CN110128491B - Antrodia camphorata galactomannan oligosaccharide derivative and preparation method and application thereof - Google Patents

Antrodia camphorata galactomannan oligosaccharide derivative and preparation method and application thereof Download PDF

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CN110128491B
CN110128491B CN201910405954.3A CN201910405954A CN110128491B CN 110128491 B CN110128491 B CN 110128491B CN 201910405954 A CN201910405954 A CN 201910405954A CN 110128491 B CN110128491 B CN 110128491B
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顾国锋
诸葛伟伟
熊成鹤
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Abstract

The invention relates to an antrodia camphorata galactomannan oligoglycoside derivative and a preparation method and application thereof. The invention provides a preparation method of an antrodia camphorata galactomannan oligosaccharide derivative, which takes various protected monosaccharide modules as raw materials and sequentially carries out glycosylation reaction through convergent glycosylation assembly to prepare the galactomannan oligosaccharide derivative, wherein the C-1 position of the reducing end galactopyranosyl of the galactomannan oligosaccharide is modified by an amino alkane long chain, so that a large amount of uniform oligosaccharide glycoside derivatives can be prepared, and the development of an immunopotentiator with the function of regulating immunity is facilitated.

Description

Antrodia camphorata galactomannan oligoglycoside derivative and preparation method and application thereof
Technical Field
The invention relates to an antrodia camphorata galactomannan oligoglycoside derivative, and a preparation method and application thereof, and belongs to the technical field of synthesis of medicinal active oligosaccharides.
Background
Antrodia camphorata, also known as antrodia camphorata, is one of the most widely studied medicinal fungi in traditional Chinese medicine, and is widely used for treating various diseases, such as liver diseases, abdominal pain, drug poisoning, diarrhea, skin pruritus, hypertension, cancer and the like. Triterpenoids, steroids, benzoquinone derivatives and polysaccharides contained in antrodia camphorata have been identified as main pharmacologically active ingredients, while crude polysaccharides thereof exhibit many biological activities. Antrodia camphorata polysaccharide has the effects of improving immunity, regulating blood pressure, reducing blood fat, inhibiting viruses, resisting allergy and radiation and the like (PloSine, 2015, e 0116192; J.Arg. food chem.2007, 5007). Recently, Perera et al isolated a cold water soluble galactomannan from Antrodia camphorata with a molecular weight of up to 70kDa and analyzed and speculated the chemical structure of the galactomannan using modern analytical techniques such as NMR spectroscopy and LC-MS, and found that the polysaccharide molecule is a polymer of repeating units of octasaccharide structure, the octasaccharide structure being α -D-mannopyranose- (1 → 3) - α -D-mannopyranose- (1 → 2) - α -D-mannopyranose- (1 → 6) - α -D-galactopyranose- (1 → 6) - [ α -D-mannopyranose- (1 → 2) - ] - α -D-mannopyranose- (1 → 6) - α -D-mannopyranose -D-galactopyranose and the glycosidic linkages between the individual monosaccharide residues are all α -glycosidic linkages (org. lett.2017,19,3486). Perera et al studied the immunostimulatory activity of Antrodia camphorata galactomannan by different immune cell models (e.g., mouse macrophage, human dendritic cell), and showed that the galactomannan was able to stimulate TNF- α (TNF- α) and interleukin-6 (IL-6) in mouse peritoneal macrophage and human dendritic cell, and further confirmed that it elicited immunostimulatory activity by phosphorylation of protein kinase C- α (PKC- α) and mitogen-activated protein kinase (MAPK).
Although the antrodia camphorata galactomannan exhibits good biological activity, the content of polysaccharide components in the primary antrodia camphorata is low, which causes difficulty in extracting and separating to obtain sufficient polysaccharide compounds, and the obtained polysaccharide molecules have high molecular weight and are microscopically heterogeneous mixtures. Studies have shown that the biological activity of polysaccharide molecules is mostly represented by their corresponding active oligosaccharide fragments. Therefore, the method for obtaining the active oligosaccharide fragment with a clear structure by using a chemical synthesis technical means has important significance for researching and developing corresponding carbohydrate drugs. At present, no relevant literature reports are found on chemical synthesis of oligosaccharide fragments related to the galactomannan based on antrodia camphorata.
