CN115073540B

CN115073540B - Synthesis method of inulin type kestose oligosaccharide monomer

Info

Publication number: CN115073540B
Application number: CN202210900500.5A
Authority: CN
Inventors: 秦勇; 薛斐; 宋颢
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2023-08-25
Anticipated expiration: 2042-07-28
Also published as: CN115073540A

Abstract

The invention belongs to the field of chemical drug synthesis, and particularly relates to a novel method for synthesizing inulin-type kestose oligosaccharide monomers (kestose to kestose). The invention uses sucrose derivative with terminal sugar C1 position as exposed hydroxyl as glycosyl acceptor, uses fructofuranose thioglycoside as glycosyl donor, builds single-configuration beta (2, 1) -D-fructofuranose glycosidic bond through high stereoselective glycosylation reaction to form trisaccharide derivative, removes terminal sugar C1 hydroxyl protecting group to form new glycosyl acceptor, continues to carry out glycosylation reaction with fructofuranose thioglycoside donor, and introduces fructofuranose building blocks to prolong sugar chains. Further repeating the reaction sequence to obtain a series of kestose oligosaccharide derivatives with different polymerization degrees, and then respectively preparing monomers from kestose to kestose through conversion such as removing protective groups and the like. The method has the characteristics of high three-dimensional selectivity, high synthesis efficiency and strong practicability of glycosylation reaction.

Description

Synthesis method of inulin type kestose oligosaccharide monomer

Technical Field

The invention belongs to the technical field of chemical drug synthesis, and particularly relates to a synthesis method of an inulin-type kestose oligosaccharide monomer.

Background

Inulin type (Inulin type) fructooligosaccharides are a group of oligosaccharide mixtures with different polymerization degrees formed by combining a sucrose molecule with a plurality of D-fructofuranose through beta (2, 1) -D-fructofuranose glycosidic bonds, and comprise kestose, and the like. The oligosaccharide is not only present in natural foods such as honey, banana, barley, etc., but also in various medicinal plants such as chicory, morinda root, dangshen and yacon. Researches show that the inulin-type kestose oligosaccharide has the activities of reducing blood pressure and blood lipid, regulating intestinal flora balance, improving cardiovascular functions, enhancing human immunity, resisting depression and the like, and is widely applied to the industries of foods and health care products. The first antidepressant traditional Chinese medicine morinda root oligosaccharide capsule approved in 2012 in China has the main active ingredient of an inulin-type kestose oligosaccharide mixture.

In recent years, the important physiological activity of kestose oligosaccharides has attracted increasing attention. Further clarifies the activity of the monomer components, and deeply researches the action mechanism of the monomer components, and has important significance for developing medicaments with definite target points and higher activity. However, at present, the method for extracting and separating inulin polysaccharide from plants, and then breaking glycosidic bonds through enzymolysis or acid hydrolysis is a main mode for obtaining inulin-type kestose oligosaccharide, the obtained products exist in a mixture form, each monomer can be obtained only through separation and purification by a chromatographic method, the purity, the yield, the efficiency and the like of the inulin polysaccharide are limited greatly, and the requirements on structural and pharmacological activity research are difficult to meet. Therefore, there is a need to develop an efficient method for obtaining high purity kestose oligosaccharide monomers by chemical synthesis means.

From the aspect of synthesis angle, the inulin-type kestose oligosaccharide can be prepared by taking fructofuranose as a glycosyl donor through glycosylation reaction. The stereoselective construction of the beta (2, 1) -D-fructofuranoside bond is a key point of synthesis, and has higher difficulty and challenge. Several strategies reported in the literature to construct β -D-fructofuranoside linkages are as follows:

(1) Oscarson et al (J.org.chem.1996, 61,4512-4513; J.org.chem.1998,63, 1780-1784) synthesized β -D-fructofuranoside linkages in good yield and stereoselectivity by means of a glycosyl internal transfer strategy using 3-O- (p-methoxybenzylidene) acetal protected fructofuranoside as donor. However, the acetal intermediate produced in this process is unstable and cannot be used for synthesizing oligosaccharides with complex structures.

(2) Oscarson et al (J.am.chem.Soc.2000, 122, 8869-8872) have developed a conformationally restricted β -configuration fructofuranoside-sulfur donor, which is protected with cyclic silicon from fructofuranose C1-OH and C4-OH, and which is restricted in the α -plane of the sugar ring to hinder attack of the acceptor from the α -direction, thereby promoting formation of β -D-fructofuranoside linkages. Using this method, they synthesized fructofuranose derivatives containing 2 beta (2, 1) -D-fructofuranoside linkages (J.org.chem.2002, 67, 8457-8462). However, because the designed glycosyl donors C1-OH and C4-OH are required to be positioned on the alpha surface of the sugar ring, only beta-configuration of the thioglycoside donor can be used in the glycosylation reaction, so that about half of alpha-configuration thioglycoside donors in the raw materials cannot be applied, and the atom availability is low. In addition, in the strategy, the product obtained after the glycosylation reaction needs to be modified by multi-step functional groups to be converted into a glycosyl acceptor to continuously participate in the subsequent glycosylation reaction of the extended sugar chain, and the steps are complicated. The above problems limit the application of this method in the synthesis of polysaccharides.

(3) The strategy of hydrogen bond mediated glycosyl transfer has been applied to the construction of beta-glycosidic bonds in many cases in recent years. Li Zhongjun et al (org. Lett.2020,22, 2967-2971) have constructed beta (2, 6) -D-fructofuranoside linkages stereoselectively by means of a hydrogen bond-mediated glycosyl transfer strategy with fructofuranoside protected by Pico as a glycosyl donor, and laid a good foundation for synthesizing timothy grass-type (levan-type) fructo-oligosaccharide.

Therefore, the method for constructing the beta-D-fructofuranoside bond is limited at present, and no report on the synthesis of an inulin-type fructo-oligosaccharide monomer by constructing the beta (2, 1) -D-fructofuranoside bond is available. Therefore, aiming at the preparation of the kestose oligosaccharide monomer, development of an efficient synthesis method is necessary.

Disclosure of Invention

The invention aims to fill the blank of the prior art and provides a method for preparing an inulin-type kestose oligosaccharide monomer. The method has the characteristics of easily available raw materials, high three-dimensional selectivity of glycosylation reaction, high synthesis efficiency and strong practicability, and is suitable for large-scale production.

The aim of the invention is realized by the following technical scheme:

the synthesis method of the inulin-type kestose oligosaccharide monomer is characterized by comprising the following steps of:

Step one: taking a sucrose derivative 1 which is a known compound as an acceptor, and a fructofuranosyl thioglycoside derivative 2 alpha or 2 beta as a donor, and forming beta (2, 1) -D-fructofuranosyl glycosidic bond through glycosylation reaction to obtain a fructosyl trisaccharide derivative 3;

wherein R protecting group in the fructofuranosyl sulfan derivative 2 alpha or 2 beta is hydroxyl protecting group, and the hydroxyl protecting group is one of triisopropyl silicon (TIPS), tert-butyl dimethyl silicon (TBS), tert-butyl diphenyl silicon (TBDPS), triethyl silicon (TES), p-methoxyphenyl (PMB), trityl (Tr), 4' -dimethoxy triphenyl (DMTR), 1-naphthylene (1-Nap) and 2-naphthylene (2-Nap);

step two: removing R protecting groups of terminal sugar C1-OH from the kestose derivative 3 to obtain kestose derivative 4;

step three: the compound synthesized in the second step is used as a new acceptor, and the first step and the second step are repeated for 0 to 60 times to obtain the kestose oligosaccharide derivative I (trisaccharide to heptasaccharide);

wherein n is 0 to 60, preferably n is 0 to 25;

step four: removing Pico protecting groups from the kestose oligosaccharide derivative I under the action of a deprotection reagent to obtain a kestose oligosaccharide derivative II;

step five: removing benzoyl protecting groups from the kestose oligosaccharide derivative II under the action of a deprotection reagent to obtain a kestose oligosaccharide derivative III;

Step six: and removing benzyl protecting groups from the kestose oligosaccharide derivative III under the action of a deprotection reagent to generate kestose oligosaccharide IV monomers.

Furthermore, the synthesis method of the inulin-type kestose oligosaccharide provided by the invention can be used for synthesizing kestose, kestose and synthesizing kestose monomers with higher polymerization degree.

Further, in the first step, the promoter in the glycosylation reaction is selected from one or two of NIS, IBr, NBS, tfOH, TMSOTf, DBDMH, preferably DBDMH; the mol ratio of the promoter to the receptor of the glycosylation reaction is 1-10;

and/or the solvent adopted in the glycosylation reaction is selected from one of toluene, dichloromethane, dichloroethane and acetonitrile, preferably dichloromethane;

and/or the temperature of the glycosylation reaction is-78 ℃ to 0 ℃, preferably-50 ℃ to-30 ℃, more preferably-35 ℃;

and/or the additive for glycosylation reaction isA molecular sieve;

and/or the molar ratio of donor to acceptor in the glycosylation reaction is 1-10.

Further, in the second step, the deprotecting reagent in the reaction for removing the R protecting group is selected from the group consisting of 3HF.Et ₃ N, HF. Pyridine, TBAF, TBAF/AcOH, KF, DDQ, HCl and H ₂ SO ₄ One of them is preferably 3HF.Et ₃ N；

And/or the solvent in the reaction for removing the R protecting group is selected from one or two of tetrahydrofuran, ethyl acetate, acetonitrile, methanol, dichloromethane and chloroform;

and/or the reaction temperature in the reaction for removing the R protecting group is 0-80 ℃, preferably 75 ℃.

Further, in the fourth step, the deprotection reagent in the Pico protecting group removal reaction is Cu (OAc) ₂ ·H ₂ O; the molar ratio of the deprotection reagent to the kestose oligosaccharide derivative I is 1-15 (the volume ratio of the deprotection reagent to the solvent is 1:1);

and/or the deprotection reaction solvent is one or two of dichloromethane, methanol, dichloroethane, ethanol, isopropanol and chloroform, preferably a mixed solvent (10:1) of dichloromethane and methanol.

Further, in the fifth step, the deprotection reagent in the reaction for removing the benzoyl protecting group is selected from one of sodium methoxide, potassium carbonate, sodium hydroxide and potassium hydroxide, and preferably sodium methoxide; the molar ratio of the deprotection reagent to the kestose oligosaccharide derivative II is 1-3, preferably 1.

Further, in the step six, the deprotection reagent in the benzyl protecting group removal reaction is selected from one of 10% palladium carbon and hydrogen, 5% palladium carbon and hydrogen, and 10% palladium hydroxide and hydrogen;

And/or the hydrogen pressure in the benzyl protecting group removal reaction is 1 to 30atm, preferably 1atm;

and/or the solvent in the benzyl protecting group removal reaction is selected from one of methanol, ethanol, isopropanol and dichloromethane/methanol, preferably methanol;

and/or the reaction temperature in the benzyl protecting group removal reaction is 25-60 ℃;

in the sixth step, the deprotection reagent in the reaction for removing the benzyl protecting group is 10% palladium carbon and hydrogen, and the mass ratio of the palladium carbon to the kestose oligosaccharide derivative III is 1-0.2.

Further, in the first step, the synthesis method of the fructofuranosyl thioglycoside derivative 2α or 2β comprises the following steps:

taking fructofuranose derivatives 13 alpha and/or 13 beta (prepared by a reference method: oscarson et al, J.Am.chem.Soc.2000,122, 8869-8872) as starting materials, and completing synthesis of fructofuranose donor 2 beta through conversion such as selective introduction and removal of different protecting groups; and according to the same synthesis steps, the synthesis of the fructofuranose donor 2 alpha is completed by selectively introducing and removing different protecting groups from the fructofuranose derivative 13 alpha.

