165.9, 165.5,165.4, 165.3, 138.9, 138.6, 138.60, 138.5, 138.4, 138.3, 138.2, 138.1,137.98, 137.92, 137.8, 133.2, 133.1, 133.0, 132.8, 132.75, 131.73, 130.3,130.2, 129.99, 129.94, 129.8, 129.73, 129.70, 129.5, 129.4, 129.3, 128.6,128.58, 128.54, 128.49, 128.44, 128.40, 128.36, 128.32, 128.29, 128.24,128.21, 128.1, 128.08, 128.03, 127.99, 127.93, 127.86, 127.82, 127.77,127.72, 127.6, 127.58, 127.53, 127.48, 127.44, 127.39, 127.33, 127.30,127.16, 126.1, 125.8, 125.6, 125.5, 99.8, 99.7, 99.6, 99.4, 98.6, 97.2, 79.6,78.3, 78.1, 77.3, 75.4, 75.3, 75.19, 75.15, 75.11, 75.0, 74.9, 74.5, 74.3,74.15, 74.10, 73.7, 73.4, 73.3, 73.2, 73.0, 72.6, 72.5, 72.38, 72.33, 72.0,71.99, 71.95, 71.87, 71.80, 71.6, 71.3, 71.0, 70.7, 70.4, 70.2, 70.0, 69.3,69.2, 69.0, 68.6, 68.4, 67.2, 62.8, 61.8, 60.4, 53.1, 51.3, 29.2, 26.6, 25.8,18.1, -4.9, -4.9, -5.4.

[0082] Under argon protection, N-phenyltrifluoroacetylimine ester (PTFAI) donor compound 22 (92 mg, 0.016 mmol, 1.2 equiv) and acceptor compound 24 (99 mg, 0.013 mmol, 1.0 equiv) were dissolved in dry Et₂O (6.0 mL, 0.002 M), and activated 4 Å MS (700 mg) was added. The mixture was stirred for 10 min, cooled to -40 ºC, and TMSOTf (1.5 mg) was added. m mol, 7.8 m The reaction was carried out at -40 ºC for 4 h. TLC monitoring showed that the reaction of the starting material was complete. The insoluble solids were removed by filtration, the filtrate was washed with DCM, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 5:1) to give a colorless paste compound 25 (mol, 0.6 equiv). α : β > 20:1, 120 mg, 9.8 m mol, 51%). 1 H NMR (500 MHz, Chloroform- d ) d 8.18–8.05 (m, 21H), 7.99 (d, J = 7.8 Hz, 3H), 7.89 (m, 10H), 7.78 (m, 7H), 7.71–6.91 (m, 332H), 6.04–5.70 (m, 20H), 5.63 (d, J = 10.0 Hz,4H), 5.54–3.28 (m, 273H), 3.24 (d, J = 7.3 Hz, 2H), 2.79–2.48 (m, 6H), 2.08(s, 4H), 1.70 (m, 4H), 1.61 (m, 3H), 1.45 (s, 6H), 0.79 (d, J = 7.6 Hz, 27H), -0.01 (dd, J = 14.8, 5.0 Hz, 17H); 13 C NMR (126 MHz, Chloroform- d ) d206.5, 172.3, 165.9, 165.69, 165.65, 165.56, 165.51, 165.4, 165.39, 165.34, 165.31, 165.26, 165.20, 165.1, 138.8, 138.68, 138.65, 138.61, 138.5, 138.3, 138.2, 138.1, 138.0, 137.96, 137.93, 137.8, 133.2, 133.1, 133.0, 132.8, 132.76, 132.73, 132.5, 131.7, 130.3, 130.2, 130.0, 129.9, 129.8, 129.7, 129.5, 129.49, 129.42, 129.3, 128.69, 128.60, 128.5, 128.45, 128.41, 128.37, 128.33, 128.30, 128.2, 128.19, 128.15, 128.09, 128.06, 128.02, 127.96, 127.91, 127.87, 127.83, 127.77, 127.72, 127.6, 127.55, 127.51, 127.48, 127.44, 127.35, 127.32, 125.7, 125.6, 125.57, 125.52, 100.7, 99.9, 99.8, 99.7, 98.8, 98.4, 79.6, 78.3, 78.1, 75.4, 75.1, 75.0, 74.9, 74.5, 74.1, 74.0, 73.7, 73.4, 73.36, 73.30, 73.2, 73.0, 72.7, 72.4, 72.3, 71.9, 71.8, 71.6, 71.4, 70.7, 70.2, 70.0, 69.1, 69.0, 68.6, 68.4, 67.3, 62.8, 62.4, 53.1, 51.3, 37.9, 29.7, 28.8, 25.8, 18.1, -4.9, -5.4.

