CN106432371A - Beta-1,2-D-oligomeric mannoprotein conjugates and preparation method and application thereof - Google Patents

Abstract

The present invention discloses a β-1,2-D-oligomannose protein conjugate as shown in formula I and its preparation method and application, In the formula, n is 0, 1 or 2, and R is the carrier protein KLH or HSA. The invention uses α-D-glucose as a raw material to synthesize phosphorylated β-1,2-D-oligomannan oligosaccharide, and couples it with a carrier protein to obtain a glycoprotein conjugate. The prepared compound was used as an anti-Candida vaccine to immunize mice, and the results showed that the compound of the present invention can induce a strong immune response in the body, wherein the immunogenicity of the β-1,2-D-mannotriose protein conjugate The strongest and produced immune serum can specifically recognize Candida albicans cell surface antigens, thereby preventing Candida albicans infection, and the β-1,2-D-oligomannoprotein conjugate of the present invention has Strong application value.

Description

β-1,2-D-oligomannose protein conjugate and its preparation method and application

technical field

The invention relates to the technical field of antifungal saccharide vaccines, in particular to β-1,2-D-oligomannose protein conjugates and a preparation method thereof. The glycoprotein conjugates are used as vaccines in the prevention and treatment of Candida albicans infection. application in therapy.

Background technique

In recent years, the mortality rate caused by Candida albicans infection has been increasing, and the bacteria have developed serious resistance to antibacterial drugs. Effective drugs and strategies for the prevention and treatment of Candida albicans infection are needed clinically. The immunotherapy of fungal infection mainly adopts active immunotherapy, which uses antigens to stimulate the body to produce a strong immune response, and achieves the purpose of clearing infected fungi or other microorganisms by producing antibodies or differentiating effector cells, with less toxic side effects and significant curative effect Features. β-1,2-mannan is a T-cell-dependent polysaccharide antigen that exists on the surface of Candida albicans cells. They are usually coupled with carrier proteins (BSA/TT) to form glycoprotein vaccines. Studies have found that such vaccines can induce The body produces protective antibodies, but the immune response is limited. We intend to couple synthetic β-1,2-mannobiose, trisaccharides and tetrasaccharides to the strong immunogenic carrier protein keyhole limpet chrysocyanin (KLH) via a phosphoethanolamine group (natural glycoprotein linkage) Form glycoprotein conjugates in order to obtain highly immunogenic glycoprotein vaccines.

Contents of the invention

The first object of the present invention is to provide a β-1,2-D-oligomannoprotein conjugate to address the deficiencies in the prior art.

The second object of the present invention is to provide a method for preparing the above-mentioned β-1,2-D-oligomannoprotein conjugate.

The third object of the present invention is to provide the use of the above-mentioned β-1,2-D-oligomannoprotein conjugate.

The fourth object of the present invention is to provide a vaccine against Candida albicans fungus.

For realizing above-mentioned first object, the technical scheme that the present invention takes is:

A β-1,2-D-oligomannose protein conjugate, the general structural formula of the β-1,2-D-oligomannose protein conjugate is as follows:

Wherein, n is 0, 1 or 2, and R is carrier protein KLH or HSA.

Further, the monosaccharide unit constituting the oligosaccharide is β-1,2-D-mannose.

Further, the β-1,2-D-oligomannose protein conjugate is prepared by coupling phosphorylated β-1,2-D-oligomannose with carrier protein.

Further, the β-1,2-D-oligomannose protein conjugate uses glutaryl as the linking group between the phosphorylated β-1,2-D-oligomannose and the carrier protein.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

The preparation method of the β-1,2-D-oligomannoprotein conjugate as described above comprises the following steps:

The first step is to synthesize β-1,2-D-mannobiose receptor 7 and β-1,2-D-mannotriose receptor 10:

Reaction conditions: a) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, 92.4%; b) CH ₃ ONa, CH ₃ OH, 94.2%; c) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 79.5% (two-step total yield); d) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, 90.3%; e) CH ₃ ONa, CH ₃ OH, 92.7%; f) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 77.6% (two-step total yield).

The second step is to synthesize phosphorylated β-1,2-D-oligomannose 19a-c:

Reaction conditions: a) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, (12a, 84.3%; 12b, 89.5%; 12c, 81.5%); b) CH ₃ ONa, CH ₃ OH, 91.3-95.5%; c) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 79.6-84.5% (two-step total yield); d) BnBr, NaH, TBAI, 0°C, 83.7-89.4%; e) TBAF, THF, 83.4-88.2%; f ) i) Tetrazole, CH ₂ Cl ₂ /CH ₃ CN; ii) tert-BuOOH, 79.3-87.5% (two-step total yield); g) DBU, CH ₂ Cl ₂ , 86.3-93.2%.

The third step is to synthesize β-1,2-D-oligomannoprotein conjugates 1a-c, 2a-c:

Reaction conditions: a) H ₂ , Pd(OH) ₂ /C, CH ₂ Cl ₂ /MeOH (1:1, v/v), 24 hours, 88.7-93.5%; b) DMF/PBS (4:1, v/v), 4 hours; c) PBS, 2.5 days.

Further, the preparation method of phosphorylated β-1,2-D-oligomannose is as follows:

(1) Preparation of β-1,2-D-mannose monosaccharide acceptor and 3,4,6-tribenzyl-2-acetoxyglucose by using α-D-glucose as raw material under the action of reaction assistant trichloroacetimide ester donor;

(2) The monosaccharide donor and monosaccharide acceptor prepared above undergo a glycosylation reaction under the action of a catalyst (TMSOTf) to prepare a disaccharide, and prepare β-1,2 by configuration inversion at the 2'-position of the disaccharide -D-mannobiose acceptor; β-1,2-D-mannobiose acceptor and monosaccharide donor prepare β-1,2-D-mannotriose acceptor by the aforementioned method;

(3) β-1,2-D-mannose acceptor and 6-tert-butyldiphenylsilyl-3,4-dibenzyl-2-acetoxyglucose trichloroacetimidate donor The corresponding β-1,2-D-oligomannose is prepared by glycosylation; the 6-position of the non-reducing end of the oligomannose is removed from the TBDPS protection and reacted with a phosphorylation reagent to obtain phosphorylated β-1,2- D-oligomannose.

Further, the method for preparing phosphorylated β-1,2-D-oligomannose comprises the following steps:

(1) react phosphorylated β-1,2-D-oligomannose with hydroxysuccinimide glutarate to prepare β-1,2-D-oligomannose activated ester;

(2) Coupling the activated β-1,2-D-oligomannose ester prepared in step (1) with a carrier protein under weak alkaline conditions to obtain β-1,2-D-oligomannose protein conjugate.

For realizing above-mentioned 3rd purpose, the technical scheme that the present invention takes is:

Use of any one of the β-1,2-D-oligomannoprotein conjugates described above in the preparation of medicaments for preventing and/or treating fungal infections. The fungus is Candida albicans.

For realizing above-mentioned 4th object, the technical scheme that the present invention takes is:

An anti-Candida albicans fungal vaccine, comprising the therapeutically effective amount of β-1,2-D-oligomannoprotein conjugate and pharmaceutically acceptable adjuvant or adjuvant.

The present invention has the advantage that:

1. The prepared compound was used as an anti-Candida vaccine to immunize mice, and the results showed that the compound of the present invention could induce a strong immune response in the body, and the immune response of the β-1,2-D-mannotriose protein conjugate The immune serum with the strongest originality and produced can specifically recognize the cell surface antigen of Candida albicans, so as to prevent the infection of Candida albicans. The β-1,2-D-oligomannoprotein conjugated It has strong application value.

Description of drawings

Accompanying drawing 1 is the general formula of β-1,2-D-oligomannoprotein conjugate.

Accompanying drawing 2 is the synthesis route of β-1,2-D-mannobiose and β-1,2-D-mannotriose receptors. Reaction conditions: a) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, 92.4%; b) CH ₃ ONa, CH ₃ OH, 94.2%; c) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 79.5% (two-step total yield); d) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, 90.3%; e) CH ₃ ONa, CH ₃ OH, 92.7%; f) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 77.6% (two-step total yield).

Accompanying drawing 3 is the synthesis route of phosphorylated β-1,2-D-oligomannose disaccharide and trisaccharide. Reaction conditions: a) TMSOTf, CH ₂ Cl ₂ , Molecular sieves, -40°C, (12a, 84.3%; 12b, 89.5%; 12c, 81.5%); b) CH ₃ ONa, CH ₃ OH, 91.3-95.5%; c) i) DMSO, Ac ₂ O; ii) L-selectride, THF, -78°C, 79.6-84.5% (two-step total yield); d) BnBr, NaH, TBAI, 0°C, 83.7-89.4%; e) TBAF, THF, 83.4-88.2%; f ) i) Tetrazole, CH ₂ Cl ₂ /CH ₃ CN; ii) tert-BuOOH, 79.3-87.5% (two-step total yield); g) DBU, CH ₂ Cl ₂ , 86.3-93.2%.

Accompanying drawing 4 is the synthetic route of β-1,2-D-oligomannose disaccharide, trisaccharide, tetraglycoprotein conjugate. Reaction conditions: a) H ₂ , Pd(OH) ₂ /C, CH ₂ Cl ₂ /MeOH (1:1, v/v), 24 hours, 88.7-93.5%; b) DMF/PBS (4:1, v/v), 4 hours; c) PBS, 2.5 days.

Accompanying drawing 5 is the MALDI-TOF-MS spectrogram of compound 2a.

Accompanying drawing 6 is the MALDI-TOF-MS spectrogram of compound 2b.

Accompanying drawing 7 is the MALDI-TOF-MS spectrogram of compound 2c.

Accompanying drawing 8 is the total antibody titer in serum detected after compound 1a-c immunized mice, wherein +Adj represents the adjuvant-added group.

Accompanying drawing 9 is the immunofluorescent picture of the combination of the serum obtained after the third immunization of mice with compound 1b and Candida albicans cells.

Accompanying drawing 10 is the flow cytogram of the combination of the serum obtained after the third immunization of mice with compound 1b and Candida albicans cells.

detailed description

The present invention will be further described below in combination with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The reaction route for the preparation of the β-1,2-D-oligomannose protein conjugate of the present invention (the general structural formula is shown in Figure 1) (Figure 2, Figure 3, Figure 4):

The first step, synthesis of β-1,2-D-mannobiose (receptor 7), β-1,2-D-mannotriose receptor (receptor 10):

The second step is to synthesize phosphorylated β-1,2-D-oligomannose 19a-c:

The third step is to synthesize glycoprotein conjugates 1a-c, 2a-c:

The synthesis of embodiment 1 compound 5

Monosaccharide acceptor 3 (1.23g, 2.5mmol) (J.Org.Chem.2001, 66, 8411.), donor 4 (1.95g, 3.0mmol) (J.Carbohydr.Chem.1994, 13, 421.) and activated Molecular sieves (1.0 g) were placed in a 100 mL round bottom flask and dried under vacuum for half an hour, then the mixture was dissolved in 50 mL of anhydrous dichloromethane. The suspension was stirred at room temperature under argon protection for half an hour, then cooled to -40°C, and then trimethylsilyl triflate (35.0 μL, 0.18 mmol) was slowly added dropwise. After half an hour, the reaction solution was neutralized and quenched with triethylamine and filtered with diatomaceous earth. After concentration, the initial product obtained was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 12:1, v/v) to obtain a white foamy solid (2.23g, 92.4%).