The method specifically comprises the following steps:

step S1: taking fructofuranose derivatives 13 alpha and/or 13 beta, selectively introducing TBDPS protecting groups into C6-OH of the fructofuranose derivatives 13 alpha and/or 13 beta, and obtaining compounds 14 alpha and/or 14 beta respectively;

Step S2: C1-OH of the compound 14 alpha and/or 14 beta is selectively introduced into DMTR protecting groups to respectively obtain the compound 15 alpha and/or 15 beta;

step S3: C3-OH and C4-OH of the compound 15 alpha and/or 15 beta are simultaneously introduced into benzyl protecting groups to respectively obtain a compound 16 alpha and/or 16 beta;

step S4: removing the C6 hydroxyl protecting group TBDPS from the compound 16 alpha and/or 16 beta under the action of a removing reagent to obtain a compound 17 alpha and/or 17 beta respectively;

step S5: the compound 17 alpha and/or 17 beta reacts with 2-picolinic acid under the action of a condensation reagent and alkali, and Pico protecting groups are introduced into C6-OH to generate 18 alpha and/or 18 beta respectively;

step S6: removing the C1 hydroxyl protecting group DMTr from the compound 18 alpha and/or 18 beta to obtain 19 alpha and/or 19 beta respectively;

step S7: the introduction of the R protecting group by C1-OH of the compound 19 alpha and/or 19 beta yields 2 alpha or 2 beta, respectively.

Further, in step S1, the protecting agent selectively introduced into the TBDPS protecting group reaction is TBDPSCl; the molar ratio of the protective reagent to the fructofuranose derivative 13 alpha and/or 13 beta is 1-2;

and/or the base selectively introduced into the TBDPS protecting group reaction is selected from one or more of triethylamine, pyridine, diisopropylethylamine and 2, 6-lutidine, preferably pyridine;

And/or the solvent selectively introduced into the TBDPS protecting group reaction is dichloromethane or directly takes the alkali as the solvent, preferably takes the alkali as the solvent.

Further, in the step S2, the reaction of selectively introducing the DMTr protecting group and the reaction of selectively introducing the TBDPS protecting group in the step S1 are continuously operated, and the protecting reagent of the reaction of selectively introducing the DMTrCl;

and/or the catalyst selectively introduced into DMTR protecting group reaction is DMAP, and the molar ratio of the catalyst to the compound 14 alpha and/or 14 beta is 0.05-0.2.

Further, in the step S3, the reagent in the reaction for introducing the benzyl protecting group is BnBr; the molar ratio of BnBr to the compound 15 alpha and/or 15 beta is 2-4;

and/or the alkali introduced into the benzyl protecting group reaction is selected from one or more of NaH, potassium carbonate, potassium tert-butoxide and sodium hydroxide, preferably NaH; the molar ratio of the alkali to the compound 15 alpha and/or 15 beta is 2-4;

and/or the solvent in the reaction for introducing the benzyl protecting group is selected from DMF, THF, DMSO and CH ₃ One or two of CN, preferably DMF.

Further, in step S4, the deprotecting reagent in the TBDPS reaction for removing the C6 hydroxyl protecting group is selected from the group consisting of 3HF.Et ₃ N, HF. Pyridine, TBAF, TBAF/AcOH and KF, preferably TBAF; the molar ratio of the deprotection reagent to the compound 16α and/or 16β is 1-3.

Further, in step S5, the condensing agent introduced into the Pico protecting group reaction is one of EDCI, DCC, DIC, TBTU, HATU and HBTU, preferably EDCI; the molar ratio of the condensation reagent to the compound 17 alpha and/or 17 beta is 1-3;

and/or, the catalyst in the Pico protecting group introduction reaction is DAMP; the molar ratio of the catalyst to the compound 17 alpha and/or 17 beta is 0.05-0.3;

and/or, the reaction solvent in the Pico protecting group introduction reaction is dichloromethane;

and/or the molar ratio of the 2-picolinic acid introduced into the Pico protecting group reaction to the compound 17 alpha and/or 17 beta is 1-3.

Further, in step S6, the reagent in the reaction for removing the C1 hydroxyl protecting group DMTR is TFA+Et ₃ SiH；

And/or the reaction solvent in the DMTR reaction for removing the C1 hydroxyl protecting group is methylene dichloride.

Further, in the step S7, the reactant introduced into the R protecting group reaction is one of TIPSOTf, TIPSCl, TBSCl, TBSOTf, TESCl, TESOTf, TBDPSCl,1-NapCl,2-NapCl,1-NapBr,2-NapBr, PMBCl, trCl and DMTrCl; the molar ratio of the reactant to the compound 19 alpha and/or 19 beta is 1-3;

And/or the alkali introduced into the R protecting group reaction is one or more of triethylamine, diisopropylethylamine, pyridine, 2, 6-lutidine, sodium hydride, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium hydroxide, potassium hydroxide and potassium phosphate; the molar ratio of the alkali to the compound 19 alpha and/or 19 beta is 1-3;

and/or the reaction solvent introduced into the R protecting group reaction is one or two of dichloromethane, tetrahydrofuran, acetonitrile, triethylamine, diisopropylethylamine, pyridine and 2, 6-lutidine.

The beneficial effects of the invention are as follows:

1. the invention provides a synthesis method of an inulin-type kestose oligosaccharide monomer (kestose to kestose), which realizes the preparation of the inulin-type kestose oligosaccharide monomer through a chemical synthesis means for the first time and makes up the defect that the monomer compound can only be obtained through an extraction and separation means at present.

2. The method provided by the invention uses sucrose derivatives with terminal sugar C1 as exposed hydroxyl as glycosyl acceptors, uses fructofuranose thioglycoside derivatives as glycosyl donors, constructs beta (2, 1) -D-fructofuranose glycosidic bonds with single configuration through highly stereoselective glycosylation reaction between the derivatives to generate the fructofuranose trisaccharide derivatives, removes hydroxyl protecting groups at the terminal sugar C1 to form new fructofuranose oligosaccharide donors, sequentially introduces fructofuranose building blocks according to the reaction sequence, prolongs sugar chains to obtain a series of fructofuranose oligosaccharide derivatives with different polymerization degrees, and further respectively prepares the fructofuranose trisaccharide to fructofuranose heptasaccharide monomers through conversion of removing protecting groups and the like. The conversion operation has the flexibility, the high efficiency and the practicability of modularized synthesis, is simple and convenient to operate, has strong universality, and is suitable for synthesizing kestose from kestose to kestose and even the kestose oligosaccharide monomer with higher polymerization degree.

3. The key glycosylation reaction adopts a hydrogen bond induced intramolecular glycosyl transfer strategy, and adopts a fructofuranosyl thio-glycoside derivative (2 alpha or 2 beta) as a glycosyl donor, and utilizes the induction effect of a C6-OH pico protecting group to form beta (2, 1) -D-fructofuranosyl glycosidic bond (beta: alpha > 20:1) with high stereoselectivity. And both 2 alpha and 2 beta can be used for glycosylation reaction, and the utilization rate of raw materials is high.

4. The furanose thioglycoside donor 2 alpha or 2 beta is used as a building block for prolonging sugar chains, and the C1-OH protecting group is different from the C3-OH, C4-OH and C6-OH protecting group, and can be selectively removed in subsequent reactions, so that the furanose thioglycoside donor has high flexibility in synthesizing the kestose oligosaccharides with different polymerization degrees. Specifically, glycosyl donor 2 alpha or 2 beta participates in glycosylation reaction to form beta (2, 1) -D-fructofuranoside bond to become terminal glycosyl of fructo-oligosaccharide, and C1-OH protecting group can be selectively removed to directly form a new glycosyl acceptor, so that the glycosylation reaction of the next extended sugar chain can be completed without complicated functional group conversion.

5. The synthesis method of the kestose oligosaccharide has the advantages of simple and easily available raw materials. Wherein the sucrose derivative 1 is prepared from sucrose which is cheap and easily available according to a literature method. The method for synthesizing the fructofuranosyl thioglycoside donor 2 alpha or 2 beta is simple and convenient to operate, and most intermediates in the synthesis can directly carry out subsequent reactions without further separation and purification, so that the continuous operation of multi-step reactions is realized, and the synthesis efficiency is improved.

6. The synthesis method of the kestose oligosaccharide has mild reaction conditions, especially the key glycosylation reaction can be implemented in a scale of tens of grams, and finally kestose oligosaccharide (trisaccharide to heptasaccharide) monomers can be obtained in a scale of grams, thereby laying a good material foundation for deep research and further development and application of pharmacological activity.

7. The reagents used in the method provided by the invention are all common chemical reagents, and special preparation is not needed; especially, the activating agent DBDMH used in the key glycosylation reaction has low price, so that the synthetic method has low cost. And the post-treatment of each reaction step is simple, a plurality of intermediates in the synthesis can be subjected to subsequent reaction without further separation and purification, the synthesis efficiency is high, and the operability is strong.

Detailed Description

The technical scheme of the present invention is described in further detail below, but the scope of the present invention is not limited to the following.

A process for the preparation of compound 2β of example 1 comprising the steps of:

compound 13β (84.0 g,0.375mol,1.0 equiv.) was dissolved in 420mL of dry pyridine, TBDPSCl (117 mL,0.450mol,1.2 equiv.) was added thereto at room temperature, and DMTrCl (152 g,0.450mol,1.2 equiv.) and a catalytic amount of DMAP (4.58 g,37.5mmol,0.1 equiv.) were added directly to the reaction after stirring overnight at room temperature. After 3h reaction at room temperature, pyridine is removed under reduced pressure, and the obtained concentrate is filtered through a silica gel pad to obtain a crude product of the compound 15 beta. The crude product was dissolved in dry DMF (100 mL), bnBr (133 mL,1.12mol,3.0 equiv.) was added thereto at 0deg.C, and NaH (60%in mineral oil,45g,1.12mol,3.0equiv.) was slowly added after stirring for 30 min. The reaction was then allowed to warm to room temperature and stirred for 3h, tlc monitoring indicated complete reaction of starting materials, Quenching the reaction with water in ice bath, extracting with EtOAc three times, combining the organic layers, washing with water twice, saturated NaCl solution once, adding anhydrous Na ₂ SO ₄ Drying and filtering, and concentrating the filtrate under reduced pressure to obtain a crude product. The crude product was dissolved in 100mL of dry THF, TBAF (117 g,0.450mol,1.2 equiv.) was added, and saturated NH was added after stirring at room temperature for 4h ₄ The Cl solution was quenched and the mixture was extracted three times with ethyl acetate and the organic phase was washed once with water, saturated NaCl solution. The organic layer was treated with anhydrous Na ₂ SO ₄ Drying and filtering, and concentrating the filtrate. The crude product obtained was dissolved in 100mL dry CH ₂ Cl ₂ To this was added 2-picolinic acid (55.4 g,0.450mol,1.2 equiv.) EDCI (86.3 g,0.450mol,1.2 equiv.) and DMAP (4.58 g,37.5mmol,0.1 equiv.) in this order at room temperature, reacted under Ar for 2 hours, the solvent was evaporated under reduced pressure, and the crude product was dissolved in EtOAc and diluted with water. The resulting mixture was filtered through celite and the aqueous layer was extracted three times with EtOAc. The organic layers were combined and washed once with water and once with saturated brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Drying, filtering and decompressing to concentrate, and the obtained crude product is directly subjected to the next reaction without separation and purification. Dissolving the crude product in CH ₂ Cl ₂ (80 mL) to which TFA (27.8 mL,0.375mol,1.0 equiv.) and Et were added sequentially at room temperature ₃ SiH (59.9 mL,0.375mol,1.0 equiv.) was reacted for 1.5h before saturated NaHCO was added under ice bath ₃ The reaction was quenched with solution. Layering, water layer with CH ₂ Cl ₂ The extraction was twice, the organic layers were combined and washed once with water and saturated brine in sequence. The organic phase was treated with anhydrous Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the crude product over a silica gel column (petroleum ether/etoac=1.5:1, v/v) afforded compound 19 β (53.4g,28%over 6steps).