Disclosure of Invention
The invention provides an antrodia camphorate galactomannan oligosaccharide glycoside derivative and a preparation method and application thereof, wherein the antrodia camphorate galactomannan oligosaccharide glycoside derivative is prepared by taking various protected monosaccharide modules as raw materials and sequentially carrying out glycosylation reaction through convergent glycosylation assembly, wherein the C-1 position of galactopyranosyl at the reduction end of galactomannan oligosaccharide glycoside is modified by an amino alkane long chain.
Description of terms:
room temperature: having a meaning well known in the art, typically 25. + -. 2 ℃.
-TBS: tert-butyl dimethylsilyl group; -Bn: a benzyl group; -STol: p-tolylthio group; -Bz: a benzoyl group.
The invention is realized by the following technical scheme:
a derivative of Antrodia camphorata galactomannan oligosaccharide glycoside has a structural general formula shown as formula I, II or III:
Figure BDA0002061221750000021
wherein R is1Is alpha-D-mannopyranosyl or alpha-D-mannopyranosyl- (1 → 2) -alpha-D-mannopyranosyl, R2Is alpha-D-mannopyranosyl or alpha-D-mannopyranosyl- (1 → 3) -alpha-D-mannopyranosyl, and n is any one integer of 0 to 4.
In the invention, the names and structural formulas of the part of preferable antrodia camphorata galactomannan-oligosaccharide glycoside derivatives are shown in the table 1.
TABLE 1A part of Antrodia camphorata galactomannan oligoglycoside derivatives
Figure BDA0002061221750000022
Figure BDA0002061221750000031
The preparation method of the antrodia camphorata galactomannan oligosaccharide glycoside derivative I comprises the following steps:
(1) taking 2-oxy-chloroacetyl-3, 4-di-oxy-benzyl-6-oxy-tert-butyl dimethyl silicon-based-alpha-D-mannopyranosyl trichloroacetimidyl ester as a glycosyl donor, taking azido alkyl 2,3, 4-tri-oxy-benzyl-alpha-D-galactopyranoside as a glycosyl acceptor, and carrying out coupling reaction a to prepare an intermediate product H1; wherein n is any integer from 0 to 4;
Figure BDA0002061221750000032
(2) taking the intermediate product H1 prepared in the step (1) to carry out silane protecting group removing reaction to prepare an intermediate product H2;
Figure BDA0002061221750000033
(3) taking 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate as a glycosyl donor, taking p-tolyl 2,3, 4-tri-oxo-benzyl-1-thio-alpha-D-galactopyranoside as a glycosyl acceptor, and carrying out coupling reaction a to obtain an intermediate product H3;
Figure BDA0002061221750000041
(4) taking the intermediate product H3 prepared in the step (3) as a glycosyl donor, taking the intermediate product H2 prepared in the step (2) as a glycosyl acceptor, and carrying out coupling reaction b to prepare an intermediate product H4;
Figure BDA0002061221750000042
(5) taking the intermediate product H4 prepared in the step (4) to carry out chloroacetyl and acetyl removal reaction to prepare an intermediate product H5;
Figure BDA0002061221750000043
(6) carrying out hydrogenolysis removal reaction on the intermediate product H5 prepared in the step (5) to prepare the antrodia camphorata galactomannan oligosaccharide glycoside derivative I,
Figure BDA0002061221750000044
according to the invention, it is preferred that in step (1) and step (3)The method of the coupling reaction a comprises the following steps: taking glycosyl donor and glycosyl acceptor with the mass ratio of (1.1-1.2):1, dissolving in dry dichloromethane, and adding
Figure BDA0002061221750000052
Stirring the molecular sieve at room temperature for 20-40 minutes under the atmosphere of nitrogen, cooling the reaction liquid to-20-0 ℃, adding trimethyl trifluoromethanesulfonate, wherein the addition amount of the trimethyl trifluoromethanesulfonate is 10-30% of the amount of the glycosyl acceptor substance, stirring for 20-40 minutes, slowly heating the reaction liquid to room temperature, neutralizing with triethylamine after complete reaction, and separating to obtain the target product.
According to the invention, the method for the silane protecting group removing reaction in the step (2) is preferably as follows: dissolving the raw material in dichloromethane, adding 90% trifluoroacetic acid solution by volume under the condition of stirring, wherein the adding amount ratio of the raw material to the trifluoroacetic acid solution is 1:5, the unit is g/ml, stirring and reacting for 20-40 minutes at room temperature, removing the solvent, and separating to obtain the target product.