[0083] Example 9:

[0084] The synthesis of tetradecanoic sugar 1, hexadecanoic sugar 2, and octadecanoic sugar compounds 3, 4, 5, and 6 is shown in the following formula:

[0085]

[0086]

[0087] Under argon protection, compounds 16, 17, 18, 19, 23, and 25 were dissolved in THF, added to an ice bath for 5 min, and reacted at room temperature for 5 h. TLC monitoring showed that the reaction of the starting materials was complete. The reaction solution was slowly poured into a saturated NaHCO3 solution to quench the reaction and extracted with EA. The organic phase was washed with brine, dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a white foamy compound.

[0088] Under argon protection, the above crude product was dissolved in DCM / MeOH ( v / v = 1:1), NaH (6.0 equiv) was added under ice bath conditions, and the reaction was carried out at room temperature for 13 h. TLC monitoring showed that the starting material had reacted completely. IR120 H was then added to the reaction solution. + The reaction is quenched by the ion exchange resin, the organic phase is filtered, and the crude product after vacuum concentration of the filtrate is directly used in the next reaction.

[0089] Under argon protection, the above crude product was dissolved in THF / i -PrOH / H2O ( v / v / v =1 / 1 / 1), add HCl (1M, 2.0 equiv), Pd / C (25% wt.) and Pd(OH)2 / C (25% wt.), evacuate the reaction system and fill it with H2, repeat three times, react at room temperature for 12 h, TLC monitoring showed that the starting material reaction was complete, filter to remove insoluble solids, concentrate the filtrate under reduced pressure and freeze dry to obtain white foamy compounds 1, 2, 3, 4, 5, 6.

[0090] Compound 1: 1 H NMR (500 MHz, Deuterium Oxide) d 5.38–4.98 (m, 27H), 4.27–3.42 (m, 159H), 2.99 (t, J = 7.6 Hz, 2H), 1.75–1.60 (m, 5H), 1.41 (s, 5H); 13 CNMR (126 MHz, Deuterium Oxide) d102.24, 102.20, 102.1, 100.5, 98.4, 98.2, 98.0, 97.9, 97.7, 97.6, 78.7, 78.5, 77.8, 73.33, 73.31, 73.2, 73.1, 72.7, 71.4, 71.0, 70.5, 70.4, 70.2, 70.1, 69.99, 69.91, 69.5, 69.4, 69.3, 69.0, 68.3, 68.1, 66.9, 66.8, 66.7, 66.5, 66.3, 65.2, 61.0, 60.9, 39.4, 27.7, 26.6, 25.3, 24.9.

[0091] Compound 2: 1 H NMR (400 MHz, Deuterium Oxide) d 5.28 (d, J = 25.8 Hz,2H), 5.20–4.91 (m, 16H), 4.23 (t, J = 2.6 Hz, 2H), 4.16–3.47 (m, 109H), 3.00(t, J = 7.6 Hz, 2H), 1.76–1.59 (m, 6H), 1.42 (s, 5H), 1.35–1.19 (m, 2H); 13 CNMR (126 MHz, Deuterium Oxide) d 102.26, 102.21, 102.1, 98.4, 78.8, 77.8, 73.3, 73.26, 73.20, 72.7, 70.2, 70.1, 69.9, 69.5, 69.46, 69.42, 69.3, 69.0, 68.3, 68.1, 66.88, 66.83, 66.78, 66.71, 66.2, 61.07, 61.04, 60.9, 39.4, 26.6, 24.9.

[0092] Compound 3: = +39.6( c = 0.10, H2O); 1 H NMR (400 MHz, D2O) d 5.22 (d, J= 1.8 Hz, 1H), 5.06 (s, 2H), 5.01 (s, 1H), 4.97–4.94 (m, 2H), 4.93 (d, J =1.9 Hz, 1H), 4.86 (d, J = 3.2 Hz, 1H), 4.15 (t, J = 2.5 Hz, 1H), 4.06–3.47(m, 49H), 2.92 (t, J = 7.6 Hz, 2H), 2.01 (s, 4H), 1.65–1.53 (m, 4H), 1.39–1.29 (m, 4H); 13 C NMR (126 MHz, D2O) d 176.7, 102.23, 102.22, 102.17, 100.7,98.3, 98.2, 98.1, 97.7, 78.8, 78.6, 78.5, 77.9, 73.3, 73.2, 72.8, 71.0, 70.6,70.3, 70.2, 70.1, 70.0, 69.9, 69.6, 69.5, 69.4, 69.0, 68.4, 68.2, 67.0, 66.9,66.8, 66.6, 66.3, 65.2, 61.5, 61.1, 61.0, 60.9, 39.4, 28.4, 27.8, 26.7, 25.4,25.0; HRMS (ESI) m / z Calcd for C 54 H 96 O 41 N [M+H] + 1414.5452, found 1414.5464。