Compound 19 beta (53.4 g,0.105mol,1.0 equiv.) is dissolved in dry CH under Ar protection ₂ Cl ₂ (150 mL) was added thereto Et at-30℃in sequence ₃ N (19.0 mL,0.136mol,1.3 equiv.) andTIPSOTf (33.8 mL,0.126mol,1.2 equiv.) was quenched with water after a further 2h reaction at this temperature. Layering, passing the aqueous layer through CH ₂ Cl ₂ Extracting for three times, mixing organic layers, and passing through anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave, after purification of the crude product by silica gel column (petroleum ether/etoac=8:1, v/v), compound 2α (66.2 g, 95%). TLC (petroleum ether/EtOAc=4:1, v/v), R _f ＝0.45； ¹ H NMR(400MHz,CDCl ₃ )δ8.78(d,J＝4.8Hz,1H),8.10(d,J＝8.0Hz,1H),7.81(td,J＝8.0,2.0Hz,1H),7.50–7.45(m,1H),7.44–7.18(m,10H),4.84(d,J＝11.6Hz,1H),4.77–4.52(m,6H),4.50–4.43(m,1H),4.34–4.26(m,1H),4.02(d,J＝11.2Hz,1H),3.90(d,J＝11.2Hz,1H),2.74–2.51(m,2H),1.15(t,J＝7.6Hz,3H),1.10–1.05(m,21H)； ¹³ C NMR(100MHz,CDCl ₃ )δ164.7,150.1,147.9,138.1,138.0,137.0,128.5,128.4,128.1,127.9,127.8,127.7,127.0,125.4,94.7,84.1,83.4,78.7,73.1,72.7,67.5,65.7,21.2,18.1,18.0,14.9,12.1.[α] _D ²⁵ ＝–46.5(c 0.87,CHCl ₃ )；IR(neat):ν _max ＝3032,2940,2865,1746,1724,1455,1303,1244,1084,994,882,799,737,695cm ^–1 ；HRMS(ESI):calcd.for C ₃₇ H ₅₁ NO ₆ SSi Na[M+Na] ⁺ m/z 688.3104；found m/z688.3099.

Example 2 a process for the preparation of compound 2α comprising the steps of:

the preparation of compound 19α in example 2 was performed in the same manner as the preparation of 19β in example 1. From compound 13α (108 g, 0.480 mol,1.0 equiv.) a six-step reaction afforded compound 19 α (63.8g,26%over 6steps). TLC (petroleum ether/EtOAc=1:2.5, v/v), R _f ＝0.55； ¹ H NMR(400MHz,CDCl ₃ )δ8.72(ddd,J＝4.8,1.6,0.8Hz,1H),8.05(dt,J＝8.0,1.2Hz,1H),7.76(td,J＝8.0,2.0Hz,1H),7.46(ddd,J＝8.0,4.8,1.2Hz,1H),7.38–7.19(m,10H),4.79(d,J＝11.2Hz,1H),4.67–4.59(m,2H),4.59–4.49(m,3H),4.34–4.29(m,2H),4.18–4.14(m,1H),3.93(d,J＝12.4Hz,1H),3.86(d,J＝12.0Hz,1H),2.75–2.59(m,2H),1.26(t,J＝7.2Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ )δ164.9,149.9,147.7,137.8,137.4,137.2,128.7,128.5,128.4,128.2,128.0,127.2,125.3,94.1,89.9,82.8,77.5,73.4,73.0,64.5,63.7,22.6,15.4.[α] _D ²⁵ ＝+95.7(c 0.33,CHCl ₃ )；IR(neat):ν _max ＝2926,2870,1725,1453,1290,1244,1109,1067,1044,995,739,697cm ^–1 ；HRMS(ESI):calcd.for C ₂₈ H ₃₁ NO ₆ SNa[M+Na] ⁺ m/z 532.1770；found m/z 532.1763.

Compound 19 alpha (72.2 g,0.142mol,1.0 equiv.) is dissolved in dry CH under Ar protection ₂ Cl ₂ (350 mL) was added thereto Et at-30℃in sequence ₃ N (25.6 mL,0.184mol,1.3 equiv.) and TIPSOTf (45.8 mL,0.170mol,1.2 equiv.) were reacted for 2h at this temperature followed by quenching with water. Layering, passing the aqueous layer through CH ₂ Cl ₂ Extracting for three times, mixing organic layers, and passing through anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave, after purification of the crude product by silica gel column (petroleum ether/etoac=8:1, v/v), compound 7 (87.7 g, 93%) as a colourless oil. TLC (petroleum ether/EtOAc=4:1, v/v), R _f ＝0.45； ¹ H NMR(400MHz,CDCl ₃ )δ8.75(d,J＝4.8Hz,1H),8.02(d,J＝8.0Hz,1H),7.69(td,J＝8.0,2.0Hz,1H),7.47–7.40(m,1H),7.36–7.21(m,10H),4.73(d,J＝11.6Hz,1H),4.65–4.47(m,5H),4.45–4.37(m,1H),4.16–4.09(m,2H),3.95(q,J＝10.4Hz,2H),2.77–2.64(m,2H),1.25(t,J＝7.2Hz,3H),1.08–1.02(m,21H). ¹³ C NMR(100MHz,CDCl ₃ )δ164.8,150.1,147.9,138.0,137.9,137.0,128.5,128.4,128.0,127.9,127.8,126.9,125.4,94.6,89.4,84.3,78.1,73.2,72.6,65.9,65.3,22.6,18.2,18.1,14.9,12.2.[α] _D ²⁵ ＝+44.5(c 0.80,CHCl ₃ )；IR(neat):ν _max ＝2941,2865,1745,1724,1455,1304,1244,1091,1068,994,883,801,742,697cm ^–1 ；HRMS(ESI):calcd.for C ₃₇ H ₅₁ NO ₆ SSiNa[M+Na] ⁺ m/z 688.3104；found m/z688.3100.

Example 3 preparation of kestose derivative 4 comprising the steps of:

glycosyl acceptor 1 (21.2 g,19.6mmol,1.0 equiv.), glycosylation promoter DBDMH (6.80 g,23.8mmol,1.2 equiv.) and freshly activatedMS (35 g) was weighed into a round bottom flask, ar was exchanged and dry toluene (concentration: 0.5M, calculated as glycosyl donor) was added. After stirring the mixture at-78 ℃ for 1 hour, it was moved to-35 ℃ and a dry toluene solution of glycosyl donor 2 (2α or 2β,21.2g,19.6mmol,1.0 equiv.) was added thereto, and the reaction was continued at-35 ℃ for 10 hours after the addition. TLC monitoring showed that after the starting material had reacted, the +.>MS, filter cake was washed with EtOAc and filtrate was taken up in saturated Na ₂ S ₂ O ₃ And NaHCO ₃ The solution was stirred at room temperature until it became pale yellow, the layers were separated, the aqueous layer was extracted three times with EtOAc, the organic layers were combined, and washed sequentially with water and saturated brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Drying, filtering, concentrating, purifying the crude product with silica gel column (petroleum ether/acetate=5:1 to 3:1, v/v) (note: crude product can also be directly used for next TIPS protection) to obtain compound 3 (29.5 g,89%, beta/alpha)>20:1)。TLC:(petroleum ether/acetone＝2:1,v/v),R _f ＝0.55； ¹ H NMR(400MHz,CDCl ₃ )δ8.71(d,J＝4.0Hz,1H),8.19(d,J＝7.2Hz,2H),8.02(d,J＝7.2Hz,2H),8.00–7.92(m,5H),7.81(t,J＝6.8Hz,6H),7.73(td,J＝7.6,1.6Hz,1H),7.57–7.10(m,32H),6.26–6.16(m,2H),6.13(d,J＝3.7Hz,1H),5.97(t,J＝7.2Hz,1H),5.74(t,J＝10.0Hz,1H),5.37(dd,J＝10.4,3.6Hz,1H),4.87(d,J＝11.6Hz,1H),4.74(dt,J＝10.0,3.2Hz,1H),4.69(d,J＝5.2Hz,2H),4.59–4.29(m,9H),4.13–4.06(m,2H),4.01(t,J＝7.2Hz,1H),3.95(d,J＝10.0Hz,1H),3.74(s,2H),0.99(s,21H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,165.7,165.7,165.5,165.2,164.5,150.0,147.8,138.3,138.1,136.9,133.5,133.5,133.4,133.3,133.1,133.0,130.3,130.1,130.0,129.9,129.9,129.8,129.8,129.7,129.7,129.4,129.3,129.2,129.0,128.9,128.8,128.5,128.4,128.4,128.4,128.3,128.3,128.1,127.7,127.6,127.5,126.9,125.4,105.2,105.1,90.5,83.2,83.0,78.5,77.4,77.3,76.0,72.5,72.3,71.2,70.6,69.2,69.0,65.9,65.5,64.7,63.3,62.5,18.1,18.0,11.9；[α] _D ²⁵ ＝+26.0(c 1.37,CHCl ₃ )；IR(neat):ν _max ＝3063,2944,2866,1723,1452,1262,1092,1068,1024,736,705cm ^–1 ；HRMS(ESI):calcd.for C ₉₆ H ₉₅ NO ₂₄ SiNa[M+Na] ⁺ m/z 1697.5945；found m/z 1697.5986.

Compound 3 (75.8 g,45.3 mmol) was dissolved in 60mL dry THF to which Et was added ₃ N.3HF (60 mL) was then allowed to react overnight at 75deg.C. TLC monitoring showed that after the starting material had reacted, saturated NaHCO was slowly added under ice bath ₃ The solution was quenched and the resulting mixture was extracted three times with EtOAc, the organic layers were combined, washed once with water and once with saturated brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Dried, filtered, concentrated under reduced pressure, and the resulting crude product was purified by column on silica gel (petroleum ether/acetone=3:1 to 2:1, v/v) to give 4 (62.5 g, 91%) as a white foam solid. TLC (petroleum ether/acetate=1.5:1, v/v), R _f ＝0.45； ¹ H NMR(400MHz,CDCl ₃ )δ8.73(d,J＝4.8Hz,1H),8.22(d,J＝7.2Hz,2H),8.07–8.01(m,3H),7.96(d,J＝7.2Hz,2H),7.90(d,J＝7.2Hz,2H),7.82–7.74(m,7H),7.61–7.46(m,6H),7.42–7.28(m,12H),7.28–7.17(m,12H),7.16–7.11(m,2H),6.20(d,J＝7.6Hz,1H),6.16–6.03(m,3H),5.73(t,J＝10.0Hz,1H),5.36–5.27(m,1H),4.92(d,J＝11.6Hz,1H),4.73(dd,J＝12.0,4.8Hz,1H),4.70–4.61(m,3H),4.60–4.51(m,2H),4.48–4.39(m,2H),4.39–4.26(m,3H),4.20–4.12(m,2H),4.05(t,J＝5.6Hz,1H),3.96–3.83(m,2H),3.53(d,J＝12.4Hz,1H),3.34–3.22(m,1H),2.76(br s,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.4,166.2,166.1,165.7,165.6,165.5,165.2,164.6,150,0,147.8,138.4,137.7,137.1,133.9,133.6,133.5,133.4,133.2,133.1,130.4,130.1,130.0,130.0,129.9,129.9,129.8,129.7,129.7,129.2,129.0,129.0,129.0,128.9,128.6,128.5,128.4,128.4,128.0,127.8,127.7,127.0,125.6,105.3,104.6,90.3,86.0,84.3,78.5,78.0,76.9,75.1,72.8,72.3,71.2,70.4,69.1,69.1,65.4,64.4,63.0,62.9,62.7；[α] _D ²⁵ ＝+11.9(c 1.33,CHCl ₃ )；IR(neat):ν _max ＝3537,2960,1722,1452,1261,1091,1067,1024,750,704cm ^–1 ；HRMS(ESI):calcd.for C ₈₇ H ₇₅ NO ₂₄ Na[M+Na] ⁺ m/z1541.4610；found m/z 1541.4646.