According to a preferred embodiment of the present invention, the coupling reaction b in step (4) is performed by: dissolving glycosyl donor and glycosyl acceptor at a ratio of (1.1-1.3):1 in dry dichloromethane, and adding
Figure BDA0002061221750000053
Stirring the molecular sieve at room temperature for 20-40 minutes under the atmosphere of nitrogen, cooling the reaction liquid to-40-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of (1-2.5) to 1, slowly heating the reaction liquid to room temperature with triethylamine after the reaction is completed, and separating to obtain the target product, wherein the mass ratio of the added N-iodosuccinimide to the added glycosyl acceptor is (1.0-1.2) to 1.
According to the present invention, the method for the removal reaction of chloroacetyl group and acetyl group in step (5) is preferably: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strongly acidic cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain the target product.
According to a preferred embodiment of the present invention, the hydrogenolysis removal reaction of benzyl group in step (6) is performed by: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
The preparation method of the antrodia camphorata galactomannan oligosaccharide glycoside derivative II comprises the following steps:
1) preparing an intermediate product H4 according to the steps (1) to (4) in the preparation method of the antrodia camphorate galactomannan oligosaccharide glycoside derivative I;
2) taking the prepared intermediate product H4 to carry out chloroacetyl removal reaction to prepare an intermediate product H6;
Figure BDA0002061221750000051
3) taking a compound H7 as a glycosyl donor, taking an intermediate product H6 prepared in the step 2) as a glycosyl acceptor, and preparing an intermediate product H8 through a coupling reaction b;
Figure BDA0002061221750000061
wherein, H7 is:
Figure BDA0002061221750000062
R3comprises the following steps:
Figure BDA0002061221750000063
4) taking the intermediate product H8 prepared in the step 3) to perform acetyl removal reaction to prepare an intermediate product H9;
Figure BDA0002061221750000064
5) carrying out hydrogenolysis removal reaction on the intermediate product H9 prepared in the step 4) to prepare an antrodia camphorate galactomannan oligosaccharide glycoside derivative II;
Figure BDA0002061221750000071
wherein R is1Comprises the following steps:
Figure BDA0002061221750000072
according to the invention, the preferred method for the chloroacetyl group removal reaction in step 2) is: dissolving raw materials in a mixed solvent of dichloromethane and anhydrous methanol, wherein the volume ratio of the dichloromethane to the anhydrous methanol is 1 (3-5), the adding amount of thiourea and 2, 6-dimethylpyridine is (4-6) to 0.1, the adding amount of the thiourea is 4-6 times of the amount of the raw materials, carrying out reflux reaction at 60-70 ℃, completely reacting, concentrating and separating to obtain a target product.
According to a preferred embodiment of the present invention, the coupling reaction b in step 3) is performed by: taking glycosyl donor and glycosyl acceptor with the mass ratio of (1.1-1.3):1, dissolving in dry dichloromethane, and adding
Figure BDA0002061221750000073
Stirring the molecular sieve at room temperature for 20-40 minutes under the nitrogen atmosphere, cooling the reaction liquid to-40-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of the substances being (1-2.5):1, wherein the mass ratio of the addition of the N-iodosuccinimide to the addition of the glycosyl acceptor is (1.0-1.2):1, stirring the mixture for 50-70 minutes, slowly raising the temperature of the reaction liquid to the room temperature, neutralizing the reaction liquid with triethylamine after the reaction is completed, and separating the target product.
According to a preferred embodiment of the present invention, the method for removing acetyl in step 4) comprises: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strongly acidic cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain the target product.