¹ HNMR (400MHz, CDCl ₃ ) δ7.40-7.19 (m, 30H, Ar), 5.88 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.37 (m, 1H, OCH ₂ CH ＝ CH ₂ ) ,5.24-5.20(m,1H,OCH ₂ CH= CH ₂ ),5.15(dd,J=7.6Hz,9.6Hz,1H,H-2'),4.86-4.75(m,6H,H-1' , CH ₂ Ph), 4.56-4.43 (m, 7H, CH ₂ Ph), 4.42-4.39 (m, 1H , OCH ₂ CH=CH ₂ ), 4.34 (s, 1H, H-1), 4.28 (d ,J=3.2Hz,1H,H-2),4.22-4.01(m,1H,OC H ₂ CH=CH ₂ ),3.82-3.74(m,3H,H-3,H-6,H-6'),3.73-3.57(m,5H,H-4,H-4',H-5',H-6'),3.52(d,J＝3.2Hz,H-3),3.44(m,1H) ,1.97(s,3H,Ac).ESI-MS: calcd.for C ₅₉ H ₆₄ O ₁₂ [M+Na] ⁺ m/z, 987.4; found, 987.9.

The synthesis of embodiment 2 compound 6

Compound 5 (1.85g, 2.0mmol) was dissolved in 100mL of anhydrous methanol, sodium methoxide (11.0mg, 0.2mmol) was added and stirred at room temperature overnight, after the reaction was completed, it was added to ion exchange resin IR 120 (H+form) And, the resin was removed by filtration, and the initial product obtained after concentration was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 4:1, v/v) to obtain a white foamy solid (1.74g, 94.2%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.55-7.23 (m, 30H, Ar), 6.05-5.93 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.40 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.29 (m, 1H, OCH ₂ CH= CH ₂ ), 5.17 (dd, J=11.2Hz, 1H, OCH ₂ Ph), 5.05-4.93 (m, 4H, OCH ₂ Ph), 4.90 ( d,J=11.2Hz,1H,OCH ₂ Ph),4.81(d,J=7.6Hz,1H,H-1'),4.72(d,J=12.0Hz,1H,OCH ₂ Ph),4.67-4.46 (m,10H,OCH ₂ Ph,O CH ₂ CH=CH ₂ ,H-1),4.36(d,J=3.2Hz,1H,H-2),4.17-4.10(m,1H,O CH ₂ CH =CH ₂ ),4.01(t,J=9.6Hz,1H,H-4),3.88-3.71(m,6H,H-2',H-3',H-6a',H-6b',H -6a,H-6b),3.70-3.57(m,3H,H-3,H-4',H-5'),3.53-3.42(m,1H,H-5). ¹³ C NMR(100MHz, CDCl ₃ )δ139.13,138.52,138.33,138.27,138.16,138.11,133.52,128.78,128.56,128.45,128.41,128.36,128.23,128.17,128.12,128.03,127.98,127.93,127.87,127.80,127.72,127.70,127.66,127.54 _{, 117.86,104.11,99.39,85.17,80.33,77.30,75.70,75.46,75.34,75.11,74.78,74.63,74.37,73.48,70.39,69.97,69.81,69.23 .} [M+K] ⁺ m/z, 962.2; found, 930.5.

The synthesis of embodiment 3 compound 7

Compound 6 (1.38 g, 1.5 mmol) was placed in a 100 mL round bottom flask, and then dimethyl sulfoxide (22.0 mL) and acetic anhydride (11.0 mL) were added. The reaction solution was stirred at room temperature for 18 hours, extracted with ethyl acetate, washed with saturated sodium carbonate and brine respectively, then dried with anhydrous sodium sulfate, filtered and concentrated to obtain the initial product, which was spun twice with toluene. The residue was dissolved in 40 mL of anhydrous tetrahydrofuran and cooled to -78°C. Under the protection of argon, L-Selectride (1 _M THF, 7.5 mL) was slowly added dropwise and stirred for fifteen minutes, then the cooling device was removed and left at room temperature to continue Stir for fifteen minutes, quench the reaction with methanol, add 50mL dichloromethane for dilution, and use hydrogen peroxide solution (5%, 30mL), sodium hydroxide solution (1 _M , 30mL), sodium thiosulfate solution (5% , 30mL) and saturated brine (30mL), washed with anhydrous magnesium sulfate, filtered and concentrated to a pale yellow oily liquid, the resulting initial product was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 4:1, v /v) A white foamy solid was obtained (1.10 g, 79.5%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.55-7.23 (m, 30H, Ar), 5.99-5.86 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.27 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.21(m, 1H, OCH ₂ CH= CH ₂ ), 5.03(s, 1H, H-1'), 5.02(dd, J=10.8Hz, 1H, OCH ₂ Ph), 4.99(dd , J＝11.2Hz, 1H, OCH ₂ Ph), 4.94 (dd, J＝10.8Hz, 1H, OCH ₂ Ph), 4.89 (dd, J＝10.8Hz, 1H, OCH ₂ Ph), 4.69 (dd, J =10.8Hz, 1H, OCH ₂ Ph), 4.68 (dd, J = 11.6Hz, 1H, OCH ₂ Ph), 4.64-4.59 (m, 2H, OCH ₂ Ph), 4.57 (d, J = 3.2Hz, 1H , H-2), 4.56-4.48(m, 4H, OCH ₂ Ph), 4.47(s, 1H, H-1), 4.46-4.43(m, 1H, OCH ₂ CH= CH ₂ ), 4.41(d ,J=3.2Hz,1H,H-2'),4.11-4.06(m,1H,O CH ₂ CH=CH ₂ ),3.98(t,J=9.2Hz,1H,H-4'),3.91- 3.70(m,5H,H-6a,H-4,H-6a',H-6b,H-6b'),3.67-3.54(m,3H,H-3',H-3,H-5' ),3.51-3.44(m,1H,H-5) ^.13 C NMR(100MHz,CDCl ₃ )δ138.44,138.31,138.18,133.83,128.38,128.27,128.16,128.01,127.95,127.78,127.72,117.615, 100.15,99.35,81.42,80.34,75.6,75.14,74.44,74.07,73.50,73.39,70.85,70.75,70.07,69.40,67.70.ESI-MS: calcd.for C ₅₇ H ₆₂ O ₁₁ [M+Li] ⁺ m /z,930.1;found,930.5.

The synthesis of embodiment 4 compound 8

Disaccharide acceptor 7 (0.95g, 1.0mmol), monosaccharide donor 4 (0.78g, 1.2mmol) and activated Molecular sieves (0.25 g) were placed in a 50 mL round bottom flask and dried under vacuum for half an hour, then the mixture was dissolved in 30 mL of anhydrous dichloromethane. The suspension was stirred at room temperature under the protection of argon for half an hour, then cooled to -40°C, and then trimethylsilyl triflate (14.0 μL, 0.075 mmol) was slowly added dropwise. After half an hour, the reaction solution was neutralized and quenched with triethylamine and filtered with diatomaceous earth. After concentration, the initial product obtained was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 12:1, v/v) to obtain a white foamy solid (1.26g, 90.3%).

¹ H NMR (600MHz, CDCl ₃ ) δ7.42-7.02 (m, 45H, Ar), 5.86 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.26 (d, J = 8.0Hz, 1H, H- 1"),5.20-5.17(m,2H,H-2",OCH ₂ CH= CH ₂ ),5.11-5.07(m,1H,OCH ₂ CH= CH ₂ ),4.98-4.94(m,3H , OCH ₂ Ph), 4.85-4.80 (m, 2H, OCH ₂ Ph), 4.75-4.69 (m, 4H, H-1', OCH ₂ Ph), 4.63 (d, J=13.2Hz, 1H, OCH ₂ Ph), 4.56-4.47(m, 9H, H-2', OCH ₂ Ph), 4.42(d, J=12.0Hz, 1H, OCH ₂ Ph), 4.41(s, 1H, H-1), 4.38- 4.35(m,1H,OCH ₂ CH=CH ₂ ),4.20(d,J=3.0Hz,1H,H-2),3.91(t,J=8.4Hz,1H,H-3"),3.82-3.66 (m,8H,H-4",H-5',H-5",H-6a",H-6a,H-6b,H-6a',H-6b'),3.62(t,J= 9.6Hz,1H,H-4'),3.54-3.46(m,3H,H-3,H-3',H-6a"),3.37(m,1H,H-5).MALDI-TOF-MS :calcd.for C ₈₆ H ₉₂ O ₁₇ [M+Na] ⁺ m/z, 1419.6, found 1420.0.

The synthesis of embodiment 5 compound 9

Compound 8 (1.02g, 0.75mmol) was dissolved in 50mL of anhydrous methanol, sodium methoxide (11.0mg, 0.2mmol) was added and stirred at room temperature overnight, after the reaction was completed, it was added to ion exchange resin IR 120 (H+form) And, the resin was removed by filtration, and the initial product obtained after concentration was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v/v) to obtain a white foamy solid (0.94g, 92.7%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.56-7.08 (m, 45H, Ar), 6.01-5.89 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.29 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.24(m,1H,OCH ₂ CH= CH ₂ ),5.16-5.02(m,6H,OCH ₂ Ph,H-1'),4.96(d,J=10.8Hz,1H,OCH ₂ Ph), 4.88 (d, J=8.0Hz, 1H, H-1"), 4.81-4.66 (m, 3H, OCH ₂ Ph), 4.65-4.41 (m, 13H, OCH ₂ Ph, H-2', H-2,O CH ₂ CH=CH ₂ ,H-1),4.15-4.07(m,1H,O CH ₂ CH=CH ₂ ),4.01-3.71(m,9H,H-2",H-3 ",H-4',H-6b",H-6a",H-6a,H-6b,H-6a',H-6b'),3.68-3.56(m,5H,1H,H-5",H-3',H-4",H-3,H-5),3.52-3.44(m,1H,H-5'). ¹³ C NMR(100MHz,CDCl ₃ )δ139.21,138.68,138.59,138.44 ,138.39,138.16,138.11,133.79,128.50,128.41,128.36,128.31,128.25,128.20,128.11,128.06,127.92,127.86,127.83,127.73,127.69,127.64,127.59,127.48,127.43,127.27,117.43,105.35,100.08 . :calcd.for C ₈₄ H ₉₀ O ₁₆ [M+Na] ⁺ m/z, 1378.617; found, 1378.115.

The synthesis of embodiment 6 compound 10

Compound 9 (678.0 mg, 0.5 mmol) was placed in a 50 mL round bottom flask, and then dimethyl sulfoxide (7.0 mL) and acetic anhydride (3.5 mL) were added. The reaction solution was stirred at room temperature for 18 hours, extracted with ethyl acetate, washed with saturated sodium carbonate and brine respectively, then dried with anhydrous sodium sulfate, filtered and concentrated to obtain the initial product, which was spun twice with toluene. The residue was dissolved in 20 mL of anhydrous tetrahydrofuran and cooled to -78°C. Under the protection of argon, L-Selectride (1 _M THF, 2.45 mL) was slowly added dropwise and stirred for fifteen minutes, then the cooling device was removed and left at room temperature to continue Stir for fifteen minutes, add 20mL dichloromethane to dilute after quenching the reaction with methanol, and use hydrogen peroxide solution (5%, 20mL), sodium hydroxide solution (1 _M , 20mL), sodium thiosulfate solution (5% , 20mL) and saturated brine (20mL), washed with anhydrous magnesium sulfate, filtered and concentrated to a pale yellow oily liquid, the resulting initial product was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v /v) A white foamy solid (526.3 mg, 77.6%) was obtained.