Example 4 preparation of kestose derivative 6 comprising the steps of:

glycosyl acceptor 4 (27.2 g,17.9mmol,1.0 equiv.), glycosylation promoter DBDMH (7.69 g,26.9mmol,1.5 equiv.) and freshly activated MS (40 g) was weighed into a round-bottomed flask, dried toluene (concentration: 0.5M calculated as glycosyl donor) was added after Ar exchange, the mixture was stirred at-78℃for 1 hour, then it was moved to-35℃to which was added a dried toluene solution of glycosyl donor (compound 2. Alpha. Or 2. Beta., 17.9g,26.9mmol,1.5 equiv.) and the reaction was continued at-35℃for 44 hours. TLC monitoring showed that after the starting material had reacted, the +.>MS, filter cake was washed with EtOAc and filtrate was taken up in saturated Na ₂ S ₂ O ₃ And NaHCO ₃ The solution was stirred at room temperature until the color became pale yellow, the layers were separated, the aqueous layer was extracted three times with EtOAc, the organic layers were combined, washed sequentially with water, saturated brine, and the organic layers were washed with anhydrous Na ₂ SO ₄ Drying, filtering, concentrating under reduced pressurePurifying the crude product with silica gel column (petroleum ether/acetate=4:1 to 3:1, v/v) (note: crude product can also be directly used for next TIPS protection) to obtain compound 5 (31.6 g,83%, beta/alpha)>20:1)。TLC:(petroleum ether/acetone＝1.5:1,v/v),R _f ＝0.48； ¹ H NMR(400MHz,CDCl ₃ )δ8.64(t,J＝4.4Hz,2H),8.14(d,J＝8.0Hz,2H),8.01(d,J＝7.6Hz,2H),7.97–7.73(m,12H),7.68–7.57(m,2H),7.53–7.44(m,4H),7.43–7.06(m,39H),6.26–6.02(m,4H),5.74(t,J＝10.0Hz,1H),5.32(dd,J＝10.4,4.0Hz,1H),4.86–4.75(m,2H),4.71–4.09(m,23H),4.05–3.94(m,3H),3.86(d,J＝10.4Hz,1H),3.79(d,J＝10.4Hz,1H),1.04–0.98(m,21H)； ¹³ C NMR(150MHz,CDCl ₃ )δ166.2,166.1,165.8,165.7,165.6,165.5,165.1,164.5,164.4,149.9,149.9,147.8,147.7,138.6,138.3,138.2,138.1,137.0,136.8,133.6,133.4,133.3,133.1,133.0,133.0,130.2,130.1,130.0,129.9,129.9,129.8,129.7,129.3,129.1,129.0,128.9,128.8,128.5,128.5,128.4,128.4,128.4,128.3,128.3,128.3,127.9,127.8,127.7,127.7,127.7,127.6,127.6,127.4,126.8,126.7,125.5,125.3,105.3,104.7,104.6,90.3,83.9,83.8,83.6,83.3,77.7,77.6,77.4,76.9,75.0,72.6,72.5,72.4,72.3,71.2,70.5,69.1,69.0,66.7,65.9,65.7,64.2,63.9,63.2,62.5,18.1,18.0,12.0；[α] _D ²⁵ ＝+19.1(c 1.67,CHCl ₃ )；IR(neat):ν _max ＝2944,2866,1724,1452,1263,1092,1068,1025,736,705cm ^–1 ；HRMS(ESI):calcd.for C ₁₂₂ H ₁₂₀ N ₂ O ₃₀ SiNa[M+Na] ⁺ m/z 2143.7593；found m/z 2143.8081.

Compound 5 (67.1 g,31.6 mmol) was dissolved in 50mL dry THF and Et was added ₃ N.3HF (50 mL) was then allowed to react overnight at 75deg.C. TLC monitoring showed that after the starting material had reacted, saturated NaHCO was slowly added under ice bath ₃ The reaction was quenched and the resulting mixture extracted three times with EtOAc, and the combined organic layers were washed once with water and brine. Adding anhydrous Na into the organic layer ₂ SO ₄ The crude product was concentrated by filtration and purified by column chromatography on silica gel (petroleum ether/acetone=3:1 to 1.5:1, v/v) to give 6 (54.7 g, 88%) as a white foam. TLC (petroleum ether/acetate=1.2:1, v/v), R _f ＝0.40； ¹ H NMR(400MHz,CDCl ₃ )δ8.65(t,J＝5.6Hz,2H),8.14(d,J＝7.6Hz,2H),8.04–7.88(m,8H),7.84–7.78(m,6H),7.71–7.59(m,2H),7.54–7.44(m,4H),7.43–7.07(m,39H),6.28–6.12(m,3H),6.01(t,J＝7.6Hz,1H),5.75(t,J＝10.0Hz,1H),5.35(dd,J＝10.4,3.6Hz,1H),4.79–4.67(m,5H),4.64–4.45(m,8H),4.39–4.10(m,11H),4.05(q,J＝6.0Hz,1H),3.99(d,J＝10.0Hz,1H),3.92(t,J＝6.4Hz,1H),3.89–3.80(m,2H),3.66(d,J＝12.0Hz,1H),3.55(d,J＝12.4Hz,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,166.1,165.8,165.7,165.7,165.5,165.2,164.7,164.4,150.0,149.9,147.7,147.6,138.4,138.0,137.9,137.8,137.1,137.0,133.7,133.4,133.4,133.1,133.0,130.2,130.1,130.0,129.9,129.9,129.8,129.7,129.3,129.2,129.1,129.0,128.9,128.8,128.5,128.5,128.4,128.4,128.4,128.4,128.3,128.3,128.1,128.0,127.8,127.8,127.7,127.7,127.6,127.0,126.9,125.4,105.0,105.0,104.8,90.6,85.4,84.4,83.5,83.4,78.3,77.9,77.33,76.6,75.7,72.8,72.7,72.4,72.2,71.2,70.5,69.1,69.1,65.7,65.3,64.6,64.03,63.7,63.4,62.5；[α] _D ²⁵ ＝+18.3(c 1.63,CHCl ₃ )；IR(neat):ν _max ＝3063,1723,1452,1263,1091,1068,1025,736,703cm ^–1 ；HRMS(ESI):calcd.for C ₁₁₃ H ₁₀₀ N ₂ O ₃₀ Na[M+Na] ⁺ m/z 1987.6259；found m/z1987.6278.

Example 5 preparation of kestose derivative 8 comprising the steps of:

glycosyl acceptor 6 (21.5 g,10.9mmol,1.0 equiv.), glycosylation promoter DBDMH (7.69 g,26.9mmol,1.5 equiv.) and freshly activatedMS (20 g) was weighed into a round-bottomed flask, dried toluene (concentration: 0.5M, calculated as glycosyl donor) was added thereto after Ar was exchanged, and the mixture was stirred at-78℃for 1 hour, then was transferred to-35℃and glycosyl donor (Compound 2αOr 2β,14.5g,21.9mmol,2.0 equiv.) in dry toluene, the reaction was continued at-35℃for 60 hours after the addition. TLC monitoring showed that after the starting material had reacted, the +.>MS, filter cake was washed with EtOAc and filtrate was taken up in saturated Na ₂ S ₂ O ₃ And NaHCO ₃ The solution was stirred at room temperature until the color became pale yellow, the layers were separated, the aqueous layer was extracted three times with EtOAc, the combined organic layers were washed sequentially with water, saturated brine, and the organic layers were washed with anhydrous Na ₂ SO ₄ Drying, filtering, concentrating under reduced pressure, purifying the crude product with silica gel column (petroleum ether/acetone=2:1, v/v) (note: crude product can also be directly used for next TIPS protection) to obtain compound 7 (21.9 g,78%, beta/alpha) >20:1)。TLC:(petroleum ether/acetone＝1.2:1,v/v),R _f ＝0.45； ¹ H NMR(400MHz,CDCl ₃ )δ8.66(m,2H),8.56(dd,J＝4.8,1.6Hz,1H),8.11(d,J＝7.6Hz,2H),8.01(d,J＝8.0Hz,2H),7.94(d,J＝7.6Hz,2H),7.91–7.75(m,11H),7.66–7.58(m,2H),7.52–7.42(m,6H),7.41–7.01(m,52H),6.25–6.04(m,4H),5.75(t,J＝10.0Hz,1H),5.32(dd,J＝10.4,4.0Hz,1H),4.88(d,J＝11.6Hz,1H),4.77(dd,J＝12.0,3.2Hz,2H),4.68–4.43(m,19H),4.41–4.14(m,12H),4.13–4.00(m,3H),3.96(dd,J＝9.6,4.8Hz,2H),3.82–3.68(m,2H),0.95(m,21H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,166.1,165.8,165.7,165.6,165.5,165.1,164.6,164.5,164.3,150.0,149.9,149.8,147.7,147.6,138.6,138.5,138.2,138.1,138.0,136.9,136.9,136.8,133.6,133.4,133.3,133.1,133.0,133.0,130.2,130.1,130.0,129.9,129.8,129.8,129.3,129.2,129.1,129.0,128.8,128.7,128.5,128.4,128.4,128.3,128.3,128.3,128.2,127.9,127.8,127.8,127.8,127.7,127.7,127.6,127.6,127.5,127.4,126.9,126.8,126.7,125.4,125.3,125.3,105.1,104.8,104.7,104.5,90.4,84.3,83.9,83.7,83.5,83.2,83.0,77.6,77.4,74.9,72.7,72.6,72.5,72.4,72.3,71.2,70.6,69.1,69.0,66.8,66.3,65.8,65.5,64.4,64.3,63.9,62.9,62.4,18.0,18.0,11.9；[α] _D ²⁵ ＝+11.4(c 1.00,CHCl ₃ )；IR(neat):ν _max ＝2922,2865,1724,1452,1260,1092,1068,1025,738,706cm ^–1 ；HRMS(ESI):calcd.for C ₁₄₈ H ₁₄₅ N ₃ O ₃₆ SiNa[M+Na] ⁺ m/z 2591.9308；found m/z 2591.9996.

Compound 7 (24.2 g,9.42 mmol) was dissolved in 20mL dry THF and Et was added ₃ N.3HF (20 mL) was then allowed to react overnight at 75deg.C. TLC monitoring showed that after the starting material had reacted, saturated NaHCO was slowly added under ice bath ₃ The reaction was quenched and the resulting mixture extracted three times with EtOAc, and the combined organic layers were washed once with water and brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave crude product which was purified by silica gel column (petroleum ether/acetone=2:1 to 1.5:1, v/v) to give 8 (19.3 g, 85%) as a white foam solid. TLC (petroleum ether/acetate=1.2:1, v/v), R _f ＝0.35； ¹ H NMR(400MHz,CDCl ₃ )δ8.65(dd,J＝8.4,4.8Hz,2H),8.60(d,J＝4.8Hz,1H),8.13(d,J＝8.0Hz,2H),8.01(d,J＝8.0Hz,2H),7.93(t,J＝7.2Hz,4H),7.86–7.76(m,9H),7.65(t,J＝8.0Hz,1H),7.57(dd,J＝14.8,7.6Hz,2H),7.52–7.42(m,4H),7.40–7.03(m,50H),6.24–6.12(m,3H),6.08(t,J＝8.0Hz,1H),5.75(t,J＝10.0Hz,1H),5.32(dd,J＝10.4,4.0Hz,1H),4.89(d,J＝11.6Hz,1H),4.79(d,J＝12.0Hz,1H),4.72(d,J＝11.6Hz,1H),4.69–4.46(m,16H),4.45–4.34(m,4H),4.31–4.10(m,11H),4.07–4.00(m,2H),3.99–3.89(m,3H),3.56–3.73(m,2H),2.90(br s,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,166.1,166.0,165.8,165.6,165.5,165.1,164.8,164.6,164.4,150.0,149.9,149.8,147.7,147.6,147.6,138.6,138.4,138.0,138.0,137.9,137.8,137.0,137.0,136.9,133.7,133.4,133.3,133.2,133.0,133.0,130.2,130.0,130.0,129.9,129.8,129.8,129.7,129.2,129.2,129.0,128.8,128.5,128.4,128.4,128.4,128.3,128.3,128.2,128.1,127.9,127.8,127.8,127.8,127.7,127.7,127.6,127.6,127.4,127.0,126.9,126.8,125.4,125.3,125.3,105.0,104.9,104.7,104.6,90.3,85.4,84.8,84.1,83.8,83.4,83.0,77.9,77.6,77.4,76.9,75.0,72.8,72.7,72.7,72.5,72.3,71.2,70.6,69.1,66.0,65.7,65.5,64.4,64.2,64.1,63.9,63.0,62.4；[α] _D ²⁵ ＝+32.0(c 0.93,CHCl ₃ )；IR(neat):ν _max ＝2941,1724,1452,1305,1264,1246,1091,1068,1026,735,698cm ^–1 ；HRMS(ESI):calcd.for C ₁₃₉ H ₁₂₅ N ₃ O ₃₆ Na[M+Na] ⁺ m/z2435.7974；found m/z 2435.8517.