[0093] Compound 4: = +34.3 ( c = 0.10, H2O); 1 H NMR (400 MHz, D2O) d 5.07 (d, J = 1.8 Hz, 1H), 5.05 (d, J = 1.7 Hz, 1H), 4.96 (d, J = 1.8 Hz, 1H), 4.93 (s,2H), 4.91 (d, J= 3.8 Hz, 1H), 4.89 (d, J = 3.7 Hz, 1H), 4.82 (s, 1H), 4.15(t, J = 2.5 Hz, 1H), 4.09–3.98 (m, 5H), 3.96–3.75 (m, 26H), 3.74–3.43 (m,25H), 2.92 (t, J = 7.6 Hz, 2H), 1.65–1.50 (m, 4H), 1.39–1.30 (m, 4H); 13 C NMR(126 MHz, D2O) d 102.3, 102.18, 102.17, 99.4, 98.18, 98.17, 97.9, 97.8, 78.8,78.6, 77.8, 73.3, 72.9, 72.8, 71.6, 70.9, 70.8, 70.5, 70.3, 70.2, 70.0, 69.9,69.6, 69.5, 69.4, 69.3, 69.1, 68.5, 68.4, 67.8, 66.9, 66.8, 66.7, 66.5, 66.4,66.3, 66.1, 65.9, 65.4, 61.1, 60.9, 60.8, 39.4, 28.4, 26.6, 25.4, 24.9; HRMS(ESI) m / z Calcd for C 54 H 96 O 41 N [M+H] + 1414.5452, found 1414.5477。

[0094] Compound 5: = +42.5 ( c = 0.10, H2O); 1 H NMR (400 MHz, D2O) d 5.06(brs, 2H), 5.01 (s, 1H), 4.98–4.94 (m, 2H), 4.93 (s, 1H), 4.89 (d, J = 3.7Hz, 1H), 4.78 (s, 1H), 4.15 (t, J = 2.5 Hz, 1H), 4.08–3.43 (m, 49H), 2.90 (t,J = 7.6 Hz, 2H), 1.66–1.48 (m, 4H), 1.39–1.25 (m, 4H); 13 C NMR (101 MHz, D2O) d 102.23, 102.22, 102.1, 99.9, 98.4, 98.1, 97.8, 97.7, 81.7, 78.8, 78.4, 77.9, 73.3, 72.8, 71.1, 70.9, 70.5, 70.4, 70.3, 70.2, 70.1, 70.0, 69.9, 69.6, 69.5, 69.4, 69.2, 69.1, 68.4, 67.9, 66.9, 66.7, 66.6, 66.4, 66.3, 65.4, 65.3, 61.1, 60.9, 39.4, 28.2, 26.6, 25.4, 24.9; HRMS (ESI) m / z Calcd for C 54 H 95 O 41 NNa[M+Na] + 1436.5272, found 1436.5254。

[0095] Compound 6: = +21.0 ( c = 0.10, H2O); 1 H NMR (400 MHz, D2O) d 5.17 (s,1H), 5.06 (br s, 2H), 4.98 (d, J = 1.8 Hz, 1H), 4.94–4.90 (m, 4H), 4.15 (t, J = 2.6 Hz, 1H), 4.07 – 3.48 (m, 49H), 2.92 (t, J = 7.6 Hz, 2H), 1.65–1.49 (m,4H), 1.36–1.27 (m, 4H); 13 C NMR (101 MHz, D2O) d102.26, 102.21, 102.17, 100.6, 98.4, 98.2, 98.1, 97.8, 78.8, 78.3, 78.0, 77.9, 73.5, 73.3, 72.8, 71.6, 71.1, 71.0, 70.7, 70.5, 70.3, 70.2, 70.0, 69.9, 69.5, 69.4, 69.2, 69.1, 68.5, 68.4, 68.0, 66.9, 66.7, 66.6, 66.3, 61.1, 60.9, 39.4, 28.2, 26.6, 25.4, 24.9; HRMS (ESI) m / z Calcd for C 54 H 96 O 41 N [M+H] + 1414.5452, found 1414.5426.