¹ H NMR (400MHz, CDCl ₃ ) δ7.66-6.94 (m, 45H, Ar), 5.95-5.84 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.25 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.20(s,1H,H-1"), 5.19(m,1H,OCH ₂ CH= CH ₂ ),5.17(s,1H,H-1'),5.06-4.96(m,4H , OCH ₂ Ph), 4.90 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.75 (br s, 1H, H-2'), 4.71-4.64 (m, 2H, OCH ₂ Ph, H-2 ), 4.61-4.42 (m, 10H, OCH ₂ Ph, O CH ₂ CH = CH ₂ , H-1), 4.38 (d, J = 10.8Hz, 1H, OCH ₂ Ph), 4.35 (br s, 1H, H-2"),4.28-4.21(m,1H,OCH ₂ Ph),4.10-3.89(m,4H,H-4",H-4',OCH ₂ Ph),3.88-3.49(m,13H, H-6a",H-6b",H-5",H-3",H-6a',H-6b',H-5',H-3',H-6a,H-6b,H- 5,H-4,H-3),3.47-3.41(m,1H,H-5). ¹³ C NMR(100MHz,CDCl ₃ )δ138.56,138.48,138.26,138.20,138.15,138.10,138.02,137.88,133.62 ,129.16,128.44,128.38,128.35,128.29,128.25,128.19,128.11,128.05,127.94,127.79,127.74,127.69,127.65,127.60,127.51,127.45,127.34,127.18,117.42,100.69,100.06,83.12,80.48,80.35 ,75.42,75.35,75.22,75.17,75.07,74.78,74.43,74.34,73.52,73.44,73.36,71.38,70.34,70.09,70.03,69.95,69.80,69.67,69.49,69.42,678.9 :calcd.for C ₈₄ H ₉₀ O ₁₆ [M+Na] ⁺ m/z, 1378.617; found, 1379.306.

The synthesis of embodiment 7 compound 12a

Monosaccharide acceptor 3 (245mg, 0.5mmol), donor 11 (471.0mg, 0.6mmol) (Eur.J.Org.Chem.1999,2523.) and activated Molecular sieves (200 mg) were placed in a 25 mL round bottom flask and dried under vacuum for half an hour, then the mixture was dissolved in 10 mL of anhydrous dichloromethane. The suspension was stirred at room temperature under the protection of argon for half an hour, then cooled to -40°C, and then trimethylsilyl triflate (6.35 μL, 0.035 mmol) was slowly added dropwise. After half an hour, the reaction solution was neutralized and quenched with triethylamine and filtered with diatomaceous earth. After concentration, the initial product obtained was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 18:1, v/v) to obtain a white foamy solid (478.5 mg, 84.3%).

¹ H NMR (600MHz, CDCl ₃ ) δ7.71-7.01 (m, 35H, Ar), 5.99-5.91 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.36 (m, 1H, OCH ₂ CH ＝ C H ₂ ),5.21(m,1H,OCH ₂ CH= CH ₂ ),5.16(dd,J=9.0,8.0Hz,1H,H-2'),5.02(d,J=8.0Hz,1H,H -1'),4.97-4.91(m,2H,OCH ₂ Ph),4.81-4.73(m,3H,OCH ₂ Ph),4.59(brs,2H),4.49-4.41(m,5H,OCH ₂ Ph, O CH ₂ CH=CH ₂ ,H-1), 4.38(d,J=3.6Hz,1H,H-2),4.08-4.01(m,2H,O CH ₂ CH=CH ₂ ,H-6a') ,3.65-3.60(m,1H,H-6b'),3.79(br d,J=9.6Hz,1H,H-6a),3.75(t,J=9.0Hz,1H,H-3'),3.64 -3.59(m,2H,H-6b,H-4),3.56-3.49(m,4H,H-3,H-4',H-5'),3.48-3.43(m,1H,H-5 ),1.95(s,3H),1.04(s,9H). ¹³ C NMR(150MHz,CDCl ₃ )δ169.9,138.5,138.4,137.9,137.7,135.7,135.6,133.9,133.4,133.3,129.7,129.6,128.3 ,128.28,128.25,128.2,128.0,127.9,127.8,127.7,127.69,127.65,127.6,127.5,127.4,116.5,100.9,99.8,83.2,79.8,77.9,76.6,75.5,775.2.5,46 , 73.2 ^, 71.7, 70.4, _69.41 , _69.37 , _64.0 , 26.9, 21.4, 19.3.

The synthesis of embodiment 8 compound 12b

Prepared from compound 7 and compound 11, the specific operation steps are the same as the synthesis of compound 12a, and the yield is 89.5%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.64-6.87 (m, 50H, Ar), 5.87-5.78 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.36 (d, J = 8.4Hz, 1H, H-1”), 5.21(dd, J=9.0, 8.4Hz, 1H, H-2”), 5.20(br d, J=10.8Hz, 1H, OCH ₂ CH= CH ₂ ), 5.09(br d , J=10.2Hz, 1H, OCH ₂ CH= CH ₂ ), 4.98-4.89 (m, 3H, OCH ₂ Ph), 4.85 (d, J=12.0Hz, 1H, OCH ₂ Ph), 4.77-4.72 ( m, 2H, OCH ₂ Ph, H-1'), 4.68 (q, J = 11.4Hz, 2H, OCH ₂ Ph), 4.63 (d, J = 12.8Hz, 1H, OCH ₂ Ph), 4.59-4.44 ( m,8H,OCH ₂ Ph,H-2'),4.43-4.36(m,3H,OCH ₂ Ph,O CH ₂ CH=CH ₂ ,H-1),4.23(d,J=2.7Hz,1H, H-2), 4.02-3.95 (m, 3H, O CH ₂ CH=CH ₂ , H-6a', H-4), 3.91 (t, J=9.0Hz, 1H, H-3"), 3.88- 3.84(m,1H,H-6b"),3.79-3.58(m,7H,H-6a',H-6b',H-6a,H-6b,H-5',H-4",H- 4'),3.54-3.45(m,3H,H-3',H-3,H-5"),3.37-3.33(m,1H,H-5),1.96(s,3H),1.02(s ,9H). ¹³ C NMR (150MHz, CDCl ₃ ) δ170.6, 138.6, 138.5, 138.3, 138.25, 138.22, 138.2, 138.1, 138.0, 135.7, 135.5, 133.9, 133.5, 133.1, 116.8, 102.2, 1309.0 , _{80.4,79.7,77.9,76.6,75.4,75.2,74.9,74.7,74.6,74.4,73.5,73.2,69.7,69.6,69.2,68.9,63.9,26.9,21.2,19.2} . H ₁₀₄ O ₁₇ Si[M+Na] ⁺ m/z, 1567.6942; found, 1567.6924.

The synthesis of embodiment 9 compound 12c

Prepared from compound 10 and compound 11, the specific operation steps are the same as the synthesis of compound 12a, and the yield is 81.5%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.46-6.74 (m, 65H, Ar), 5.79-5.73 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.56 (d, J = 7.8Hz, 1H, H-1"'), 5.23(t, J=9.0, 7.8Hz, 1H, H-2"'), 5.16(s, 1H, H-1"), 5.16-5.09(m, 3H, OCH ₂ CH =CH ₂ , H -1",H-1'),5.06(m,1H,OCH ₂ CH= CH ₂ ),5.02(d,J=11.4Hz,2H,OCH ₂ Ph),4.95(d ,J=12.0Hz,1H,OCH ₂ Ph), 4.91(d,J=10.8Hz,1H,OCH ₂ Ph),4.85(d,J=10.8Hz,1H,OCH ₂ Ph),4.83(d,J =3.0Hz, 1H, H-2"), 4.80-4.73 (m, 3H, OCH ₂ Ph), 4.66 (q, J = 11.4Hz, 2H, OCH ₂ Ph), 4.57-4.60 (m, 3H, OCH ₂ Ph,H-2'),4.52-4.36(m,10H,OCH ₂ Ph,H-2),4.34-4.30(m,1H,O CH ₂ CH=CH ₂ ),4.27-4.24(m,2H ,OCH ₂ Ph,H-1),4.01-3.97(m,2H,H-4',H-4"'),3.90-3.85(m,2H,O CH ₂ CH=CH ₂ ,H-3"'),3.82-3.60(m,14H),3.56-3.54(m,1H,H-3'),3.44-3.38(m,2H,H-3,H-5'),3.31-3.28(m, 1H,H-5),1.94(s,3H),0.94(s,9H). ¹³ C NMR(150MHz,CDCl ₃ )δ170.6,138.67,138.6,138.5,138.4,138.3,138.2,137.9,137.8,137.3, 135.64,135.62,133.5,133.1,132.8,129.4,129.3,128.4,128.3,128.3,128.22,128.20,128.17,128.11,128.1,127.9,127.88,127.86,127.8,127.7,127.6,127.5,127.49,127.44,127.40, 127.36, 127.33, 127.2, 126.9, 117.2, 101.8, 100.7, 100.5, 1 00.4,84.0,81.90,80.6,77.4,75.8,75.5,75.3,75.2,75.1,75.00,74.9,74.8,74.7,74.5,74.4,73.65,73.4,73.3,73.1,72.7,71.2,70.6,70.2,69. 69.4, 69.3, 69.0, 68.1, 62.8, 26.9, 21.2, 19.2. HRMS (ESI): calcd. for C ₁₂₂ H ₁₃₂ O ₂₂ Si[M+Na] ⁺ m/z, 1999.8877; found, 1999.8931.

The synthesis of embodiment 10 compound 13a

Compound 12a (470mg, 4.22mmol) was dissolved in 15mL of anhydrous methanol, sodium methoxide (4.5mg, 0.84mmol) was added and stirred overnight at room temperature, after the reaction was completed, ion exchange resin IR 120 (H+form) was added to neutralize , filtered to remove the resin, and the initial product obtained after concentration was subjected to silica gel column chromatography (n-hexane/acetone, 14:1, v/v) to obtain a white foamy solid (417.5 g, 92.3%).

¹ H NMR (600MHz, CDCl ₃ ) δ7.77-7.06 (m, 35H, Ar), 6.02-5.95 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.39 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.28 (m, 1H, OCH ₂ CH= CH ₂ ), 5.13 (dd, J=11.4Hz, 1H, OCH ₂ Ph), 4.99 (d, J=10.8Hz, 1H, OCH ₂ Ph) ,4.95(d,J=12.0Hz,1H,OCH ₂ Ph),4.90(d,J=12.0Hz,1H,OCH ₂ Ph),4.88(d,J=7.6Hz,1H,H-1'), 4.85 (d, J = 11.4Hz, 1H, OCH ₂ Ph), 4.71 (d, J = 12.0Hz, 1H, OCH ₂ Ph), 4.62-4.50 (m, 6H, OCH ₂ Ph, O CH ₂ CH = CH ₂ ,H-1),4.43(d,J=3.6Hz,1H,H-2),4.15-4.10(m,1H,O CH ₂ CH=CH ₂ ),4.06(d,J=10.8Hz,1H ,H-6a'),4.02(t,J=9.6Hz,1H,H-4),3.96-3.92(m,1H,H-6b'),3.86-3.80(m,3H,H-6a,H -6b,H-2'),3.76-3.71(m,1H,H-3'),3.65-3.52(m,3H,H-3,H-4',H-5'),3.50-3.46( m,1H,H-5),1.09(s,9H). ¹³ C NMR(150MHz,CDCl ₃ )δ139.2,138.5,138.25,138.21,137.8,135.7,135.6,133.57,133.51,133.5,129.64,129.60,128.4 ,128.34,128.29,128.1,128.0,127.99,127.8,127.73,127.70,127.6,127.57,127.5,117.8,103.7,99.6,85.2,80.3,76.9,75.7,75.2,74.9,493.8,37 , 70.0, 69.1, 63.9, 26.9, 19.3. HRMS (ESI): calcd. for C ₆₆ H ₇₄ O ₁₁ Si [M+Na] ⁺ m/z, 1093.4898; found, 1093.4884.