According to a preferred embodiment of the invention, the hydrogenolysis removal reaction of the benzyl group in step 5) is carried out by: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
The preparation method of the antrodia camphorata galactomannan oligosaccharide glycoside derivative III comprises the following steps:
(i) preparing an intermediate product H9 according to the steps 1) to 4) in the preparation method of the antrodia camphorata galactomannan oligosaccharide glycoside derivative II;
(ii) taking a compound H10 as a glycosyl donor, taking the intermediate product H9 prepared in the step (i) as a glycosyl acceptor, and performing coupling reaction b to prepare an intermediate product H11;
Figure BDA0002061221750000081
wherein, H10 is:
Figure BDA0002061221750000082
R4comprises the following steps:
Figure BDA0002061221750000083
(iii) (iii) taking the intermediate product H11 prepared in the step (ii) to carry out acetyl and benzoyl removal reaction to prepare an intermediate product H12;
Figure BDA0002061221750000084
wherein R is5Comprises the following steps:
Figure BDA0002061221750000085
(iv) (iv) performing hydrogenolysis removal reaction on the intermediate product H12 prepared in the step (iii) to prepare an antrodia camphorata galactomannan oligosaccharide glycoside derivative III,
Figure BDA0002061221750000091
wherein R is1Comprises the following steps:
Figure BDA0002061221750000092
wherein R is2Comprises the following steps:
Figure BDA0002061221750000093
according to a preferred embodiment of the present invention, the coupling reaction b in step (ii) is carried out by: dissolving glycosyl donor and glycosyl acceptor at a ratio of (1.1-1.3):1 in dry dichloromethane, and adding
Figure BDA0002061221750000094
Stirring the molecular sieve at room temperature for 20-40 minutes under the nitrogen atmosphere, cooling the reaction liquid to-40-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of the substances being (1-2.5):1, wherein the mass ratio of the addition of the N-iodosuccinimide to the addition of the glycosyl acceptor is (1.0-1.2):1, stirring the mixture for 50-70 minutes, slowly raising the temperature of the reaction liquid to the room temperature, neutralizing the reaction liquid with triethylamine after the reaction is completed, and separating the target product.
According to a preferred embodiment of the present invention, the acetyl group and benzoyl group removal reaction in step (iii) is performed by: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strongly acidic cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain the target product.
According to a preferred embodiment of the present invention, the hydrogenolysis removal reaction of the benzyl group in step (iv) is carried out by: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
The antrodia camphorate galactomannan oligosaccharide glycoside derivative is applied to antifungal vaccines as an immunopotentiator.
The experimental procedures not described in detail in the present invention were carried out according to the routine procedures in the art.
Advantageous effects
The invention provides a preparation method of the antrodia camphorata galactomannan oligosaccharide glycoside derivative, which takes various protected monosaccharide modules as raw materials and carries out glycosylation reaction in sequence through convergent glycosylation assembly to prepare the galactomannan oligosaccharide glycoside derivative, wherein the C-1 position of the galactopyranosyl at the reduction end of the galactomannan oligosaccharide glycoside is modified by long-chain aminoalkanes. The invention solves the problems of low content of polysaccharide components in the primary antrodia camphorata and difficulty in extracting and separating to obtain enough polysaccharide compounds, can prepare a large amount of uniform oligosaccharide glycoside derivatives, is beneficial to developing immunopotentiators with the function of regulating immunity, and has important significance for developing corresponding saccharide medicaments.
Drawings
FIG. 1 shows the preparation of the galactomannan octaose derivative GM-4 of Antrodia camphorata1H NMR spectrum;
FIG. 2 shows the preparation of the galactomannan octaose derivative GM-4 from Antrodia camphorata13C NMR spectrum.
Detailed Description
The invention will be further illustrated with reference to the following examples and figures, without however limiting the scope of protection of the invention thereto.
Room temperature: having the meaning well known in the art, typically 25. + -. 2 ℃; the pharmaceutical products referred to in the present invention are all common commercial products, and the compounds not specifically mentioned are all substances which are commercially available or can be prepared by reference to the literature, and the procedures not specifically mentioned are carried out according to the routine procedures in the art.
The type of strong acid cation exchange resin referred to in the examples is amberlite IR-120, or Amberlyst-15.