[0096] Example 10: Cellular Immunoassay

[0097] (1) Experimental method:

[0098] Based on the reported potent endotoxin tolerance-like effects of structural mimics, this invention further evaluated the inhibitory levels of the aforementioned synthetic galactomannan oligosaccharides and polysaccharides on cytokine production. To assess the immunomodulatory activity of the synthetic galactomannan oligosaccharides or polysaccharides, RAW264.7 cells were cultured at 3.0 × 10⁶ cells per well. 4 The cells were seeded at a density of [number] cells / well in 96-well plates and incubated for 24 hours. The culture medium was then replaced with a medium supplemented with 25 [units of something unspecified]. m M or 100 m M-synthesized compounds 1-6 were cultured in fresh DMEM medium for 24 hours, followed by stimulation with lipopolysaccharide (5.0 ng / mL) for 4 hours. The culture supernatant was collected and centrifuged at 12,000 × g for 5 minutes at 4°C. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were quantitatively determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (MULTISKAN GO, Thermo Fisher Scientific).

[0099] (2) Experimental results:

[0100] The results are as follows Figure 1 and Figure 2 As shown: From Figure 1It can be seen that the octasaccharide 3–6 has a significantly stronger inhibitory effect on IL-6 secretion than the tetracosaccharide 1 and hexadecaccharide 2, with an inhibition rate of up to 90.76% compared to the control group LPS. Figure 2 It can be seen that the octasaccharide 3–6 has a stronger inhibitory effect on TNF-α secretion than the tetracosaccharide 1 and hexadecaccharide 2, and its inhibition rate on TNF-α production can reach 57.35% compared with the control group LPS. The above results show that the synthesis of galactomannan oligosaccharides or polysaccharides (25... m M and 100 m M) significantly inhibited LPS-induced IL-6 and TNF-α production in a concentration-dependent manner.

[0101] Furthermore, this invention evaluated the ability of the aforementioned compounds to inhibit LPS-induced pathway activation. RAW264.7 cells were first treated with octasaccharide compound 4 (100 μM) for 24 hours, then stimulated with 5 ng / mL LPS for 45 minutes. Western blot analysis was performed on p38, p-p38, p65, and p-p65 in the RAW264.7 cell lysates. As shown in Figure 3, the experimental results showed that LPS significantly increased the levels of p-p38 and p-p65, but did not affect their total protein expression. Therefore, *Antrodia camphorata* oligosaccharides and polysaccharides, without affecting total protein expression, can significantly inhibit LPS-induced IL-6 and TNF-α production in a concentration-dependent manner, providing a promising glycosyl therapy for alleviating the severe inflammatory response caused by endotoxins. This provides crucial evidence for the mechanism of interaction between structurally precise *Antrodia camphorata* galactomannan and immune receptors, and promotes the design of immunotherapeutic drugs based on glycosides.

[0102] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for synthesizing Ganoderma annosum galactomannan oligosaccharides and polysaccharides, characterized in that, The method comprises the following steps: Step one: synthesis of glycosyl donor 13 and glycosyl acceptor 14, 15 The protected tetrasaccharide compound 7 is hydrolyzed to remove the isopropylthio group at the anomeric position, and then reacted with N-phenyltrifluorimine chloride to obtain the tetrasaccharide donor 13; The protected tetrasaccharide compound 8 is hydrolyzed to remove the isopropylthio group at the anomeric position to obtain the compound 14; the protected tetrasaccharide compound 8 is directly reacted with the azido hexanol acceptor under the catalysis of a promoter to obtain the acceptor 15 containing a linking arm; ； Step two: synthesis of octasaccharide 16, 17, 18, 19, 20 and hexadecasaccharide 23, tetracosasaccharide 25 The glycosyl donor 7, 13 or 22 and the glycosyl acceptor 8, 9, 10, 11, 14, 21 or 24 are dissolved in a solvent, molecular sieves and a promoter are added, and then reacted to obtain the octasaccharide 16, 17, 18, 19, 20 and the hexadecasaccharide 23, tetracosasaccharide 25, respectively; ； Step three: removal of the protecting group The octasaccharide 16, 17, 18, 19 and the hexadecasaccharide 23, tetracosasaccharide 25 containing the linking arm are first subjected to hydrofluoric acid to remove the silicon group, then subjected to NaH to remove the ester group, and finally subjected to Pd catalytic hydrogenation to remove the benzyl group and the 2-naphthylmethyl group, so as to obtain the octasaccharide 3, 4, 5, 6 and the hexadecasaccharide 2, tetracosasaccharide 1 with all hydroxyl groups exposed; 。 2. The method for synthesizing Antrodia camphorata galactomannan oligosaccharides and polysaccharides as described in claim 1, characterized in that, The reagent for the hydrolysis in step one is dibromohydantoin.

3. The method for synthesizing Antrodia camphorata galactomannan oligosaccharides and polysaccharides as described in claim 1, characterized in that, The promoter in step one and step two is selected from one or more of trimethylsilyl triflate and N-iodosuccinimide.

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