The synthesis of embodiment 11 compound 13b

Prepared from compound 12b and sodium methoxide, the specific operation steps are the same as the synthesis of compound 13a, and the yield is 95.5%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.71-6.93 (m, 50H, Ar), 5.92-5.86 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.22 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.16(m, 1H, OCH ₂ CH= CH ₂ ), 5.04(d, J=11.4Hz, 1H, OCH ₂ Ph), 4.95(d, J=10.8Hz, 1H, OCH ₂ Ph) ,4.98-4.91(m,4H,OCH ₂ Ph,H-1'),4.84(d,J=7.8Hz,1H,H-1"),4.78(d,J=10.8Hz,1H,OCH ₂ Ph ), 4.63 (d, J = 12.6Hz, 1H, OCH ₂ Ph), 4.57 (d, J = 11.4Hz, 2H, OCH ₂ Ph), 4.54 (d, J = 3.0Hz, 1H, H-2') ,4.51(d,J=3.0Hz,1H,H-2),4.49-4.35(m,9H,OCH ₂ Ph,O CH ₂ CH=CH ₂ ,H-1),4.12-4.02(m,3H, O CH ₂ CH=CH ₂ ,H-6a",H-4),3.93-3.87(m,1H,H-6b"),3.83-3.72(m,5H,H-2",H-4', H-6a, H-6b, H-6a'), 3.71-3.64(m, 2H, H-6a', H-3"), 3.62-3.58(m, 1H, H-5'), 3.57-3.39 (m,5H,H-3',H-3,H-5',H-4",H-5),0.99(s,9H). ¹³ C NMR(150MHz,CDCl ₃ )δ139.1,138.57,138.54 ,138.1,137.9,135.6,133.7,133.5,133.4,129.6,128.8,128.4,128.3,128.29,128.23,128.22,128.17,128.13,128.01,128.0,127.77,127.72,127.7,127.56,127.52,127.4,127.3,127.2 . .HRMS(ESI):calcd.for C ₉₃ H ₁₀₂ O ₁₆ S i[M+Na] ⁺ m/z, 1525.6835; found, 1525.6777.

The synthesis of embodiment 12 compound 13c

Prepared from compound 12c and sodium methoxide, the specific operation steps are the same as those of compound 13a, and the yield is 91.3%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.64-6.86 (m, 65H, Ar), 5.83-5.76 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.31 (s, 1H, H-1") ,5.15(m,1H,OCH ₂ CH= CH ₂ ),5.09(m,1H,OCH ₂ CH= CH ₂ ),5.06(d,J=11.4Hz,1H,OCH ₂ Ph),5.01(d ,J=10.8Hz,1H,OCH ₂ Ph),4.98(s,1H,H-1'),4.97(d,J=10.8Hz,1H,OCH ₂ Ph),4.90(d,J=11.4Hz, 1H, OCH ₂ Ph), 4.85(d, J=3.0Hz, 1H, H-2'), 4.84-4.78(m, 3H, OCH ₂ Ph), 4.72(d, J=7.8Hz, 1H, H- 1"'), 4.62 (d, J=12.0Hz, 1H, OCH ₂ Ph), 4.59 (d, J=11.4Hz, 2H, OCH ₂ Ph), 4.52-4.39 (m, 10H, OCH ₂ Ph, H -2'), 4.37-4.32(m, 3H, OCH ₂ Ph, O CH ₂ CH=CH ₂ , H-1), 4.22(d, J=12.0Hz, 1H, OCH ₂ Ph), 4.18(d, J=10.8Hz, 1H, OCH ₂ Ph), 4.02(t, J=9.6Hz, 1H, H-4"), 3.95-3.88(m, 4H, O CH ₂ CH=CH ₂ , H-4', H-6a"',H-6b"'),3.85-3.80(m,2H,H-6a",H-6b"),3.75-3.55(m,10H),3.54-3.46(m,4H,H -3,H-2,H-5"',H-5"),3.33-3.30(m,1H,H-5),0.96(s,9H). ¹³ C NMR(150MHz,CDCl ₃ )δ139. 1,138.8,138.6,138.5,138.34,138.30,138.2,138.1,138.0,135.7,135.6,133.7,133.5,133.3,129.5,128.4,128.4,128.3,128.26,128.24,128.2,128.14,128.09,128.0,127.97,127.93, 127.8, 127.77, 127.73, 127.6, 127.5, 127.49, 127.4, 127.36, 127.3, 127.2, 127.0, 117.2, 105 .7, 101.4, 100.3, 99.7, 86.2, 81.0, 80.3, 79.8, 76.6, 75.8, 75.7, 75.6, 75.5, 75.4, 75.34, 75.32, 75.2, 74.8, 74.7, 74.5, 73.4, 72.2, 70.2, 70.1, 70.9 , 69.91, 69.7, 69.4, 69.2, 63.9, 26.9, 19.3. HRMS (ESI): calcd. for C ₁₂₀ H ₁₃₀ O ₂₁ Si[M+Na] ⁺ m/z, 1957.8772;

The synthesis of embodiment 13 compound 14a

Compound 13a (380.0 mg, 0.36 mmol) was placed in a 25 mL round bottom flask, then dimethyl sulfoxide (5.0 mL) and acetic anhydride (2.5 mL) were added. The reaction solution was stirred at room temperature for 18 hours, extracted with ethyl acetate, washed with saturated sodium carbonate and brine respectively, then dried with anhydrous sodium sulfate, filtered and concentrated to obtain the initial product, which was spun twice with toluene. The residue was dissolved in 20 mL of anhydrous tetrahydrofuran and cooled to -78°C. Under the protection of argon, L-Selectride (1 _M THF, 1.75 mL) was slowly added dropwise and stirred for fifteen minutes, then the cooling device was removed and left at room temperature to continue Stir for fifteen minutes, add 20mL dichloromethane to dilute after quenching the reaction with methanol, and use hydrogen peroxide solution (5%, 20mL), sodium hydroxide solution (1 _M , 20mL), sodium thiosulfate solution (5% , 20mL) and saturated brine (20mL), dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a light yellow oily liquid, the initial product was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v/ v) White foamy solid (323.5 mg, 84.5%) was obtained.

¹ H NMR (600MHz, CDCl ₃ ) δ7.69-7.07 (m, 35H, Ar), 5.92-5.84 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.26 (m, 1H, OCH ₂ CH ＝ C H ₂ ),5.16(m,1H,OCH ₂ CH= CH ₂ ),5.12(br s,1H,H-1'),4.96-4.90(m,2H,OCH ₂ Ph),4.88-4.82(m ,2H,OCH ₂ Ph),4.69(d,J=3.6Hz,1H,H-2),4.67-4.62(m,2H,OCH ₂ Ph)4.57(d,J=12.0Hz,1H,OCH ₂ Ph ),4.51-4.37(m,5H,OCH ₂ Ph,O CH ₂ CH=CH ₂ ,H-1),4.36(d,J=3.0Hz,1H,H-2'),4.09-4.03(m, 2H,O CH ₂ CH=CH ₂ ,H-6a'),3.91-3.79(m,4H,H-6b',H-4,H-4',H-6a),3.76-3.72(m,1H ,H-6b),3.64-3.57(m,2H,H-3',H-3),3.51-3.43(m,2H,H-5',H- ⁵ ),1.03(s,9H). C NMR(150MHz,CDCl ₃ )δ139.1,138.6,138.4,138.24,138.20,133.9,128.8,128.49,128.42,128.4,128.2,128.12,128.10,127.8,127.76,127.72,127.6,127.5,127.4,117.2,102.3, 100.1, 81.7, 80.7, 75.9, 75.5, 75.24, _75.20 , _74.46 , _74.42 , 74.3, 73.8, 73.6, 73.4, 70.8, 70.4, 70.0, 69.3, 62.5. [M+Na] ⁺ m/z, 1093.4898; found, 1093.4882.

The synthesis of embodiment 14 compound 14b

It is prepared from compound 13b by oxidation of acetic anhydride/dimethyl sulfoxide and reduction of L-Selectride. The specific operation steps are the same as those of compound 14a, and the yield is 81.5%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.65-6.89 (m, 50H, Ar), 5.88-5.82 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.29 (s, 1H, H-1") ,5.20(m,1H,OCH ₂ CH= CH ₂ ),5.16(m,1H,OCH ₂ CH= CH ₂ ),5.12(s,1H,H-1'),4.94(d,J=10.8 Hz, 2H, OCH ₂ Ph), 4.90 (d, J = 12.0Hz, 1H, OCH ₂ Ph), 4.85 (d, J = 10.8Hz, 1H, OCH ₂ Ph), 4.77 (d, J = 10.8Hz, 1H, OCH ₂ Ph), 4.73 (d, J=3.0Hz, 1H, H-2'), 4.63 (d, J=3.0Hz, 1H, H-2), 4.51-4.42 (m, 6H, OCH ₂ Ph, H-1), 4.41-4.37 (m, 3H, OCH ₂ Ph, O CH ₂ CH=CH ₂ ), 4.34 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.32 (d, J= 2.4Hz, 1H, H-2"), 4.22 (d, J=10.8Hz, 2H, OCH ₂ Ph), 4.06-3.97 (m, 3H, OCH ₂ Ph, O CH ₂ CH=CH ₂ , H-6a "),3.92-3.86(m,2H,H-4",H-4'),3.80-3.72(m,2H,H-6a',H-6b'),3.69-3.55(m,5H,H -6a, H-6b, H-4, H-3', H-3), 3.54-3.47(m, 3H, H-5", H-5', H-3"), 3.41-3.37(m ,1H,H-5) ^.13C NMR(150MHz,CDCl ₃ )δ138.5,138.4,138.2,138.1,138.1,138.07,138.03,137.9,135.63,135.61,133.6,133.5,133.4,129.5,128.9,128. ,128.3,128.2,128.2,128.1,128.0,127.9,127.85,127.82,127.7,127.69,127.65,127.63,127.5,127.4,127.4,127.2,127.1,117.3,100.6,100.3,99.6,83.1,80.4,79.9,76.6 ,75.4,75.3,75.1,74.8,74.8,74.5,74.1,73.4,70.8,70.3 ,70.2,70.1,69.9,69.6,69.3,69.0,67.5,64.1,26.9,19.3.HRMS(ESI):calcd.for C ₉₃ H ₁₀₂ O ₁₆ Si[M+Na] ⁺ m/z,1525.6835; found, 1525.6805.