Example 6 preparation of kestose derivative 10 comprising the steps of:

glycosyl acceptor 8 (20.9 g,8.66mmol,1.0 equiv.), glycosylation promoter DBDMH (4.95 g,17.3mmol,2.0 equiv.) and freshly activatedMS (25 g) was weighed into a round-bottomed flask, dried toluene (concentration: 0.5M calculated as glycosyl donor) was added after Ar was exchanged, the mixture was stirred at-78℃for 1 hour, then it was moved to-35℃to which was added a dried toluene solution of glycosyl donor (Compound 2. Alpha. Or 2. Beta., 11.5g,17.3mmol,2.0 equiv.) and the reaction was continued at-35℃for 80 hours after the addition. TLC monitoring showed that after the starting material had reacted, the +. >MS, filter cake was washed with EtOAc and filtrate was taken up in saturated Na ₂ S ₂ O ₃ And NaHCO ₃ The solution was stirred at room temperature until the color became pale yellow, the layers were separated, the aqueous layer was extracted three times with EtOAc, the combined organic layers were washed sequentially with water, saturated brine, and the organic layers were washed with anhydrous Na ₂ SO ₄ Drying, filtering, concentrating under reduced pressure, purifying the crude product with silica gel column (petroleum ether/acetate=7:4 to 5:4, v/v) (note: crude product can also be directly used for next TIPS protection) to obtain compound 9 (19.6 g,75%, beta/alpha)>20:1)。TLC:(petroleum ether/acetone＝1:1,v/v),R _f ＝0.58； ¹ H NMR(400MHz,CDCl ₃ )δ8.64(dd,J＝9.2,4.8Hz,2H),8.58(d,J＝4.8Hz,1H),8.52(d,J＝4.4Hz,1H),8.08(d,J＝7.6Hz,2H),8.00(d,J＝7.6Hz,2H),7.93(d,J＝7.6Hz,2H),7.89(d,J＝7.6Hz,1H),7.87–7.73(m,10H),7.70(d,J＝8.0Hz,1H),7.64(td,J＝8.0,2.0Hz,1H),7.58(td,J＝7.6,1.6Hz,1H),7.51–6.94(m,67H),6.27–6.04(m,4H),5.75(t,J＝10.0Hz,1H),5.32(dd,J＝10.0,3.6Hz,1H),4.89(d,J＝11.6Hz,1H),4.80–4.73(m,2H),4.69–3.95(m,45H),3.90(dd,J＝9.6,5.2Hz,2H),3.77–3.65(m,2H),0.98–0.92(m,21H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,166.1,165.9,165.8,165.6,165.5,165.1,164.6,164.6,164.6,164.4,150.0,149.9,149.9,149.8,147.7,147.7,147.6,147.6,138.6,138.5,138.3,138.2,138.1,138.1,138.0,137.0,136.9,136.9,136.8,133.5,133.4,133.3,133.1,133.0,133.0,130.2,130.1,130.0,129.9,129.8,129.8,129.7,129.3,129.2,129.1,129.0,128.9,128.8,128.5,128.4,128.4,128.3,128.3,128.3,128.2,128.0,128.0,127.8,127.7,127.7,127.6,127.6,127.5,127.5,127.4,126.9,126.8,126.8,126.6,125.5,125.3,125.3,125.3,105.0,104.7,104.6,104.6,104.6,90.3,84.5,84.0,83.5,83.4,83.4,83.0,77.7,77.6,77.4,77.3,77.3,74.7,72.7,72.7,72.6,72.5,72.5,72.4,72.3,71.2,70.6,69.1,69.0,67.0,66.4,66.1,65.8,65.3,64.6,64.3,64.3,63.8,62.9,62.4,18.1,18.0,11.9；[α] _D ²⁵ ＝+9.75(c 1.07,CHCl ₃ )；IR(neat):ν _max ＝2924,1724,1452,1305,1264,1093,1069,1025,734,700cm ^–1 ；HRMS(ESI):calcd.for C ₁₇₄ H ₁₇₀ N ₄ O ₄₂ SiNa[M+Na] ⁺ m/z 3038.0957；found m/z 3038.0962.

Compound 9 (34.0 g,11.3 mmol) was dissolved in 30mL dry THF and Et was added ₃ N.3HF (30 mL) was then allowed to react overnight at 75deg.C. TLC monitoring showed that after the starting material had reacted, saturated NaHCO was slowly added under ice bath ₃ The reaction was quenched and the resulting mixture was extracted three times with EtOAc, and the combined organic layers were washed once with water and brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Dried, filtered, concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (petroleum ether/acetone=1.5:1, v/v) to give 16 (26.4 g, 82%) as a white foam solid. TLC (petroleum ether/acetate=1:1, v/v), R _f ＝0.45； ¹ H NMR(400MHz,CDCl ₃ )δ8.64–8.58(m,3H),8.55(d,J＝4.8Hz,1H),8.08(d,J＝7.6Hz,1H),8.01(d,J＝7.6Hz,2H),7.96(d,J＝8.0Hz,2H),7.90(d,J＝8.0Hz,3H),7.88–7.71(m,10H),7.63(td,J＝7.6,1.6Hz,1H),7.58(td,J＝7.6,1.6Hz,1H),7.53(td,J＝7.6,1.6Hz,1H),7.50–7.03(m,67H),6.30–6.13(m,3H),6.07(t,J＝8.0Hz,1H),5.77(t,J＝10.0Hz,1H),5.35(dd,J＝10.4,3.6Hz,1H),4.87(d,J＝11.6Hz,1H),4.81–3.94(m,47H),3.91–3.81(m,2H),3.60(d,J＝11.6Hz,1H),3.51(d,J＝12.0Hz,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,166.0,165.9,165.7,165.6,165.5,165.08,164.7,164.6,164.5,164.4,150.0,149.9,149.8,149.7,147.6,147.5,147.5,138.6,138.4,138.3,138.1,138.0,137.9,137.8,137.0,136.9,136.9,136.8,133.5,133.4,133.3,133.1,133.0,130.2,130.0,129.9,129.9,129.8,129.8,129.7,129.7,129.2,129.2,129.0,128.8,128.7,128.4,128.4,128.4,128.3,128.3,128.2,128.1,128.0,127.9,127.9,127.8,127.8,127.8,127.8,127.7,127.7,127.7,127.6,127.6,127.5,127.4,127.4,127.0,126.9,126.8,126.6,125.4,125.3,125.3,125.2,104.9,104.7,104.6,104.6,104.5,90.4,85.2,85.0,84.1,83.6,83.6,83.4,83.0,82.9,77.8,77.7,77.6,77.4,77.2,77.0,74.9,72.8,72.6,72.5,72.4,72.4,71.1,70.6,69.1,66.3,65.9,65.7,65.3,64.5,64.4,64.3,64.0,63.8,62.7,62.3；[α] _D ²⁵ ＝+7.23(c 2.93,CHCl ₃ )；IR(neat):ν _max ＝3063,2923,1723,1452,1304,1284,1246,1091,1068,1025,734,698cm ^–1 ；HRMS(ESI):calcd.for C ₁₆₅ H ₁₅₀ N ₄ O ₄₂ Na[M+Na] ⁺ m/z 2882.9656；found m/z 2882.9904.

Example 7 preparation of kestose derivative 12 comprising the steps of:

glycosyl acceptor 10 (20.2 g,7.06mmol,1.0 equiv.), glycosylation promoter DBDMH (10.1 g,35.3mmol,5.0 equiv.) and freshly activated MS (35 g) was weighed into a round-bottomed flask, dried toluene (concentration: 0.5M calculated as glycosyl donor) was added after Ar was exchanged, the mixture was stirred at-78℃for 1 hour, then was moved to-35℃and glycosyl donor (Compound 2. Alpha. Or 2. Beta., 23)5g,35.3mmol,5.0 equiv.) of dry toluene solution, the reaction was continued at-35℃for 90 hours after addition. TLC monitoring showed that after the starting material had reacted, the +.>MS, filter cake was washed with EtOAc and filtrate was taken up in saturated Na ₂ S ₂ O ₃ And NaHCO ₃ The solution was stirred at room temperature until the color became pale yellow, the layers were separated, the aqueous layer was extracted three times with EtOAc, the combined organic layers were washed sequentially with water, saturated brine, and the organic layers were washed with anhydrous Na ₂ SO ₄ Drying, filtering, concentrating under reduced pressure, purifying the crude product with silica gel column (petroleum ether/acetate=2:1 to 1.5:1, v/v) (note: crude product can also be directly used for next TIPS protection) to obtain compound 11 (15.4 g,63%, beta/alpha)>20:1)。TLC:(petroleum ether/acetone＝1:1,v/v),R _f ＝0.52； ¹ H NMR(400MHz,CDCl ₃ )δ8.64(dd,J＝13.6,4.8Hz,2H),8.59–8.50(m,3H),8.08(d,J＝8.0Hz,2H),8.01(d,J＝7.6Hz,2H),7.94(d,J＝7.6Hz,2H),7.89(d,J＝8.0Hz,1H),7.79(m,10H),7.70–7.61(m,2H),7.56(td,J＝8.0,1.6Hz,1H),7.52–6.99(m,77H),6.97–6.88(m,3H),6.27–6.04(m,4H),5.76(t,J＝9.6Hz,1H),5.33(dd,J＝10.4,4.0Hz,1H),4.89(d,J＝11.6Hz,1H),4.79(d,J＝11.6Hz,1H),4.76–4.37(m,36H),4.36–4.17(m,10H),4.15–3.96(m,10H),3.90(d,J＝9.6Hz,1H),3.80(m,2H),3.70(q,J＝10.4Hz,2H),0.95(m,21H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,166.1,165.9,165.8,165.6,165.5,165.1,164.6,164.6,164.6,164.5,164.3,150.0,149.9,149.8,149.8,147.7,147.6,147.6,147.5,138.6,138.5,138.4,138.3,138.2,138.1,138.1,138.0,138.0,137.0,136.9,136.8,136.7,133.6,133.3,133.1,133.0,130.2,130.0,130.0,129.9,129.8,129.8,129.7,129.3,129.2,129.0,129.0,128.8,128.8,128.4,128.4,128.4,128.3,128.3,128.3,128.2,128.2,128.2,128.0,128.0,127.9,127.8,127.7,127.7,127.7,127.6,127.6,127.6,127.5,127.5,127.5,127.3,126.9,126.8,126.7,126.6,125.5,125.3,125.3,125.2,125.2,105.0,104.8,104.6,104.5,104.5,104.5,90.2,84.7,84.0,83.6,83.5,83.4,83.3,82.9,82.9,82.8,77.7,77.6,77.4,77.3,76.7,74.6,72.7,72.6,72.5,72.5,72.5,72.4,72.3,72.3,71.1,70.6,69.1,69.0,66.9,66.4,66.3,65.9,65.8,65.1,64.5,64.2,64.0,63.9,63.7,62.8,62.4,18.0,17.9,11.9；[α] _D ²⁵ ＝+7.74(c 1.27,CHCl ₃ )；IR(neat):ν _max ＝2920,2866,1724,1265,1246,1092,1068,1026,734,698cm ^–1 ；HRMS(ESI):calcd.for C ₂₀₀ H ₁₉₅ N ₅ O ₄₈ SiNa[M+Na] ⁺ m/z 3487.2706；found m/z3487.2816.