Example 1: general synthetic methods
A-1: coupling reaction a (glycosidation reaction)
Taking glycosyl donor and glycosyl acceptor with the mass ratio of (1.1-1.2):1, dissolving in dry dichloromethane, and adding
Figure BDA0002061221750000101
Stirring the molecular sieve at room temperature for 30 minutes under the nitrogen atmosphere, cooling the reaction liquid to-15 ℃, adding trimethyl trifluoromethanesulfonate (TMSOTf, the adding amount is 20 percent of the amount of the glycosyl acceptor substance), stirring the mixture for 30 minutes, slowly heating the reaction liquid to room temperature, neutralizing the mixture with triethylamine after TLC (thin layer chromatography) detects that the raw materials completely react, filtering the mixture to remove insoluble solids, spin-drying the filtrate, separating the crude product by a silica gel column to obtain a target product, and gradually changing the volume ratio of Petroleum Ether (PE) to Ethyl Acetate (EA) from 8:1 to 2:1 by using eluent;
a-2: coupling reaction b (glycosidation reaction)
Taking glycosyl donor and glycosyl acceptor with the mass ratio of (1.1-1.3):1, dissolving in dry dichloromethane, and adding
Figure BDA0002061221750000102
Stirring the molecular sieve for 30 minutes at room temperature under a nitrogen atmosphere, cooling the reaction liquid to-15 ℃, adding N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf) according to the mass ratio of (1-2.5) to 1, stirring the mixture for 60 minutes, slowly raising the temperature of the reaction liquid to the room temperature, neutralizing the mixture by triethylamine after TLC detects that the raw materials completely react, filtering the mixture to remove insoluble solids, spin-drying the filtrate, and separating the crude product by a silica gel column to obtain a target product (the target product)Using eluent as a mixed solution of Petroleum Ether (PE) and Ethyl Acetate (EA) with the volume ratio gradually changed from 8:1 to 2:1, unless otherwise specified);
b: reaction for removing silane protecting group
Dissolving a raw material in dichloromethane, adding a trifluoroacetic acid (TFA) solution with the volume percentage of 90 percent under the stirring condition, wherein the adding amount ratio of the raw material to the trifluoroacetic acid solution is 1:5, the unit is g/ml, stirring and reacting for 30 minutes at room temperature, distilling under reduced pressure to remove the solvent, and separating a crude product through a silica gel column to obtain a target product (eluent is a mixed solution of Petroleum Ether (PE) and Ethyl Acetate (EA) with the volume ratio of 6:1 gradually changed to 2:1 from 6:1 unless otherwise specified);
c: chloroacetyl group removal reaction
Dissolving a raw material in a mixed solvent of dichloromethane and anhydrous methanol, wherein the volume ratio of the dichloromethane to the anhydrous methanol is 1:4, adding thiourea and 2, 6-lutidine with the mass ratio of 5:0.1, wherein the addition amount of the thiourea is 5 times of the mass of the raw material, carrying out reflux reaction at 60-70 ℃, detecting by TLC that the raw material is completely reacted, concentrating, and separating a crude product by a silica gel column to obtain a target product (the eluent is a mixed solution in which the volume ratio of Petroleum Ether (PE) to Ethyl Acetate (EA) is gradually changed from 4:1 to 2:1 unless otherwise specified);
d: chloroacetyl, acetyl and benzoyl removal reactions
Dissolving raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 10, reacting for 1 hr, detecting the reaction completion, and adding strong acid cation exchange resin (H)+) Neutralizing, filtering the reaction solution, concentrating, and separating the crude product by a silica gel column to obtain a target product (the eluent is a mixed solution of Petroleum Ether (PE) and Ethyl Acetate (EA) with the volume ratio gradually changed from 8:1 to 2:1, unless otherwise specified);
e: hydrogenolysis removal reaction of benzyl group
Dissolving raw materials (10 parts by mass) in deionized water, adding catalytic palladium carbon (10% Pd/C, 1 part by mass) under the protection of nitrogen, introducing hydrogen into a reaction bottle, replacing the nitrogen, stirring reaction liquid at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering the reaction liquid to remove the palladium carbon, carrying out reduced pressure distillation to remove a solvent, passing an obtained crude product through a gel chromatography BioGelP-2 column, and freeze-drying obtained fractions to obtain a target product, wherein the mobile phase is deionized water.