The synthesis of embodiment 15 compound 14c

It is prepared from compound 13c by oxidation of acetic anhydride/dimethyl sulfoxide and reduction of L-Selectride. The specific operation steps are the same as those of compound 14a, and the yield is 79.6%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.69-6.95 (m, 65H, Ar), 5.85-5.78 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.47 (s, 1H, H-1"'),5.26(s,1H,H-1"),5.19(m,1H,OCH ₂ CH= CH ₂ ),5.11(m,1H,OCH ₂ CH= CH ₂ ),5.01(s,1H, H-1'),4.98(d,J＝10.8Hz,1H,OCH ₂ Ph),4.94-4.91(m,2H,OCH ₂ Ph,H-2'),4.94-4.91(m,4H,OCH ₂ Ph), 4.68 (d, J=12.0Hz, 1H, OCH ₂ Ph), 4.61-4.57 (m, 3H, OCH ₂ Ph, H-2"'), 4.50-4.39 (m, 7H, OCH ₂ Ph) ,4.38(d,J=3.0Hz,1H,H-2"),4.36-4.31(m,4H,OCH ₂ Ph,H-2,H-1,O CH ₂ CH=CH ₂ ),4.24-4.21 (m,3H,OCH ₂ Ph),4.09-4.01(m,5H,OCH ₂ Ph,H-6a"',H-6b"',H-4"',H-4"),3.95-3.92( m,1H,O CH ₂ CH=CH ₂ ),3.62-3.60(m,10H),3.56-3.49(m,5H,H-3",H-6a,H-5',H-5",H -3),3.35-3.32(m,1H,H-5),1.03(s,9H). ¹³ CNMR(150MHz,CDCl ₃ )δ138.9,138.69,138.49,138.4,138.24,138.20,138.1,138.04,138.01, 138.0,135.7,135.6,133.7,133.6,133.4,129.48,129.5,129.0,128.4,128.33,128.32,128.30,128.27,128.22,128.20,128.1,128.0,127.9,127.8,127.79,127.76,127.73,127.64,127.62, 127.5, 127.45, 127.42, 127.3, 127.2, 127.1, 127.0, 117.1, 101.8, 100.4, 100.2, 99.8, 83.1, 80.7, 80.6, 79.7, 76.6, 75.5, 75.44, 75.3, 75.2, 7475, 7.1, 7 .9,73.5,73.4,72.2,72.0,70.3,70.0,69.9,69.88,69.8,69.7,69.1,69.0,68.7,67.6,63.9,26.9,19.3.HRMS(ESI):calcd.for C ₁₂₀ H ₁₃₀ O ₂₁ Si[M+Na] ⁺ m/z, 1957.8772; found, 1957.8750.

The synthesis of embodiment 16 compound 15a

Compound 14a (85.0mg, 0.08mmol) was dissolved in 25mL N'N-dimethylformamide and tetrabutylammonium iodide (1.0mg, 0.003mmol) and benzyl bromide (19μL, 0.16mmol) were added, the reaction solution After cooling to 0°C, sodium hydride (4.8 mg, 0.12 mmol) was added and the reaction was continued for one hour under the protection of argon. After the reaction was quenched with methanol, 50 mL of dichloromethane was added to dilute, and then washed with 30 mL of saturated brine. The organic layer was washed without Dried over magnesium sulfate, filtered and concentrated to obtain the primary product, the primary product was subjected to silica gel column chromatography (n-hexane/acetone, 18:1, v/v) to obtain a white foamy solid (77.0mg, 83.7%)

¹ H NMR (600MHz, CDCl ₃ ) δ7.79-7.10 (m, 40H, Ar), 6.02-5.93 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.36 (m, 1H, OCH ₂ CH ＝ C H ₂ ), 5.25(m,1H,OCH ₂ CH= CH ₂ ),5.24(br s,1H,H-1'),5.21(d,J=10.8Hz,1H,OCH ₂ Ph),5.03- 4.91(m,3H,OCH ₂ Ph),4.79(d,J＝3.0Hz,1H,H-2),4.71-4.58(m,4H,OCH ₂ Ph,H-1),4.57-4.53(m, 1H,O CH ₂ CH=CH ₂ ), 4.52-4.44(m,1H,OCH ₂ Ph),4.30(d,J=3.0Hz,1H,H-2'),4.18-4.13(m,2H,O CH ₂ CH=CH ₂ ,H-6a'), 4.07-4.03(m,1H,O CH ₂ CH=CH ₂ ,H-6b'),3.95-3.84(m,3H,H-4,H-4 ',H-6a),3.80-3.76(m,1H,H-6b),3.73(d,J=9.2,3.6Hz,1H,H-3),3.67(d,J=9.2,3.2Hz,1H ,H-3'),3.65-3.60(m,1H,H-5'),3.58-3.54(m,1H,H-5),1.12(s,9H). ¹³ C NMR(150MHz,CDCl ₃ ) δ139.5,138.53,138.51,138.5,138.4,135.7,135.6,133.9,133.7,133.5,129.6,129.58,128.7,128.6,128.37,128.35,128.3,128.2,128.04,128.02,127.9,127.72,127.70,127.6,127.5, 127.4, 127.1, 117.1, 100.3, 100.8, 82.3, 80.5, 77.4, 75.6, 75.2, 75.1, 75.0, 74.2, 74.1, 73.7, 73.5, 70.7, 70.6, 70.2, 69.6, 69.5, 64.4, 26.9, 19.3. ESI): calcd.for C ₇₃ H ₈₀ O ₁₁ Si[M+Na] ⁺ m/z, 1183.5368; found, 1183.5321.

The synthesis of embodiment 17 compound 15b

Prepared by the reaction of compound 14b and benzyl bromide, the specific operation steps are the same as those of compound 15a, and the yield is 89.4%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.69-6.93 (m, 55H, Ar), 5.93-5.85 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.41 (s, 1H, H-1") ,5.24(m,1H,OCH ₂ CH= CH ₂ ),5.21-5.15(m,3H,OCH ₂ CH= CH ₂ ,OCH ₂ Ph,H-1'),4.94(d,J=11.4Hz ,1H,OCH ₂ Ph),4.96(d,J=10.8Hz,1H,OCH ₂ Ph),4.90-4.84(m,4H,OCH ₂ Ph),4.81-4.75(m,2H,OCH ₂ Ph,H -2'), 4.65(d, J=3.0Hz, 1H, H-2), 4.57(d, J=11.4Hz, 1H, OCH ₂ Ph), 4.52(d, J=12.0Hz, 1H, OCH ₂ Ph),4.47-4.30(m,9H,H-1,OCH ₂ Ph),4.20(br s,1H,H-2"),4.17-4.12(m,1H,OCH ₂ Ph),4.08-3.98( m, 4H, H-6a", H-6b", OCH ₂ CH =CH ₂ , OCH ₂ Ph), 3.91(t, J=10.8, 9.6Hz, 1H, H-4"), 3.84-3.76( m,2H,H-4',H-6a),3.73-3.66(m,4H,H-4,H-6b,H-6a',H-6b'),3.65-3.57(m,4H,H -3',H-3,H-3",H-5"),3.56-3.51(m,1H,H-5'),3.45-3.41(m,1H,H-5),1.01(s, 9H). ¹³ C NMR (150MHz, CDCl ₃ ) δ140.0, 138.8, 138.6, 138.53, 138.50, 138.3, 138.2, 138.1, 137.9, 135.7, 133.8, 133.7, 133.6, 129.4, 128.6, 128.4, 128.2 128.17,128.15,128.1,127.99,127.95,127.8,127.7,127.66,127.60,127.56,127.54,127.51,127.46,127.3,127.2,127.0,126.8,117.2,101.5,100.7,83.4,80.6,80.2,75.37,75.34, 75.2,75.1,74.9,74.8,74.8,74.6,74.4 ,74.3,73.4,73.3,70.8,70.5,70.2,70.18,69.9,69.7,69.1,68.9,64.4,26.9,19.3.HRMS(ESI):calcd.for C ₁₀₀ H ₁₀₈ O ₁₆ Si[M+Na] ⁺ m/z, 1615.7340; found, 1615.7274.

The synthesis of embodiment 18 compound 15c

Prepared by the reaction of compound 14c and benzyl bromide, the specific operation steps are the same as those of compound 15a, and the yield is 87.9%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.69-6.97 (m, 70H, Ar), 5.85-5.78 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.49 (s, 1H, H-1"'),5.40(s,1H,H-1"),5.25(m,1H,OCH ₂ CH= CH ₂ ),5.18(m,1H,OCH ₂ CH= CH ₂ ),5.11(d,J= 10.8Hz,1H,OCH ₂ Ph),5.03(s,1H,H-1'),4.99(d,J=11.4Hz,1H,OCH ₂ Ph),4.94-4.91(m,2H,OCH ₂ Ph, H-2'),4.87-4.77(m,6H,OCH ₂ Ph),4.65(d,J=3.0Hz,1H,H-2"'),4.59(d,J=12.0Hz,1H,OCH ₂ Ph), 4.54-4.34 (m, 13H, OCH ₂ Ph, O CH ₂ CH=CH ₂ , H-2, H-1), 4.32 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.26 ( d,J=3.0Hz,1H,H-2"),4.24(d,J=10.8Hz,1H,OCH ₂ Ph),4.13-4.17(m,2H,OCH ₂ Ph),4.10-4.04(m, 4H, OCH ₂ Ph, H-6a"', H-6b"', H-4"), 3.97-3.92 (m, 1H, OCH ₂ Ph), 3.86 (t, J=9.6Hz, 1H, H- 4'),3.80-3.59(m,11H),3.58-3.48(m,4H,H-6a,H-5"',H-5",H-3),3.37-3.33(m,1H,H -5),0.98(s,9H) ^.13 C NMR(150MHz,CDCl ₃ )δ140.3,138.9,138.9,138.7,138.6,138.4,138.3,138.2,138.1,138.0,137.98,137.92,135.74,135.70,133.8 133.7,133.4,129.4,129.3,128.7,128.5,128.38,128.4,128.3,128.29,128.27,128.24,128.20,128.0,127.96,127.92,127.90,127.87,127.86,127.77,127.73,127.7,127.62,127.60,127.56, 127.50, 127.4, 127.3, 127.2, 127.1, 127 .0,126.9,126.7,117.2,101.8,100.4,100.3,83.5,80.9,80.7,79.5,75.4,75.39,75.33,75.2,75.0,74.9,74.8,74.7,74.5,74.4,73.4,73.369,73.9 ,70.0,69.9,69.7,69.7,69.1,68.9,68.7,64.0,26.9,19.3.HRMS(ESI):calcd.for C ₁₂₇ H ₁₃₆ O ₂₁ Si[M+Na] ⁺ m/z,2047.9241;found, 2047.9266.