Compound 11 (15.0 g,4.33 mmol) was dissolved in 20mL dry THF and Et was added ₃ N.3HF (20 mL) was then allowed to react overnight at 75deg.C. TLC monitoring showed that after the starting material had reacted, saturated NaHCO was slowly added under ice bath ₃ The reaction was quenched and the resulting mixture extracted three times with EtOAc, and the combined organic layers were washed once with water and brine. The organic layer was treated with anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave crude product which was purified by silica gel column (petroleum ether/acetone=1.5:1, v/v) to give 12 (12.5 g, 88%) as a white foamy solid. TLC (petroleum ether/acetate=1:1, v/v), R _f ＝0.42； ¹ H NMR(400MHz,CDCl ₃ )δ8.64(dd,J＝14.4,4.8Hz,2H),8.57(dd,J＝11.2,4.8Hz,2H),8.51(d,J＝4.8Hz,1H),8.08(d,J＝8.0Hz,2H),8.01(d,J＝7.6Hz,2H),7.98–7.88(m,4H),7.84–7.74(m,10H),7.68–7.62(m,2H),7.59(t,J＝7.6Hz,1H),7.55–7.02(m,76H),7.01–6.90(m,3H),6.29–6.19(m,2H),6.18–6.06(m,2H),5.76(t,J＝9.6Hz,1H),5.33(dd,J＝10.4,3.6Hz,1H),4.91(d,J＝11.6Hz,1H),4.81–4.72(m,3H),4.71–4.33(m,36H),4.31–4.17(m,8H),4.16–4.02(m,8H),3.99(d,J＝9.6Hz,1H),3.94–3.81(m,4H),3.61(dd,J＝12.0,6.4Hz,1H),3.50(dd,J＝12.0,6.0Hz,1H),3.13(t,J＝6.8Hz,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,166.1,166.0,165.6,165.6,165.5,165.1,164.8,164.6,164.6,164.5,164.3,150.0,149.9,149.9,149.8,149.7,147.7,147.6,147.6,147.5,138.6,138.5,138.3,138.3,138.1,138.0,138.0,137.9,137.9,137.1,137.0,136.8,136.8,133.6,133.3,133.1,133.0,133.0,130.2,130.0,130.0,129.9,129.9,129.8,129.7,129.7,129.3,129.2,129.0,129.0,128.8,128.8,128.5,128.4,128.4,128.4,128.4,128.3,128.3,128.3,128.2,128.0,127.9,127.8,127.8,127.8,127.7,127.7,127.6,127.6,127.5,127.4,127.4,127.0,126.9,126.8,126.8,126.6,125.5,125.4,125.3,125.3,125.2,104.9,104.8,104.7,104.6,104.5,104.5,90.3,85.3,85.0,84.2,83.7,83.6,83.5,83.4,83.0,83.0,77.8,77.6,77.6,77.4,74.6,72.8,72.7,72.6,72.6,72.5,72.5,72.4,71.1,70.6,69.1,69.0,66.5,66.1,65.8,65.7,65.3,64.8,64.2,64.1,64.0,63.9,62.7,62.4；[α] _D ²⁵ ＝+5.58(c 1.43,CHCl ₃ )；IR(neat):ν _max ＝2941,1724,1305,1264,1246,1091,1068,1025,735,698cm ^–1 ；HRMS(ESI):calcd.for C ₁₉₁ H ₁₇₅ N ₅ O ₄₈ Na[M+Na] ⁺ m/z 3331.1371；found m/z 3331.1456.

Example 8 preparation of kestose IVa comprising the steps of:

compound 4 (8.50 g,5.60mmol,1.0 equiv.) was dissolved in CH ₂ Cl ₂ To MeOH (10:1, c=0.1M) mixed solvent, cu (OAc) was added thereto at room temperature ₂ ·H ₂ O (1.68 g,8.40mmol,1.5 equiv.) was stirred. TLC monitoring showed that after the starting material had reacted, saturated NH was added ₄ Quenching with Cl solution, filtering to remove insoluble solids, and filtering the filtrate with CH ₂ Cl ₂ Diluted, then washed twice with 1M HCl, saturated NaHCO in turn ₃ Washing once, washing once with water and washing once with saturated NaCl. Combining the aqueous layers with CH ₂ Cl ₂ Extracting twice, mixing organic layers, adding anhydrous Na ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain crude product. After dispersing the crude product in MeOH (c=0.1M), naOMe (5M/L in MeOH,1.12ml,5.60mmol,1.0 equiv.) was added thereto at room temperature and stirred for 30min, acidic ion exchange resin Amberlite IR 120 (H ⁺ ) Neutralization reaction, regulating pH to neutral or weak acidity, filtering resin, vacuum pumping the filtrate to remove solvent, purifying the crude product by silica gel column Chromatography (CH) ₂ Cl ₂ Meoh=6:1 to 4:1, v/v) to give glycoside IIIa (3.14g,82%over two steps). TLC (CH) ₂ Cl ₂ /MeOH＝4:1,v/v),R _f ＝0.40； ¹ H NMR(400MHz,CD ₃ OD)δ7.44–7.40(m,2H),7.37–7.22(m,8H),5.41(d,J＝4.0Hz,1H),4.82(s,1H),4.66–4.52(m,3H),4.36–4.29(m,2H),4.15(t,J＝7.2Hz,1H),4.10–4.03(m,1H),3.92–3.64(m,14H),3.42–3.33(m,2H)； ¹³ C NMR(150MHz,CD ₃ OD)δ139.5,139.5,129.4,129.3,129.3,128.8,128.7,128.7,106.3,105.1,93.9,85.7,83.7,83.4,81.5,78.4,74.9,74.6,74.2,73.6,73.4,73.1,71.4,65.4,63.4,63.2,63.0,62.2；[α] _D ²⁵ ＝+1.89(c 1.38,MeOH)；IR(neat):ν _max ＝3332,2940,2834,1453,1108,1016,738,698,554cm ^–1 ；HRMS(ESI):calcd.for C ₃₂ H ₄₄ O ₁₆ Na[M+Na] ⁺ m/z 707.2527；found m/z 707.2525.

Compound IIIa (3.00 g,4.38 mmol) and 10% Pd/C (1.20g,40%weight of IIIa) were dispersed in MeOH, the H was exchanged ₂ (1 atm) followed by reaction at room temperature for 11 hours. TLC monitoring showed that the starting material had disappeared, pd/C was removed by filtration through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH/H ₂ O=12:6:1 to 5:5:1) and purified by reverse phase silica gel column (H ₂ O) kestose IVa (1.70 g, 77%). TLC (CH) ₂ Cl ₂ /MeOH/H ₂ O＝5:5:1,v/v),R _f ＝0.40, ¹ H NMR(400MHz,D ₂ O,acetone as internal standard,at 2.22ppm)δ5.42(d,J＝3.6Hz,1H),4.27(d,J＝8.8Hz,1H),4.18(d,J＝8.4Hz,1H),4.10–4.00(m,2H),3.89–3.62(m,14H),3.53(dd,J＝10.0,3.6Hz,1H),3.46(t,J＝9.2Hz,1H)； ¹³ C NMR(100MHz,D ₂ O,acetone as internal standard,at 30.89ppm)δ104.4,103.9,93.1,81.9,81.8,77.3,77.3,75.1,74.5,73.2,73.1,71.8,69.8,63.0,62.8,61.5,61.0,60.7；[α] _D ²⁵ ＝+25.7(c 1.25,H ₂ O)；IR(neat):ν _max ＝3275,2932,1641,1417,1112,1021,988,925cm ^–1 ；HRMS(ESI):calcd.for C ₁₈ H ₃₂ O ₁₆ Na[M+Na] ⁺ m/z527.1588；found m/z 527.1585.

Example 9 preparation of kestose IVb comprising the following steps

Compound 10 (17.0 g,8.65mmol,1.0 equiv.) is dissolved in CH ₂ Cl ₂ To MeOH (10:1, c=0.1M) mixed solvent, cu (OAc) was added thereto at room temperature ₂ ·H ₂ O (4.32 g,21.6mmol,2.5 equiv.) was stirred. TLC monitoring showed that after the starting material had reacted, saturated NH was added ₄ Quenching with Cl solution, filtering to remove insoluble solids, and filtering the filtrate with CH ₂ Cl ₂ Diluted, then washed twice with 1M HCl, saturated NaHCO in turn ₃ The solution was washed once, water was washed once, and saturated NaCl solution was washed once. Combining the aqueous layers with CH ₂ Cl ₂ Extracting twice, mixing organic layers, adding anhydrous Na ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain crude product. After dispersing the crude product in MeOH (c=0.1M), naOMe (5M/L in MeOH,1.73ml,8.65mmol,1.0 equiv.) was added thereto at room temperature and stirred for 30min, acidic ion exchange resin Amberlite IR 120 (H ⁺ ) Neutralization reaction, regulating pH to neutral or weak acidity, filtering resin, vacuum pumping the filtrate to remove solvent, purifying the crude product by silica gel column Chromatography (CH) ₂ Cl ₂ Meoh=6:1 to 4:1, v/v) to give glycoside IIIb (6.65g,75%over two steps). TLC (CH) ₂ Cl ₂ /MeOH＝3.5:1,v/v),R _f ＝0.50； ¹ H NMR(400MHz,CD ₃ OD)δ7.39–7.11(m,20H),5.41(d,J＝4.0Hz,1H),4.81(d,J＝12.0Hz,1H),4.74(d,J＝11.6Hz,1H),4.64–4.50(m,6H),4.43(d,J＝7.2Hz,1H),4.34(t,J＝7.2Hz,2H),4.17–4.02(m,3H),3.93–3.58(m,19H),3.42–3.33(m,2H)； ¹³ C NMR(100MHz,CD ₃ OD)δ139.7,139.7,139.6,139.5,129.4,129.3,129.0,128.9,128.8,128.7,128.6,128.6,128.5,106.0,105.7,105.2,94.0,85.8,85.7,83.7,83.6,83.5,82.1,81.4,78.4,75.0,74.6,74.3,73.7,73.6,73.4,73.3,73.2,71.4,65.1,64.7,63.9,63.5,63.1,63.0,62.2；[α] _D ²⁵ ＝-8.0(c 0.95,MeOH)；IR(neat):ν _max ＝3349,2928,1454,1016,929,735,696cm ^–1 ；HRMS(ESI):calcd.for C ₅₂ H ₆₆ O ₂₁ Na[M+Na] ⁺ m/z 1049.3994；found m/z 1049.3990.

The compound is preparedIIIb (5.20 g,5.07 mmol) and 10% Pd/C (1.56g,30%weight of IIIb) were dispersed in MeOH, H was exchanged ₂ (1 atm) followed by reaction at room temperature for 24 hours. TLC monitoring showed that the starting material had disappeared, pd/C was removed by filtration through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH/H ₂ O=16:6:1 to 5:5:1) and purified by reverse phase silica gel column (H ₂ O) kestose IVb (2.83 g, 84%). TLC (CH) ₂ Cl ₂ /MeOH/H ₂ O＝5:5:1,v/v),R _f ＝0.45； ¹ H NMR(400MHz,D ₂ O,acetone as internal standard,at 2.22ppm)δ5.43(d,J＝3.2Hz,1H),4.29–4.15(m,3H),4.14–3.99(m,3H),3.90–3.64(m,19H),3.53(dd,J＝10.0,3.6Hz,1H),3.46(t,J＝10.0Hz,1H)； ¹³ C NMR(100MHz,D ₂ O,acetone as internal standard,at 30.89ppm)δ104.3,103.8,103.7,93.1,81.9,81.7,81.7,78.1,77.4,77.4,75.1,74.9,74.5,73.2,73.1,71.8,69.8,62.9,62.9,62.8,61.7,61.5,61.0,60.7；[α] _D ²⁵ ＝+9.80(c 1.72,H ₂ O)；IR(neat):ν _max ＝3283,2930,1642,1415,1335,1107,1016,988,926cm ^–1 ；HRMS(ESI):calcd.for C ₂₄ H ₄₂ O ₂₁ Na[M+Na] ⁺ m/z689.2116；found m/z 689.2116.