Example 2: preparation of 3-aminopropyl alpha-D-mannopyranosyl- (1 → 6) -alpha-D-galactopyranosyl- (1 → 6) -alpha-D-mannopyranosyl- (1 → 6) -alpha-D-galactopyranoside (GM-1)
Coupling reaction of 2-oxy-chloroacetyl-3, 4-di-oxy-benzyl-6-oxy-tert-butyldimethylsilyl-alpha-D-mannopyranosyl trichloroacetimidate and 3-azido-1-propyl 2,3, 4-tri-oxy-benzyl-alpha-D-galactopyranoside at-15 ℃ under the catalysis of trimethyl trifluoromethanesulfonate (TMSOTf) to generate disaccharide H1-1 (yield 65%); then, the 6-position silane protecting group (TBS) was selectively removed with 90% by volume of trifluoroacetic acid to obtain disaccharide acceptor H2-1 (yield 96%); meanwhile, at the temperature of-15 ℃, 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate and p-tolyl 2,3, 4-tri-oxo-benzyl-1-thio-beta-D-galactopyranoside are subjected to coupling reaction under the catalysis of trimethyl trifluoromethanesulfonate (TMSOTf) to generate a glucosinolate disaccharide donor H3 (yield of 75%); then, under the temperature of minus 15 ℃, disaccharide donor H3 and disaccharide acceptor H2-1 are catalyzed by N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf) in ether solution to carry out coupling reaction to obtain tetrasaccharide H4-1; finally, H4-1 selectively removed chloroacetyl and acetyl protecting groups with 1M sodium methoxide in methanol solution (to obtain H5-1), and then the benzyl and azido protecting groups were catalytically hydrogenolyzed with palladium on carbon under hydrogen atmosphere to prepare the desired product GM-1 of tetrasaccharide (73% yield in two steps). The synthetic route of the antrodia camphorata galactomannan tetraside derivative GM-1 is as follows:
Figure BDA0002061221750000121
(1) 3-azido-1-propyl 2-oxo-chloroacetyl-3, 4-di-oxo-benzyl-6-oxo-tert-butyldimethylsilyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (H1-1)
Taking 2-oxy-chloroacetyl-3, 4-di-oxy-benzyl-6-oxy-tert-butyl dimethyl silicon base-alpha-D-mannopyranosyl trichloro chlorideImide ester (388 mg, 0.56 mmol) as glycosyl donor and 3-azido-1-propyl 2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (266 mg, 0.50 mmol) as glycosyl acceptor were coupled as in the synthesis of a-1 in example 1 to afford intermediate H1-1(346 mg, 65% yield).1H NMR(600MHz,CDCl3):δ7.43–7.40(m,2H,ArH),7.38–7.30(m,14H,ArH),7.30–7.26(m,9H,ArH),5.23(t,J＝3.0,1.8Hz,1H,H-2),4.95(d,J＝11.4Hz,1H,-CH2Ph),4.89(d,J＝11.4Hz,1H,-CH2Ph),4.83(t,J＝11.4Hz,2H,-CH2Ph),4.78–4.75(m,2H,H-1,-CH2Ph),4.66(t,J＝12.0,10.8Hz,2H,-CH2Ph),4.61(d,J＝10.8Hz,1H,-CH2Ph),4.57(d,J＝12.0Hz,1H,-CH2Ph),4.53–4.49(m,2H,H-1′,-CH2Ph),4.18–4.06(m,2H,-OCOCH2Cl),4.03(dd,J＝10.2,3.6Hz,1H),3.92–3.84(m,4H),3.83–3.82(m,1H),3.77–3.73(m,2H),3.67–3.62(m,2H),3.58–3.54(m,1H),3.44–3.39(m,1H),3.38–3.31(m,2H),3.31–3.27(m,1H),1.89–1.75(m,2H),0.90(s,9H),0.06(d,J＝6.6Hz,6H)；13C NMR(150MHz,CDCl3):δ166.9,97.7,96.7,79.0,77.8,76.6,75.3,74.6,74.5,73.7,73.5,73.4,72.5,72.0,70.6,68.8,65.9,64.8,61.8,48.3,48.2,40.9,28.7,25.9,18.3,-5.1,-5.3.
(2) 3-azido-1-propyl-2-oxo-chloroacetyl-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (H2-1)
Intermediate H1-1(200 mg, 0.18 mmol) prepared in step (1) was subjected to deprotection of the silane group according to general procedure B in example 1 to give intermediate H2-1(164 mg, 96% yield).1H NMR(600MHz,CDCl3):δ7.44–7.41(m,2H,ArH),7.39–7.30(m,15H,ArH),7.30–7.26(m,8H,ArH),5.26(dd,J＝3.0,1.8Hz,1H,H-2),4.97(d,J＝11.4Hz,1H,-CH2Ph),4.90(d,J＝12.0Hz,1H,-CH2Ph),4.87(d,J＝10.8Hz,1H,-CH2Ph),4.82(d,J＝12.0Hz,1H,-CH2Ph),4.79–4.76(m,2H,H-1),4.67(t,J＝11.4Hz,2H,-CH2Ph),4.59(t,J＝11.4Hz,2H,-CH2Ph),4.54–4.50(m,2H,H-1′),4.15(d,J＝1.2Hz,2H,-OCOCH2Cl),4.03(dd,J＝10.2,3.6Hz,1H),3.94–3.88(m,2H),3.83(dd,J＝3.0,1.2Hz,1H),3.80–3.71(m,4H),3.69–3.61(m,3H),3.46–3.41(m,1H),3.39–3.33(m,2H),3.33–3.29(m,1H),1.90–1.79(m,2H),1.79–1.75(m,1H,-OH)；13C NMR(150MHz,CDCl3):δ166.7,97.7,97.1,79.0,77.8,76.6,75.3,74.5,74.4,73.6,73.5,73.5,71.9,71.8,70.3,68.8,66.5,64.9,61.8,48.3,40.9,28.7.