The synthesis of embodiment 19 compound 16a

Compound 15a (183.0 mg, 0.16 mmol) was dissolved in 5 mL of tetrahydrofuran, then tetrabutylammonium fluoride (0.63 mL, 1 _M ) was added, stirred at room temperature for one day, diluted with 30 mL of dichloromethane, and then washed with 30 mL of saturated brine , the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to obtain the initial product, and the initial product was subjected to silica gel column chromatography (n-hexane/acetone, 10:1, v/v) to obtain a white foamy solid (77.0mg, 83.4% )

¹ H NMR (600MHz, CDCl ₃ ) δ7.63-7.15 (m, 30H, Ar), 5.97-5.88 (m, 1H, OCH ₂ CH ＝ CH ₂ ), 5.28 (m, 1H, OCH ₂ CH ＝ C H ₂ ),5.21(m,1H,OCH ₂ CH= CH ₂ ),5.15(d,J=12.0Hz,1H,OCH ₂ Ph),5.04-4.96(m,5H,OCH ₂ Ph,H-1 '), 4.93 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.71 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.66-4.60 (m, 2H, OCH ₂ Ph), 4.58- 4.47(m,5H,OCH ₂ Ph,H-1),4.43(d,J=12.0Hz,1H,OCH ₂ Ph),4.39(d,J=3.0Hz,1H,H-2),4.29(d ,J=3.0Hz,1H,H-2),4.10-4.07(m,1H,O CH ₂ CH=CH ₂ ),4.06(t,J=9.6Hz,1H,H-4'),3.95(t , J=9.6Hz, 1H, H-4), 3.92-3.84(m, 2H, H-6a', H-6b'), 3.82(d, J=10.8Hz, 1H, H-6a), 3.76( dd,J=10.8,5.0Hz,1H,H-6b),3.66(dd,J=9.3,2.9Hz,1H,H-3),3.59(dd,J=9.6,3.0Hz,1H,H-3 '),3.52-3.47(m,1H,H-5),3.44-3.41(m,1H,H-5'), ¹³ C NMR(150MHz,CDCl3)δ139.10,138.56,138.36,138.24,138.20,133.89, 128.78,128.49,128.42,128.35,128.18,128.12,128.10,127.83,127.76,127.72,127.61,127.52,127.37,117.20,102.25,100.15,81.69,80.68,77.42,77.21,77.00,75.98,75.46,75.24,75.20, 74.46, 74.42, 74.33, 73.80, 73.56, 73.43, 70.81, 70.42, 70.00, 69.30, _62.49 . HRMS (ESI): _{calcd.forC57H62O11} [M+Na] ⁺ m/z, _945.4190 ; .4161.

The synthesis of embodiment 20 compound 16b

Prepared by the reaction of compound 15b and tetrabutylammonium fluoride, the specific operation steps are the same as those of compound 16a, and the yield is 88.2%.

¹ H NMR (600MHz, CDCl ₃ ) δ7.51-6.95 (m, 45H, Ar), 5.92-5.84 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.29 (s, 1H, H-1") ,5.24(m,1H,OCH ₂ CH= CH ₂ ),5.19-5.15(m,2H,OCH ₂ CH=CH ₂ , H -1'),5.07(d,J=12.0Hz,1H,OCH ₂ Ph), 4.98 (d, J=11.4Hz, 1H, OCH ₂ Ph), 4.93 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.89-4.84 (m, 3H, OCH ₂ Ph), 4.71 (d, J = 12.0Hz, 1H, OCH ₂ Ph), 4.71 (d, J = 3.0Hz, 1H, H-2'), 4.65 (d, J = 12.0Hz, 1H, OCH ₂ Ph), 4.61 ( d,J=3.0Hz,1H,H-2), 4.56(d,J=10.8Hz,1H,OCH ₂ Ph),4.46-4.40(m,6H,OCH ₂ Ph,H-1,O CH ₂ CH =CH ₂ ), 4.29 (d, J = 10.8Hz, 1H, OCH ₂ Ph), 4.15 (d, J = 3.0Hz, 1H, H-2"), 4.13 (d, J = 11.4Hz, 1H, OCH ₂ Ph), 4.07(d, J=11.4Hz, 1H, OCH ₂ Ph), 4.03-3.98(m, 1H, O CH ₂ CH=CH ₂ ), 3.94(t, J=9.6Hz, 1H, H- 4"),3.86(t,J＝9.6Hz,1H,H-4'),3.76-3.80(m,2H,H-6a",H-6a'),3.75-3.67(m,5H,H- 6b",H-4,H-6a,H-6b,H-6b'),3.66-3.63(m,1H,H-3'),3.63-3.58(m,1H,H-3),3.55- 3.51(m,2H,H-3",H-5'),3.42-3.35(m,2H,H-5,H-5"). ¹³ C NMR(150MHz,CDCl ₃ )δ139.6,138.7,138.5, 138.4,138.4,138.2,138.1,138.0,137.7,133.6,128.6,128.41,128.40,128.4,128.3,128.29,128.23,128.2,128.04,128.00,127.8,127.7,127.69,127.65,127.61,127.6,127.4,12 7.1,127.1,117.4,101.6,100.7,100.4,83.0,80.7,80.6,75.5,75.4,75.2,75.1,74.7,74.66,74.62,74.6,74.5,73.6,73.4,71.6,70.5,70.4,69.8,9, 69.6, 68.7, 62.4. HRMS (ESI): calcd. for C ₈₄ H ₉₀ O ₁₆ [M+Na] ⁺ m/z, 1377.6127; found, 1377.6112.

The synthesis of embodiment 21 compound 16c

Prepared by the reaction of compound 15c and tetrabutylammonium fluoride, the specific operation steps are the same as those of compound 16a, and the yield is 87.2%.

¹ H NMR (500MHz, CDCl ₃ ) δ7.52-6.02 (m, 60H, Ar), 5.92-5.83 (m, 1H, OCH ₂ CH ＝CH ₂ ), 5.53 (s, 1H, H-1"'),5.32(s,1H,H-1"),5.24(m,1H,OCH ₂ CH= CH ₂ ),5.18(m,1H,OCH ₂ CH= CH ₂ ),5.15(s,1H, H-1'), 4.99(d, J=12.0Hz, 1H, OCH ₂ Ph), 5.00-4.88(m, 5H, OCH ₂ Ph, H-2'), 4.83(d, J=10.8Hz, 1H , OCH ₂ Ph), 4.75 (d, J=12.0Hz, 1H, OCH ₂ Ph), 4.87-4.77 (m, 15H, OCH ₂ Ph, H-2, H-2"', O CH ₂ CH=CH ₂ ), 4.25 (d, J=3.0Hz, 1H, H-2”), 4.18-4.10 (m, 2H, OCH ₂ Ph), 4.05-3.96 (m, 3H, O CH ₂ CH=CH ₂ , OCH ₂ Ph),3.87-3.54(m,15H),3.48-3.40(m,2H,H-5,H-5”). ¹³ C NMR(125MHz, CDCl ₃ )δ139.6,138.75,138.72,138.6,138.5, 138.4,138.2,138.16,138.1,137.9,137.8,133.6,128.62,128.60,128.5,128.45,128.4,128.3,128.32,128.3,128.25,128.22,128.21,128.2,128.1,128.04,128.02,128.0,127.8,127.72, 127.7, 127.6, 127.5, 127.4, 127.3, 127.1, 127.0, 117.3, 101.7, 101.2, 100.7, 100.2, 82.9, 81.8, 80.7, 80.4, 75.6, 75.5, 75.3, 75.2, 75.1, 75.0, 74.8 74.4,73.6,73.5,73.45,72.6,70.5,70.5,70.3,70.1,69.8,69.6,69.6,69.5,69.3,68.8,62.4.MALDI-TOF-MS: calcd.for C ₁₁₁ H ₁₁₈ O ₂₁ [M+ Na] ⁺ m/z, 1811.10; found, 1810.76.

The synthesis of embodiment 22 compound 18a

Compound 16a (50.0mg, 0.06mmol) and phosphorylation reagent 17 (138mg, 0.26mmol) (Synthesis.1992, 1269.) were placed in a 10mL round bottom flask and dissolved in 3.0mL dichloromethane, and the solution was cooled to 0°C Add 1H-tetrazole solution (0.6mL, 0.45 _M ) dropwise, react for fifteen minutes under the protection of argon, remove the cooling device and continue stirring at room temperature for two hours, then cool to -40°C and add tert-butanol peroxide dropwise (0.07mL, 5.5 _M ) solution, remove the cooling device and continue to stir at room temperature for three hours, add 15mL of 10% sodium thiosulfate solution to quench the reaction, the reaction solution is extracted with 30mL of dichloromethane, and then successively with saturated sodium bicarbonate solution (30mL) and brine (30mL), and the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to obtain the initial product, and the initial product was subjected to silica gel column chromatography (n-hexane/ethyl acetate, 3:2, v/v ) to obtain a diastereomeric mixture (66.7 mg, 82.0%).

Selected signals: ¹ H NMR(500MHz,CDCl ₃ )δ4.97(br s,1H),4.41(br s,1H). ¹³ CNMR(125MHz,CDCl ₃ )δ102.14,102.09,100.31,100.26. ³¹ P NMR( 160MHz, CDCl ₃ )δ-0.61, -0.76. HRMS (ESI): calcd. for C ₈₁ H ₈₄ NO ₁₆ P[M+Na] ⁺ m/z, 1380.5425; found, 1380.5381.

The synthesis of embodiment 23 compound 18b

Prepared by the reaction of compound 16b and phosphorylation reagent 17, the specific operation steps are the same as the synthesis of compound 18a, and the yield is 87.5%.

Selected signals: ¹ HNMR(500MHz,CDCl ₃ )δ5.40(br s,1H),5.18(br s,1H),4.46(br s,1H). ¹³ C NMR(125MHz,CDCl ₃ )δ101.62,101.52, 100.79, 100.70, 100.65, 100.54. ³¹ PNMR (160MHz, CDCl ₃ ) δ-0.50, -0.70. HRMS (ESI): calcd. for C ₁₀₈ H ₁₁₂ NO ₂₁ P[M+Na] ⁺ m/z, 1812.7362; found, 1812.7319.

The synthesis of embodiment 24 compound 18c

Prepared by the reaction of compound 16c and phosphorylation reagent 17, the specific operation steps are the same as the synthesis of compound 18a, and the yield is 79.3%.