Example 10 preparation of kestose IVc comprising the following steps

Compound 8 (20.0 g,8.29mmol,1.0 equiv.) was dissolved in CH ₂ Cl ₂ To MeOH (10:1, c=0.1M) mixed solvent, cu (OAc) was added thereto at room temperature ₂ ·H ₂ O (7.45 g,37.3mmol,4.5 equiv.) was stirred. TLC monitoring showed that after the starting material had reacted, saturated NH was added ₄ Quenching with Cl solution, filtering to remove insoluble solids, and filtering the filtrate with CH ₂ Cl ₂ Diluted, then washed twice with 1M HCl, saturated NaHCO in turn ₃ The solution was washed once, water was washed once, and saturated NaCl solution was washed once. Combining the aqueous layers with CH ₂ Cl ₂ Extracting twice, mixing organic layers, adding anhydrous N a ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain crude product. After dispersing the crude product in MeOH (c=0.1M), naOMe (5M/L in MeOH,1.66ml,8.29mmol,1.0 equiv.) was added thereto at room temperature and stirred for 30min, acidic ion exchange resin Amberlite IR 120 (H ⁺ ) Neutralization reaction, regulating pH to neutral or weak acidity, filtering resin, vacuum pumping the filtrate to remove solvent, purifying the crude product by silica gel column Chromatography (CH) ₂ Cl ₂ Meoh=8:1 to 6:1, v/v) to give glycoside IIIc (9.08g,80%over two steps). TLC (CH) ₂ Cl ₂ /MeOH＝4:1,v/v),R _f ＝0.60； ¹ H NMR(400MHz,CD ₃ OD)δ7.38–7.13(m,30H),5.42(d,J＝4.0Hz,1H),4.83–4.69(m,3H),4.63–4.47(m,9H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.8Hz,2H),4.20–4.02(m,4H),3.99–3.63(m,23H),3.57(d,J＝12.0Hz,1H),3.44–3.33(m,2H)； ¹³ C NMR(100MHz,CD ₃ OD)δ139.9,139.8,139.7,139.6,139.6,139.5,129.4,129.3,129.3,129.0,128.9,128.9,128.8,128.7,128.6,128.6,128.6,128.5,128.4,106.0,105.7,105.4,105.2,94.0,86.2,86.0,85.5,83.7,83.6,83.6,83.5,82.1,81.9,81.4,78.4,75.1,74.6,74.3,73.9,73.7,73.6,73.4,73.4,73.2,73.1,71.4,65.1,64.8,64.5,63.8,63.6,63.4,63.1,62.2；[α] _D ²⁵ ＝-16.5(c 1.82,MeOH)；IR(neat):ν _max ＝3345,2936,1454,1018,933,735,696cm ^–1 ；HRMS(ESI):calcd.for C ₇₂ H ₈₈ O ₂₆ Na[M+Na] ⁺ m/z 1391.5462；found m/z 1391.5458.

Compound IIIc (9.00 g,6.57 mmol) and 10% Pd/C (2.70g,30%weight of IIIc) were dispersed in MeOH, the H was exchanged ₂ (1 atm) followed by reaction at 40℃overnight. TLC monitoring showed that the starting material had disappeared, pd/C was removed by filtration through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH/H ₂ O=20:7:1 to 4:3:1) and purified by reverse phase silica gel column (H ₂ O) kestose IVc (4.24 g, 78%). TLC (CH) ₂ Cl ₂ /MeOH/H ₂ O＝5:5:1,v/v),R _f ＝0.40； ¹ H NMR(400MHz,D ₂ O,acetone as internal standard,at 2.22ppm)δ5.43(d,J＝4.0Hz,1H),4.29–4.16(m,4H),4.13–4.01(m,4H),3.90–3.65(m,24H),3.53(dd,J＝10.0,3.6Hz,1H),3.46(t,J＝9.5Hz,1H)； ¹³ C NMR(100MHz,D ₂ O,acetone as internal standard,at 30.89ppm)δ104.3,103.8,103.7,103.7,93.1,81.9,81.7,81.7,81.7,78.2,78.0,77.4,77.4,75.1,75.0,74.9,74.5,73.2,73.1,71.8,69.8,62.9,62.8,62.8,62.8,61.6,61.5,61.3,61.1,60.8；[α] _D ²⁵ ＝+0.71(c 1.12,H ₂ O)；IR(neat):ν _max ＝3301,2930,1643,1414,1329,1112,1015,987,928cm ^–1 ；HRMS(ESI):calcd.for C ₃₀ H ₅₂ O ₂₆ Na[M+Na] ⁺ m/z 851.2645；found m/z 851.2647.

Example 11 preparation of Saccharohexaose IVd comprising the following steps

Compound 10 (18.0 g,6.29mmol,1.0 equiv.) is dissolved in CH ₂ Cl ₂ To MeOH (10:1, c=0.1M) mixed solvent, cu (OAc) was added thereto at room temperature ₂ ·H ₂ O (7.54 g,37.8mmol,6.0 equiv.) was stirred. TLC monitoring showed that after the starting material had reacted, saturated NH was added ₄ Quenching with Cl solution, filtering to remove insoluble solids, and filtering the filtrate with CH ₂ Cl ₂ Diluted, then washed twice with 1M HCl, saturated NaHCO in turn ₃ The solution was washed once, water was washed once, and saturated NaCl solution was washed once. Combining the aqueous layers with CH ₂ Cl ₂ Extracting twice, mixing organic layers, adding anhydrous Na ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain crude product. After dispersing the crude product in MeOH (c=0.1M), naOMe (5M/L in MeOH,1.26ml,6.29mmol,1.0 equiv.) was added thereto at room temperature and stirred for 30min, acidic ion exchange resin Amberlite IR 120 (H ⁺ ) Neutralization reaction, regulating pH to neutral or weak acidity, filtering resin, vacuum pumping the filtrate to remove solvent, purifying the crude product by silica gel column Chromatography (CH) ₂ Cl ₂ Meoh=6:1 to 5:1, v/v) to give glycoside IIId (7.76g,72%over two steps). TLC (CH) ₂ Cl ₂ /MeOH＝5:1,v/v),R _f ＝0.40； ¹ H NMR(400MHz,CD ₃ OD)δ7.37–7.11(m,40H),5.42(d,J＝4.0Hz,1H),4.84–4.66(m,4H),4.64–4.42(m,15H),4.35(dd,J＝8.0,3.6Hz,2H),4.20–4.09(m,4H),4.07(t,J＝8.0Hz,1H),4.04–3.62(m,28H),3.54(d,J＝12.0Hz,1H),3.43–3.33(m,2H)； ¹³ C NMR(100MHz,CD ₃ OD)δ140.0,139.9,139.7,139.7,139.7,139.6,139.6,139.5,129.4,129.3,129.3,129.0,128.9,128.9,128.9,128.8,128.7,128.6,128.6,128.4,106.0,105.7,105.5,105.4,105.2,94.0,86.1,86.1,85.9,85.4,83.8,83.6,83.6,83.5,83.4,82.1,81.9,81.8,81.4,78.4,75.1,74.7,74.3,73.9,73.8,73.7,73.6,73.4,73.4,73.3,73.2,73.2,71.4,65.0,64.8,64.7,64.6,63.8,63.5,63.4,63.3,63.1,63.0,62.2；[α] _D ²⁵ ＝-20.6(c 1.28,MeOH)；IR(neat):ν _max ＝3357,2928,1453,1018,935,734,696cm ^–1 ；HRMS(ESI):calcd.for C ₉₂ H ₁₁₀ O ₃₁ Na[M+Na] ⁺ m/z1733.6929；found m/z 1733.7068.

Compound IIId (6.70 g,3.91 mmol) and 10% Pd/C (2.34g,35%weight of IIId) were dispersed in MeOH (65 mL) and H was exchanged ₂ (1 atm) followed by reaction at 40℃for 17h. TLC monitoring showed that the starting material had disappeared, pd/C was removed by filtration through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH/H ₂ O=20:7:1 to 2.5:3:1) and purified by reverse phase silica gel column (H ₂ O) kestose IVd (3.64 g, 94%). TLC (CH) ₂ Cl ₂ /MeOH/H ₂ O＝2.5:3:1,v/v),R _f ＝0.40； ¹ H NMR(400MHz,D ₂ O,MeOH as internal standard,at 3.34ppm)δ5.42(d,J＝4.0Hz,1H),4.29–4.16(m,5H),4.12–4.00(m,5H),3.93–3.64(m,29H),3.53(dd,J＝10.0,4.0Hz,1H),3.46(t,J＝9.6Hz,1H)； ¹³ C NMR(100MHz,D ₂ O,MeOH as internal standard,at 49.5ppm)δ104.3,103.8,103.7,103.7,103.6,93.1,81.8,81.7,81.7,81.7,81.7,78.0,78.0,77.9,77.4,77.3,75.0,75.0,74.9,74.9,74.4,73.2,73.0,71.8,69.8,62.9,62.8,62.8,62.8,62.7,61.6,61.4,61.3,61.3,61.1,60.7；[α] _D ²⁵ ＝-7.96(c 2.25,H ₂ O)；IR(neat):ν _max ＝3343,2929,1415,1112,1026,931cm ^–1 ；HRMS(ESI):calcd.for C ₃₆ H ₆₂ O ₃₁ Na[M+Na] ⁺ m/z 1013.3173；found m/z 1013.3176.

Example 12 preparation of kestose IVe comprising the following steps

Compound 12 (12.2 g,3.69mmol,1.0 equiv.) is dissolved in CH ₂ Cl ₂ To MeOH (10:1, c=0.1M) mixed solvent, cu (OAc) was added thereto at room temperature ₂ ·H ₂ O (5.52 g,27.7mmol,7.5 equiv.) was stirred. TLC monitoring showed that after the starting material had reacted, saturated NH was added ₄ Quenching with Cl solution, filtering to remove insoluble solids, and filtering the filtrate with CH ₂ Cl ₂ Diluted, then washed twice with 1M HCl, saturated NaHCO in turn ₃ The solution was washed once, water was washed once, and saturated NaCl solution was washed once. Combining the aqueous layers with CH ₂ Cl ₂ Extracting twice, mixing organic layers, adding anhydrous Na ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain crude product. After dispersing the crude product in MeOH (c=0.1M), naOMe (5M/L in MeOH,0.74ml,3.69mmol,1.0 equiv.) was added thereto at room temperature and stirred for 30min, acidic ion exchange resin Amberlite IR 120 (H ⁺ ) Neutralization reaction, regulating pH to neutral or weak acidity, filtering resin, vacuum pumping the filtrate to remove solvent, purifying the crude product by silica gel column Chromatography (CH) ₂ Cl ₂ Meoh=10:1 to 8:1, v/v) to give glycoside IIIe (5.30g,70%over two steps). TLC (CH) ₂ Cl ₂ /MeOH＝6:1,v/v),R _f ＝0.60； ¹ H NMR(400MHz,CD ₃ OD)δ7.38–7.09(m,50H),5.41(d,J＝3.6Hz,1H),4.85–4.66(m,5H),4.63–4.44(m,19H),4.34(t,J＝8.4Hz,2H),4.21–3.58(m,40H),3.53(d,J＝12.0Hz,1H),3.43–3.32(m,2H)； ¹³ C NMR(100MHz,CD ₃ OD)δ140.0,139.9,139.7,139.7,139.7,139.6,139.6,139.6,139.5,129.4,129.4,129.4,129.4,129.3,129.3,129.0,129.0,129.0,128.9,128.9,128.9,128.8,128.7,128.6,128.6,128.5,128.4,106.1,105.7,105.5,105.4,105.4,105.2,94.0,85.9,85.8,85.8,85.5,83.8,83.6,83.6,83.5,83.4,83.2,82.1,81.8,81.8,81.8,81.4,78.4,75.1,74.7,74.3,73.9,73.8,73.8,73.6,73.6,73.4,73.4,73.3,73.3,73.3,73.2,71.4,65.1,64.9,64.8,64.8,64.7,63.8,63.5,63.4,63.4,63.4,63.0,63.0,62.2；[α] _D ²⁵ ＝-24.5(c 1.25,MeOH)；IR(neat):ν _max ＝3358,3032,2932,1454,1019,937,735,696cm ^–1 ；HRMS(ESI):calcd.for C ₁₁₂ H ₁₃₂ O ₃₆ Na[M+Na] ⁺ m/z 2075.8396；found m/z 2075.8388。