(3) P-toluenesulfonyl 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl-beta-D-galactopyranoside (H3)
Coupling was performed according to the synthesis method of A-1 in example 1 using 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate (565 mg, 0.89 mmol) as a glycosyl donor and p-tolyl 2,3,4, -tri-oxo-benzyl-1-thio-alpha-D-galactopyranoside (434 mg, 0.78 mmol) as a glycosyl acceptor to obtain intermediate H3(602 mg, 75% yield).1H NMR(600MHz,CDCl3):δ7.46(d,J＝7.8Hz,2H,ArH),7.42(d,J＝7.4Hz,2H,ArH),7.40–7.26(m,22H,ArH),7.17–7.13(m,2H,ArH),7.02–6.98(m,2H,ArH),5.28–5.25(m,1H,H-2),4.99(d,J＝11.4Hz,1H,-CH2Ph),4.84(t,J＝10.8,10.2Hz,2H,-CH2Ph),4.76(s,2H),4.75(d,J＝10.2Hz,1H,-CH2Ph),4.70(t,J＝12.6,10.8Hz,2H,-CH2Ph),4.66(d,J＝1.5Hz,1H),4.60(d,J＝11.4Hz,1H,-CH2Ph),4.55–4.52(m,2H),4.48(dd,J＝12.0,10.8Hz,2H,-CH2Ph),3.96–3.87(m,3H),3.86–3.77(m,4H),3.70(d,J＝9.4Hz,1H),3.59(dd,J＝9.6,2.4Hz,1H),3.52–3.44(m,2H),2.22(s,3H),2.17(s,3H)；13C NMR(150MHz,CDCl3):δ170.46,98.0,88.2,84.2,78.1,76.5,75.7,75.2,74.3,74.1,73.5,73.3,73.0,71.9,71.5,68.7,68.5,66.6,21.2,21.1.
(4) 3-azido-1-propyl-2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) -2-oxo-chloroacetyl-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (H4-1)
Taking the product prepared in step (3)Intermediate H3(24 mg, 0.023 mmol) was used as the glycosyl donor and intermediate H2-1(19 mg, 0.020 mmol) prepared in step (2) was used as the glycosyl acceptor, and the coupling reaction was carried out according to the method for the synthesis of A-2 in example 1 to obtain intermediate H4-1 (17 mg of tetrasaccharide in alpha configuration, 38% yield).1H NMR(600MHz,CDCl3):δ7.41–7.37(m,2H,ArH),7.36–7.25(m,36H,ArH),7.24–7.13(m,15H,ArH),7.13–7.09(m,2H,ArH),5.24–5.22(m,2H),5.09(d,J＝3.6Hz,1H),4.94(t,J＝12.6,11.4Hz,2H,-CH2Ph),4.86(d,J＝12.0Hz,1H,-CH2Ph),4.81(d,J＝10.2Hz,1H,-CH2Ph),4.78–4.75(m,3H),4.75–4.73(m,2H),4.71(d,J＝4.8Hz,2H),4.69(d,J＝6.0Hz,1H),4.66(d,J＝3.0Hz,1H),4.65–4.63(m,1H),4.62–4.57(m,3H),4.57–4.52(m,3H),4.51–4.47(m,2H),4.46–4.40(m,3H),4.03(dd,J＝10.2,3.0Hz,1H),3.99(dd,J＝10.2,3.0Hz,1H),3.93–3.78(m,8H),3.76–3.71(m,3H),3.70–3.63(m,5H),3.62–3.56(m,3H),3.48(d,J＝5.5Hz,2H),3.39(t,J＝9.6,7.8Hz,1H),3.33–3.23(m,4H),2.13(s,3H),1.77–1.65(m,2H)；13C NMR(150MHz,CDCl3):δ170.3,166.9,97.8,97.7,97.6,97.0,79.0,78.4,78.1,78.0,76.7,76.6,75.3,75.2,74.8,74.6,74.5,74.4,74.0,73.4,73.38,73.0,72.7,72.1,71.9,71.5,70.3,68.8,68.6,68.4,66.1,66.0,65.6,64.8,48.2,40.5,28.6,21.1.