NMR signals for 18c(isomer 1): ¹ H NMR (600MHz, CDCl ₃ ) δ7.73-6.93(m, 73H, Ar), 5.85-5.78(m, 1H, OCH ₂ CH ＝CH ₂ ), 5.48( s,1H,H-1"'),5.30(s,1H,H-1"),5.19(m,1H,OCH ₂ CH= CH ₂ ),5.14-5.10(m,1H,OCH ₂ Ph) ,5.09(s,1H,H-1'),5.05-5.00(m,1H,OCH ₂ Ph),4.97-4.82(m,9H,OCH ₂ Ph,H-2'),4.71(d,J= 12.0Hz, 1H, OCH ₂ Ph), 4.62 (d, J = 12.0Hz, 1H, OCH ₂ Ph), 4.59 (d, J = 12.0 Hz, 1H, OCH ₂ Ph), 4.56 (d, J = 10.8 Hz ,1H,OCH ₂ Ph),4.57(d,J=12.0 Hz,1H,OCH ₂ Ph),4.53-4.44(m,6H,OCH ₂ Ph,H-1,H-2"'),4.41-4.32 (m,8H),4.29(d,J=3.6 Hz,1H,H-2"),4.26(d,J=10.8 Hz,1H,OCH ₂ Ph),4.15-4.06(m,4H),4.03- 3.93(m,4H),3.92-3.75(m,5H),3.72-3.58(m,8H),3.55(dd,J=9.6,3.6 Hz,1H,OCH ₂ Ph),3.52-3.43(m,2H ),3.37-3.32(m,1H),3.26-3.18(m,1H),3.08-2.99(m,1H). ¹³ C NMR(150 MHz,CDCl ₃ )δ156.18,144.08,143.92,141.16,141.12,139.49 ,138.64,138.52,138.43,138.34,138.21,138.05,138.02,137.99,137.84,137.75,135.80,136.76,133.59,128.64,128.57,128.52,128.45,128.32,128.28,128.26,128.22,128.18,128.16,128.12,128.04 ,128.02,127.97,127.96,127.88,127.85,127.81,127.70,127.64,127.56,127.54,127.50 ,127.43,127.39,127.24,127.11,127.06,126.97,126.95,125.21,125.12,119.77,117.31,102.08,101.31,100.55,100.29,83.10,81.97,80.53,80.35,75.52,75.31,75.27,75.12,75.05,74.69 , 74.58,74.24,74.20,73.61,73.56,73.73.21,70.92,70.54,70.18,69.69.60, ^{69.36.15,689.77,66.85,47,47,45,45,45,45,45,45,45,45,45,45,45,45,45,45,45,45,45,666.4} . PNMR (160 MHz, CDCl ₃ ) δ-0.67. MALDI-TOF-MS: calcd. for C ₁₀₈ H ₁₁₂ NO ₂₁ P[M+H] ⁺ m/z, 2222.947; found, 2223.236. NMR signals for 18c (isomer 2): ¹ H NMR (600 MHz, CDCl ₃ ) δ7.73-6.93(m,73H,Ar),5.87-5.78(m,1H,OCH ₂ CH ＝CH ₂ ),5.48(s,1H,H -1"'),5.32(s,1H,H-1"),5.19(m,1H,OCH ₂ CH= CH ₂ ),5.15-5.11(m,2H,OCH ₂ Ph),5.07(s, 1H,H-1'),5.06-5.00(m,3H,OCH ₂ Ph),4.97-4.82(m,8H,OCH ₂ Ph,H-2'),4.74(d,J＝11.4 Hz,1H, OCH ₂ Ph), 4.69 (d, J=12.0 Hz, 1H, OCH ₂ Ph), 4.66 (d, J=12.0 Hz, 1H, OCH ₂ Ph), 4.62 (d, J=10.8 Hz, 1H, OCH ₂ Ph), 4.57(d, J=12.0 Hz, 1H, OCH ₂ Ph), 4.54(d, J=12.0 Hz, 1H, OCH ₂ Ph), 4.51(br s, 1H, H-1), 4.49(d _{( _} d, J=3.0 Hz, 1H, H-2"), 4.22 (d,J=10.8 Hz,2H,OCH ₂ Ph),4.13-4.04(m,3H),4.03-3.93(m,3H),3.87-3.83(m,3H),3.68-3.58(m,9H) ,3.55(dd,J＝9.6,3.6 Hz,1H,OCH ₂ Ph),3.52-3.47(m,2H),3.37-3.34(m,1H),3.14-3.08(m,2H). ¹³ CNMR(150 MHz,CDCl ₃ )δ156.22,144.01,143.97,141.19,139.59,138.68,138.60,138.33,138.26,138.11,138.08,138.04,137.88,137.78,136.08,136.04,133.61,128.67,128.52,128.46,128.42,128.37,128.30 ,128.29,128.23,128.22,128.17,128.09,128.05,128.01,127.98,127.96,127.92,127.79,127.72,127.66,127.63,127.58,127.53,127.40,127.21,127.18,127.09,127.05,127.00,126.57,125.16,119.81 ,117.27,101.97,101.47,100.52,100.27,83.12,81.73,80.62,80.31,75.47,75.35,75.28,75.23,75.09,75.01,74.97,74.94,74.70,74.51,74.30,74.26,73.60,73.53,73.40,73.37 , ^{72.86,71.25,70.43,70.15,70.12,69.70,69.63,69.59,69.57,69.42,69.27,69.24,68.83,66.70,66.66,66.59,47.11,41.16,41.12} _. 0.52. HRMS (ESI): calcd. for C ₁₀₈ H ₁₁₂ NO ₂₁ P [M+Na] ⁺ m/z, 2244.9293; found, 2244.9475.

The synthesis of embodiment 25 compound 19a

Compound 18a (50.0mg, 0.037mmol) was placed in a 5mL eggplant bottle and dissolved in 0.8mL dichloromethane, stirred at room temperature and added dropwise 5 drops of 1,8-diazabicycloundec-7-ene ( DBU), after two minutes, add 5 drops of acetic acid to neutralize the reaction, remove the solvent under low temperature and low pressure, and obtain the initial product through silica gel column chromatography (dichloromethane/methanol, 30:1, v/v) to obtain a diastereomeric mixture (38.0 mg, 90.4%).

Selected signals: ¹ H NMR(600MHz,CDCl3)δ4.96(br s,1H),4.40(br s,1H).13CNMR(125MHz,CDCl3)δ101.84,101.69,100.14,99.86.HRMS(ESI):calcd. for C66H75NO14P[M+H]+m/z, 1136.4920; found, 1136.4925.

The synthesis of embodiment 26 compound 19b

Prepared by the reaction of compound 18b and DBU, the specific operation steps are the same as the synthesis of compound 19a, and the yield is 93.2%.

Selected signals: ¹ H NMR(600MHz,CDCl ₃ )δ5.31(s,1H),5.13(s,1H),4.44(s,1H). ¹³ CNMR(150MHz,CDCl ₃ )δ101.27,100.78,100.67,100.55 ,100.22,100,08.HRMS(ESI):calcd.for C ₉₃ H ₁₀₂ NO ₁₉ P[MH] ^- m/z,1566.6711;found,1566.6705.

Synthesis of Example 27 Compound 19c

Prepared by the reaction of compound 18c and DBU, the specific operation steps are the same as the synthesis of compound 19a, and the yield is 86.3%.

NMR signals for 19c(isomer 1): ¹ H NMR (600MHz, CDCl ₃ )δ7.50-6.92(m,65H,Ar),5.85-5.77(m,1H,OCH ₂ CH ＝CH ₂ ),5.46( s,1H,H-1"'),5.31(s,1H,H-1"),5.18(m,1H,OCH ₂ CH= CH ₂ ),5.04(s,1H,H-1'), 5.03-4.97(m,5H,OCH ₂ Ph),4.90-4.78(m,5H,OCH ₂ Ph),4.67-4.60(m,3H),4.59-4.45(m,6H,OCH ₂ Ph),4.43- 4.33(m,8H,OCH ₂ Ph,H-1),4.31-4.19(m,3H),4.09(d,J=11.2Hz,1H,OCH ₂ Ph),4.04(d,J=11.2Hz,1H ,OCH ₂ Ph),3.98-3.88(m,3H),3.84-3.68(m,6H),3.67-3.56(m,6H),3.54(dd,1H,J＝9.6,3.0Hz),3.51-3.45 (m,1H),3.44-3.40(m,1H),3.37-3.32(m,1H). ¹³ C NMR(150MHz,CDCl ₃ )δ138.78,138.49,138.24,138.16,138.11,138.05,137.99,137.87,137.71 ,133.59,128.76,128.56,128.50,128.42,128.40,128.31,128.30,128.24,128.17,128.16,128.11,128.07,128.04,128.00,127.98,127.90,127.85,127.76,127.73,127.69,127.64,127.51,127.40,127.20 ,127.15,127.10,117.25,101.65,101.59,100.40,100.10,83.33,81.50,80.66,80.26,75.46,75.36,75.27,75.23,75.03,74.99,74.95,74.73,74.59,74.35,73.58,73.51,73.38,70.25 ,70.08,69.78,69.52,69.48,69.40,69.03,68.83,66.60.NMR signals for 19c(isomer 2): ¹ H NMR(600MHz, CDCl ₃ )δ7.55-6.93(m,65H,Ar),5.85-5.79(m,1H,OCH ₂ CH ＝CH ₂ ),5.53(s,1H,H-1"'), 5.38(s,1H,H-1"),5.18(m,1H,OCH ₂ CH= CH ₂ ),5.15-5.11(m,2H,OCH ₂ Ph,H-1'),5.10(s,1H ,H-1'),5.01-4.91(m,5H,OCH ₂ Ph),4.89(d,J=3.0Hz,1H),4.85-4.78(m,3H),4.66(d,J=10.8Hz, 1H, OCH ₂ Ph), 4.61-4.57 (m, 2H), 4.47-4.37 (m, 14H, OCH ₂ Ph, H-2, H-1), 4.29 (d, J=10.8Hz, 1H, OCH ₂ Ph), 4.25-4.14(m, 3H), 4.07(d, J=11.2Hz, 1H, OCH ₂ Ph), 3.98-3.75(m, 7H), 3.71-3.55(m, 9H), 3.51-3.44( m,2H),3.39-3.34(m,2H). ¹³ C NMR(150MHz,CDCl ₃ )δ138.94,138.20,138.13,138.05,138.02,138.00,137.98,137.91,137.84,137.71,133.61,128.193,12 ,128.56,128.52,128.46,128.37,128.34,128.30,128.25,128.21,128.07,128.04,128.02,127.95,127.93,127.92,127.87,127.84,127.74,127.67,127.60,127.55,127.50,127.35,127.24,126.92,117.26 ,101.32,101.15,100.46,99.78,82.15,80.61,80.54,75.58,75.39,75.30,75.20,75.09,75.02,74.93,74.82,74.65,74.49,73.61,73.48,73.43,71.13,70.38,70.30,70.12,70.04 ,69.33,69.16,69.09,69.05,68.89,66.31,60.03.HRMS(ESI):calcd.for C ₁₂₀ H ₁₃₁ NO ₂₄ P[M+H] ⁺ m/ z, 2000.8793; found, 2000.8799.

The synthesis of embodiment 28 compound 20a

Compound 19a (20.0mg) and palladium hydroxide on carbon (10Wt.%, 10.0mg) were placed in a 5mL test tube and methanol/dichloromethane mixed solution (2mL, 1:1, v:v) was added, and then the test tube was placed Put it into a hydrogenation reactor and shake it under 40 atmospheres for one day. After the reaction, the suspension is filtered with diatomaceous earth, the filter cake is washed with methanol, the washings are combined and concentrated, and the residue is washed with ethyl acetate and then subjected to LH- 20 column chromatography gave a white solid (8.2 mg, 91.5%).

¹ H NMR (600MHz, CDCl ₃ )δ4.84(s,1H,H-1'),4.58(s,1H,H-1),4.26-4.21(m,1H,),4.14-4.07(m, 4H,H-2),4.05-4.02(m,1H,H-2'),3.90-3.84(m,2H,O CH ₂ CH ₂ CH ₃ ),3.76-3.68(m,2H,H-4' ),3.57-3.45(m,3H,O CH ₂ CH ₂ CH ₃ ),3.42-3.38(m,1H,H-3'),3.33(s,1H,),3.30-3.27(m,3H,) ,3.24-3.12(m,4H,H-5'),1.64-1.57(m,2H,OCH ₂ CH ₂ CH ₃ ),0.94(t,J=7.8Hz,1H,OCH ₂ CH ₂ CH ₃ ). ¹³ C NMR (150MHz, CDCl ₃ ) δ101.33, 100.47, 78.16, 76.91, 75.69, 73.53, 72.83, 70.86, 70.61, 66.99, 66.12, 64.48, 61.82, 61.11, 40.05, 22.59, 9.60.HRcaldMS (ESI forC ₁₇ H ₃₃ NO ₁₄ P[MH] ^- m/z, 506.1639; found, 506.1628

The synthesis of embodiment 29 compound 20b

Prepared from compound 19b by catalytic reduction reaction of palladium hydroxide on carbon, the specific operation steps are the same as the synthesis of compound 20a, and the yield is 88.7%.