Compound IIIe (5.10 g,2.48 mmol) and 10% Pd/C (5.10g,100%weight of IIIe) were dispersed in MeOH, the H was exchanged ₂ (1 atm) followed by reaction at 40℃for 20h. TLC monitoring showed that the starting material had disappeared, pd/C was removed by filtration through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (CH ₂ Cl ₂ /MeOH/H ₂ O=20:7:1 to 2.5:3:1) and purified by reverse phase silica gel column (H ₂ O) kestose IVe (4.24 g, 78%). TLC (CH) ₂ Cl ₂ /MeOH/H ₂ O＝2.5:3:1,v/v),R _f ＝0.40； ¹ H NMR(400MHz,D ₂ O,MeOH as internal standard,at 3.34ppm)δ5.42(d,J＝4.0Hz,1H),4.30–4.15(m,6H),4.14–4.00(m,6H),3.94–3.65(m,34H),3.53(dd,J＝10.0,4.0Hz,1H),3.46(t,J＝9.6Hz,1H)； ¹³ C NMR(100MHz,D ₂ O,MeOH as internal standard,at 49.5ppm)δ104.3,103.8,103.7,103.7,103.7,103.7,93.1,81.9,81.7,81.7,81.7,81.7,81.7,78.0,78.0,77.8,77.8,77.3,77.3,75.0,75.0,74.9,74.8,74.8,74.5,73.2,73.0,71.8,69.8,62.9,62.8,62.8,62.8,62.7,62.7,61.6,61.4,61.4,61.4,61.3,61.1,60.7；[α] _D ²⁵ ＝-12.0(c 1.45,H ₂ O)；IR(neat):ν _max ＝3327,2931,1415,1113,1026,933cm ^–1 ；HRMS(ESI):calcd.for C ₄₂ H ₇₂ O ₃₆ Na[M+Na] ⁺ m/z1175.3701；found m/z 1175.3701.

The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Claims

1. The synthesis method of the inulin-type kestose oligosaccharide monomer is characterized by comprising the following steps of:

step one: taking the sucrose derivative 1 as an acceptor, taking the fructofuranosyl thioglycoside derivative 2 alpha or 2 beta as a donor, and forming beta (2, 1) -D-fructofuranosyl glycosidic bond through glycosylation reaction to obtain the fructosyl trisaccharide derivative 3;

wherein R protecting group in the fructofuranosyl thio-glycoside derivative 2 alpha or 2 beta is hydroxyl protecting group, and the hydroxyl protecting group is one of triisopropyl silicon, tert-butyl dimethyl silicon, tert-butyl diphenyl silicon, triethyl silicon, p-methoxyphenyl, trityl, 4' -dimethoxy triphenyl, 1-naphthalene methylene and 2-naphthalene methylene;

step three: the compound synthesized in the second step is used as a new receptor, and the first step and the second step are repeated for 0 to 60 times to obtain the kestose oligosaccharide derivative I;

wherein n is 0 to 60;

step six: removing benzyl protecting groups of the kestose oligosaccharide derivative III under the action of a deprotection reagent to generate kestose oligosaccharide IV monomers;

2. the synthesis method according to claim 1: the method is characterized in that in the first step, the promoter in the glycosylation reaction is one or two selected from NIS, IBr, NBS, tfOH, TMSOTf, DBDMH; the mol ratio of the promoter to the receptor of the glycosylation reaction is 1-10;

the solvent adopted in the glycosylation reaction is selected from one of toluene, dichloromethane, dichloroethane and acetonitrile;

the temperature of the glycosylation reaction is-78-0 ℃;

The additive for the glycosylation reaction isA molecular sieve;

the molar ratio of the donor to the acceptor in the glycosylation reaction is 1-10.

3. The synthesis method according to claim 1: characterized in that in the second step, the deprotection reagent in the reaction for removing the R protecting group is selected from the group consisting of 3HF.Et ₃ N, HF. Pyridine, TBAF, TBAF/AcOH, KF, DDQ, HCl and H ₂ SO ₄ One of the following;

the solvent in the reaction for removing the R protecting group is one or two selected from tetrahydrofuran, ethyl acetate, acetonitrile, methanol, dichloromethane and chloroform;

the reaction temperature in the reaction for removing the R protecting group is 0-80 ℃.

4. The synthesis method according to claim 1: the method is characterized in that in the fourth step, the deprotection reagent in the Pico protecting group removal reaction is Cu (OAc) ₂ ·H ₂ O; the molar ratio of the deprotection reagent to the kestose oligosaccharide derivative I is 1-15;

the deprotection reaction solvent is one or two of dichloromethane, methanol, dichloroethane, ethanol, isopropanol and chloroform.

5. The synthesis method according to claim 1: the method is characterized in that in the fifth step, the deprotection reagent in the reaction for removing the benzoyl protecting group is selected from one of sodium methoxide, potassium carbonate, sodium hydroxide and potassium hydroxide; the molar ratio of the deprotection reagent to the kestose oligosaccharide derivative II is 1-3.

6. The synthesis method according to claim 1: the method is characterized in that in the step six, a deprotection reagent in the benzyl protecting group removal reaction is selected from one of 10% palladium carbon and hydrogen, 5% palladium carbon and hydrogen and 10% palladium hydroxide and hydrogen;

the hydrogen pressure in the benzyl protecting group removal reaction is 1-30 atm;

the solvent in the benzyl protecting group removal reaction is selected from one of methanol, ethanol, isopropanol and dichloromethane/methanol;

the reaction temperature in the benzyl protecting group removal reaction is 25-60 ℃.

7. The synthesis method according to claim 6: the method is characterized in that in the step six, the deprotection reagent in the benzyl protecting group removal reaction is 10% of palladium carbon and hydrogen, and the mass ratio of the palladium carbon to the kestose oligosaccharide derivative III is 1-0.2.

8. The synthetic method according to any one of claims 1 to 7, wherein in the first step, the synthetic method of fructofuranosyl thioglycoside derivative 2α or 2β comprises the steps of:

step S1: taking fructofuranose derivatives 13 alpha or 13 beta, selectively introducing TBDPS protective groups into C6-OH of the fructofuranose derivatives 13 alpha or 13 beta, and obtaining compounds 14 alpha or 14 beta respectively;

step S2: C1-OH of the compound 14 alpha or 14 beta is selectively introduced into DMTr protecting groups to obtain a compound 15 alpha or 15 beta respectively;

Step S3: C3-OH and C4-OH of the compound 15 alpha or 15 beta are simultaneously introduced into benzyl protecting groups to respectively obtain a compound 16 alpha or 16 beta;

step S4: removing the C6 hydroxyl protecting group TBDPS from the compound 16 alpha or 16 beta under the action of a removing reagent to obtain a compound 17 alpha or 17 beta respectively;

step S5: the compound 17 alpha or 17 beta reacts with 2-picolinic acid under the action of a condensation reagent and alkali, and Pico protecting groups are introduced into C6-OH to generate 18 alpha or 18 beta respectively;

step S6: removing the C1 hydroxyl protecting group DMTR from the compound 18 alpha or 18 beta to obtain 19 alpha or 19 beta respectively;

step S7: introducing R protecting groups into C1-OH of the compound 19 alpha or 19 beta to generate 2 alpha or 2 beta respectively;

9. the synthetic method according to claim 8, wherein in step S1, the protecting reagent selectively introduced into the TBDPS protecting group reaction is TBDPSCl; the molar ratio of the protective reagent to the fructofuranose derivative 13 alpha or 13 beta is 1-2;

the base selectively introduced into the TBDPS protecting group reaction is selected from one or more of triethylamine, pyridine, diisopropylethylamine and 2, 6-lutidine;

the solvent selectively introduced into the TBDPS protecting group reaction is dichloromethane or directly takes the alkali as the solvent.

10. The synthetic method according to claim 9, wherein in step S2, the selectively introduced DMTr protecting group reaction and the selectively introduced TBDPS protecting group reaction in step S1 are operated continuously, and the protecting agent of the selectively introduced DMTr protecting group reaction is DMTrCl;

The catalyst for selectively introducing DMTR protecting group is DMAP, and the molar ratio of the catalyst to the compound 14 alpha or 14 beta is 0.05-0.2.

11. The synthetic method according to claim 8, wherein in step S3, the reagent for introducing benzyl protecting group is BnBr; the molar ratio of BnBr to the compound 15 alpha or 15 beta is 2-4;

the alkali introduced into the benzyl protecting group reaction is one or more selected from NaH, potassium carbonate, potassium tert-butoxide and sodium hydroxide; the molar ratio of the alkali to the compound 15 alpha or 15 beta is 2-4;

the solvent in the benzyl protecting group introduction reaction is selected from DMF, THF, DMSO and CH ₃ One or two of the CNs.

12. The method according to claim 8, wherein in step S4, the deprotecting reagent in the reaction for removing the C6 hydroxy protecting group TBDPS is selected from the group consisting of 3HF.Et ₃ N, HF. Pyridine, TBAF, TBAF/AcOH and KF; the molar ratio of the deprotection reagent to the compound 16α or 16β is 1-3.

13. The synthetic method according to claim 8, wherein in step S5, the condensing agent introduced into the Pico protecting group reaction is one of EDCI, DCC, DIC, TBTU, HATU and HBTU; the molar ratio of the condensation reagent to the compound 17 alpha or 17 beta is 1-3;

The catalyst for introducing Pico protecting group reaction is DAMP; the molar ratio of the catalyst to the compound 17 alpha or 17 beta is 0.05-0.3;

the reaction solvent in the Pico protecting group introduction reaction is methylene dichloride;

the molar ratio of the 2-picolinic acid introduced into the Pico protecting group reaction to the compound 17 alpha or 17 beta is 1-3.

14. The method according to claim 8, wherein in step S6, the reagent in the reaction for removing C1 hydroxy protecting group DMTR is TFA+Et ₃ SiH；

The reaction solvent in the DMTR reaction for removing the C1 hydroxyl protecting group is methylene dichloride.

15. The synthetic method according to claim 8, wherein in the step S7, the reactant introduced into the R protecting group reaction is one of TIPSOTf, TIPSCl, TBSCl, TBSOTf, TESCl, TESOTf, TBDPSCl,1-NapCl,2-NapCl,1-NapBr,2-NapBr, PMBCl, trCl and DMTrCl; the mol ratio of the reactant to the compound 19 alpha or 19 beta is 1-3;

the alkali introduced into the R protecting group reaction is one or more of triethylamine, diisopropylethylamine, pyridine, 2, 6-lutidine, sodium hydride, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium hydroxide, potassium hydroxide and potassium phosphate; the molar ratio of the alkali to the compound 19 alpha or 19 beta is 1-3;

The reaction solvent introduced into the R protecting group reaction is one or two of dichloromethane, tetrahydrofuran, acetonitrile, triethylamine, diisopropylethylamine, pyridine and 2, 6-lutidine.