¹ H NMR (600MHz, CDCl ₃ ) δ5.02(s, 1H, H-1"), 4.81(s, 1H, H-1'), 4.57(s, 1H, H-1), 4.31-4.25( m,1H,H-6"a),4.22(br s,1H,H-2'),4.20-4.15(m,1H,H-6"a),4.14-4.10(m,3H,H-2 ", CH ₂ OP), 4.05 (d, J=3.0Hz, 1H, H-2), 3.89-3.84 (m, 3H, H-6a', O CH ₂ CH ₂ CH ₃ ), 3.76-3.68 (m ,3H,H-6b',H-4",H-4'),3.55-3.42(m,6H),3.26-3.14(m,4H,H-5",H-5, CH ₂ NH ₂ ) ,1.64-1.59(m,2H,OCH ₂ CH ₂ CH ₃ ),0.94(t,J=7.8Hz,1H,OCH ₂ CH ₂ CH ₃ ). ¹³ C NMR(150MHz,CDCl ₃ )δ101.4,101.3,100.3 ,79.4,77.7,76.86,75.4,75.3,73.3,72.8,70.8,70.4,67.7,66.4,66.1,64.7,64.6,61.9,61.1,60.8,40.0,22.6,9.6. ₂₃ H ₄₄ NO ₁₉ P[MH] ^- m/z, 668.2167; found, 668.2166.

The synthesis of embodiment 30 compound 20c

It is prepared from compound 19c by catalytic reduction reaction of palladium hydroxide on carbon, and the specific operation steps are the same as the synthesis of compound 20a, and the yield is 93.5%.

¹ H NMR (600MHz, CDCl ₃ ) δ5.06(s, 1H, H-1"'), 5.01(s, 1H, H-1"), 4.75(s, 1H, H-1'), 4.57( s,1H,H-1),4.49(d,J=3.0Hz,1H,H-2"),4.39-4.33(m,1H,H-6a"'),4.18-4.06(m,5H,H -6b"', H-2', H-2"', CH ₂ OP), 4.05 (d, J=3.0Hz, 1H, H-2"), 3.89-3.81 (m, 5H), 3.76-3.39 (m,14H),3.37-3.29(m,2H,),3.24-3.11(m,5H,). ¹³ C NMR(150MHz,CDCl ₃ )δ102.4,102.0,100.9,100.4,81.3,80.1,77.1,76.9 ,75.4,75.4,73.5,72.8,72.7,72.6,70.8,70.68,67.4,67.3,66.4,64.5,64.49,61.7,61.6,61.1,60.6,40.1,40.0,22.7,9.7. for C ₂₉ H ₅₄ NO ₂₄ P[MH] ^- m/z, 830.2695; found, 830.2676.

Synthesis of Example 31 Compound 22a

Dissolve compound 20a and succinimide glutarate 21 (DSG, 15equiv) in DMF/PBS (4:1, 0.1 _M PBSbuffer) mixed solution, stir at room temperature for four hours, remove solvent under low temperature and low pressure and strip with toluene After spinning twice, the residue was washed with excess ethyl acetate for several times, and after drying, the pure activated ester was obtained with a yield of 78.2%.

HRMS(ESI): calcd.for C ₂₆ H ₄₂ N ₂ O ₁₉ P[MH] ^- m/z, 717.2125, found, 717.2119.

Synthesis of Example 32 Compound 22b

Prepared by the reaction of compound 20b and succinimide glutarate 21, the specific operation steps are the same as the synthesis of compound 22a, and the yield is 81.5%.

HRMS(ESI): calcd.for C ₃₂ H ₅₂ N ₂ O ₂₄ P[MH] ^- m/z, 879.2653, found, 879.2656.

Synthesis of Example 33 Compound 22c

Prepared by the reaction of compound 20c and succinimide glutarate 21, the specific operation steps are the same as the synthesis of compound 22a, and the yield is 82.3%.

HRMS(ESI): calcd.for C ₃₈ H ₆₂ N ₂ O ₂₉ P[MH] ^- m/z, 1041.3187, found, 1041.3176.

Synthesis of Example 34 Glycoprotein Conjugates (1a-c, 2a-c)

Dissolve compound 22 and carrier protein KLH or HAS in a molar ratio of 30:1 (activated ester: carrier protein) in 0.1 _M PBS buffer solution, and stir at room temperature for two and a half days under the protection of argon. The reaction solution was subjected to Biogel A 0.5 column chromatography (0.1 _M PBS buffer solution, pH=7.8), deionized water dialysis and freeze-drying to obtain pure glycoprotein conjugates 1a-c and 2a-c (see Figure 5 for molecular weight -7).

Example 35 Immunological activity test of compounds of the present invention

1. Experimental materials and sources

Compounds to be tested: glycoprotein compounds 1a-c prepared in Example 34 of the present invention.

2. Test method

(1) Mouse immunization scheme

1) Select 6-8 week old female Balb/c mice (purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences), and group them according to the vaccine+adjuvant and vaccine-only immunization schemes, and the control group is the adjuvant-only group, with 6 mice in each group mice;

2) The glycoprotein compound 1a-c and its corresponding adjuvant group (dissolved in PBS) suspension were subcutaneously injected into mice, and each immunized glycoprotein contained 2 μg of sugar, and the same method was used to boost the immunization every 2 weeks , a total of 4 times of immunization. Blood was collected before immunization, 13 days after the second immunization, 13 days after the third immunization, and 14 days after the fourth immunization, and the serum was separated and stored in a -80°C refrigerator for testing.

(2) Determination of antibody titer in serum of mice before and after immunization

The mixed serum titer of each mouse in each experimental group was detected by ELISA method.

1) Coating plate: Compounds 2a-c were diluted with pH 9.6 carbonate buffer solution to 5ug/ml and coated on the enzyme labeling strip. Combine at 37°C for 2 hours, then use blocking solution (15% skimmed milk) 200ul/well overnight at 4°C;

2) After washing with PBST for 3 times, add 100ul of doubly diluted mouse serum in each group to each well, and incubate at 37°C for 2 hours;

3) After washing with PBST for 5 times, add 100ul of 1:2000 diluted HRP-labeled rabbit anti-mouse IgG secondary antibody (Invitrogen), and incubate at 37°C for 45 minutes;

4) After washing with PBST for 5 times, the A450 value was read by a microplate reader (MultiscanMK3, Thermo) after TMB color development, and the serum that was 3 times higher than the negative control was defined as positive serum.

(3) Combination of serum taken from mice after immunization with Candida albicans cells

1) Pick a monoclonal colony of Candida albicans SC5314 and enrich it with YPD liquid medium for 24 hours. At this time, it is a yeast-like cell in the logarithmic phase of growth. Candida albicans hyphae phase induction method: resuspend and wash Candida albicans in the logarithmic phase of growth twice in PBS, and place them in 1640 medium containing 10% calf serum for static culture at 37°C for 90 minutes;

2) Resuspend Candida albicans in the hyphae phase or yeast phase twice in PBS, fix with 4% paraformaldehyde at -20°C for 10 minutes, centrifuge and resuspend in PBS again for three times;

3) The sera of the experimental group and the control group were diluted 1:100 with PBS containing 10% calf serum and incubated with the fixed bacteria for 40 minutes, and then centrifuged and resuspended in PBS for 3 times;

4) Co-incubate the bacteria with 488-labeled goat anti-mouse IgG antibody for 30 minutes, then centrifuge with PBS and resuspend 3 times;

5) Put the resuspended bacteria in a 96-well plate and let it stand for 10 minutes, and observe it under 488 fluorescence with a fluorescence microscope;

6) Place the resuspended bacteria in a flow cytometry centrifuge tube and directly test them on the flow cytometer, select FITC as the excitation light, use the negative control Candida albicans to adjust to zero, and test the bacteria after co-incubating with the fluorescent antibody again , 10,000 cells were read per loading.

3. Experimental results

The KLH-glycoprotein conjugates 1a-c prepared by the present invention subcutaneously immunize mice, all of which can induce a strong immune response in the body, wherein compound 1b has the strongest immunogenicity and the immune serum produced can specifically recognize Rosary albicans Bacterial cell surface polysaccharide antigen, the experimental results are shown in Figures 8-10.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (10)

1. A β-1,2-D-oligomannose protein conjugate, characterized in that, the general structural formula of the β-1,2-D-oligomannose protein conjugate is: Wherein, n is 0, 1 or 2, and R is carrier protein KLH or HSA. 2. The β-1,2-D-oligomannose protein conjugate according to claim 1, wherein the monosaccharide unit constituting the oligosaccharide is β-1,2-D-mannose. 3. β-1,2-D-oligomannose protein conjugate according to claim 1, is characterized in that, by phosphorylated β-1,2-D-oligomannose and carrier protein conjugate Jointly made. 4. The β-1,2-D-oligomannose protein conjugate according to claim 3, wherein the glutaryl group is used as the phosphorylated β-1,2-D-oligomannose Linking group with carrier protein. 5. The preparation method of the β-1,2-D-oligomannoprotein conjugate of claim 1, characterized in that, comprising the steps of: The first step is to synthesize β-1,2-D-mannobiose receptor 7 and β-1,2-D-mannotriose receptor 10: The second step is to synthesize phosphorylated β-1,2-D-oligomannose 19a-c: The third step is to synthesize β-1,2-D-oligomannoprotein conjugates 1a-c, 2a-c: 6. according to the preparation method of the β-1,2-D-oligomannose protein conjugate of claim 5, it is characterized in that, the preparation method of the phosphorylated β-1,2-D-oligomannose as follows: (1) Preparation of β-1,2-D-mannose monosaccharide acceptor and 3,4,6-tribenzyl-2-acetoxyglucose by using α-D-glucose as raw material under the action of reaction assistant trichloroacetimide ester donor; (2) The monosaccharide donor and monosaccharide acceptor prepared above undergo a glycosylation reaction under the action of a catalyst (TMSOTf) to prepare a disaccharide, and prepare β-1,2 by configuration inversion at the 2'-position of the disaccharide -D-mannobiose acceptor; β-1,2-D-mannobiose acceptor and monosaccharide donor prepare β-1,2-D-mannotriose acceptor by the aforementioned method; (3) β-1,2-D-mannose acceptor and 6-tert-butyldiphenylsilyl-3,4-dibenzyl-2-acetoxyglucose trichloroacetimidate donor The corresponding β-1,2-D-oligomannose is prepared by glycosylation; the 6-position of the non-reducing end of the oligomannose is removed from the TBDPS protection and reacted with a phosphorylation reagent to obtain phosphorylated β-1,2- D-oligomannose. 7. according to the preparation method of the described β-1,2-D-oligomannoprotein conjugate of claim 5, it is characterized in that, comprises the steps: (1) react phosphorylated β-1,2-D-oligomannose with hydroxysuccinimide glutarate to prepare β-1,2-D-oligomannose activated ester; (2) Coupling the activated β-1,2-D-oligomannose ester prepared in step (1) with a carrier protein under weak alkaline conditions to obtain β-1,2-D-oligomannose protein conjugate. 8. Use of the β-1,2-D-oligomannoprotein conjugate according to any one of claims 1-4 in the preparation of a medicament for preventing and/or treating fungal infections. 9. The application according to claim 8, characterized in that the fungus is Candida albicans. 10. An anti-Candida fungal vaccine, comprising the β-1,2-D-oligomannoprotein conjugate described in any one of claims 1-4 and pharmaceutically acceptable adjuvant or adjuvant.