(5) 3-aminopropyl alpha-D-mannopyranosyl- (1 → 6) -alpha-D-galactopyranosyl- (1 → 6) -alpha-D-mannopyranosyl- (1 → 6) -alpha-D-galactopyranoside (GM-1)
Taking the intermediate product H4-1(21 mg, 0.011 mmol) prepared in the step (4) to perform chloroacetyl and acetyl removal reactions according to the general method D in the example 1 to prepare an intermediate product H5-1; the hydrogenolysis removal of the benzyl group of the H5-1 thus obtained was carried out in accordance with general procedure E in example 1 to produce the desired product, GM-1(6 mg, 76%).1H NMR(600MHz,D2O):δ4.81(d,J＝3.6Hz,1H),4.80(s,1H),4.74(s,1H),4.73(s,1H),3.99(dd,J＝8.4,3.6Hz,1H),3.92(dd,J＝7.8,3.6Hz,1H),3.86–3.43(m,24H),3.06–2.91(m,2H),1.90–1.81(m,2H)；13C NMR(150MHz,D2O):δ99.52,99.07,98.36,97.61,74.15,72.86,70.86,70.39,69.94,69.42,69.32,69.22,68.72,68.46,68.27,67.92,66.63,66.35,66.27,66.06,65.85,65.32,60.82(2C),37.62,26.34.
Example 3: preparation of 3-aminopropyl α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranosyl- (1 → 6) - [ α -D-mannopyranosyl- (1 → 2) - ] - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranoside (GM-3)
The chloroacetyl in tetrasaccharide H4-1 is selectively removed by thiourea in dichloromethane-anhydrous methanol solution (v/v ═ 1: 4) to prepare a tetrasaccharide acceptor H6-1 with 90% yield; subsequently, at-15 deg.C, the thiobiose donor H7 and the tetrasaccharide acceptor H6-1 were coupled in ether solution with N-iodosuccinimide (NIS) and AgOTf as catalysts to obtain the hexasaccharide H8-1, and each J in H8-1C-1,H-1The coupling constant values are all larger than 169 Hz, so that all glycosidic bonds are determined to be alpha bonds; finally, H8-1 was selectively freed from the acetyl protecting group with 1M sodium methoxide in methanol solution (yielding H9-1), and then the benzyl and azido protecting groups were subjected to catalytic hydrogenolysis with palladium on carbon under hydrogen atmosphere to produce GM-3, the hexasaccharide target product (77% yield over two steps). The synthetic route of the antrodia camphorata galactomannan hexaglucoside derivative GM-3 is as follows:
Figure BDA0002061221750000151
(1) 3-azido-1-propyl-2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) -2-hydroxy-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (H6-1)
Intermediate H4-1(156 mg, 0.08 mmol) prepared in step (4) of example 2 was subjected to chloroacetyl group removal as in general procedure C of example 1 to give intermediate H6-1(135 mg, 90% yield).1H NMR(600MHz,CDCl3):δ7.41–7.38(m,2H,ArH),7.37–7.34(m,6H,ArH),7.34–7.26(m,33H,ArH),7.25–7.16(m,12H,ArH),7.15–7.12(m,2H,ArH),5.27–5.24(m,1H,H-2),5.08(d,J＝3.6Hz,1H),4.92(dd,J＝12.0,11.4Hz,2H,-CH2Ph),4.85(dd,J＝11.4,10.2Hz,2H,-CH2Ph),4.81–4.78(m,3H),4.77–4.70(m,5H),4.69–4.65(m,2H),4.64–4.57(m,5H),4.57–4.51(m,4H),4.49(d,J＝11.4Hz,1H,-CH2Ph),4.47–4.43(m,2H),4.04–3.97(m,2H),3.95–3.85(m,6H),3.84–3.77(m,4H),3.76–3.57(m,11H),3.40(dd,J＝9.6,7.8Hz,1H),3.35–3.22(m,4H),2.55(d,J＝4.8Hz,1H),2.15(s,3H,-(C＝O)CH3),1.77–1.65(m,2H)；13C NMR(150MHz,CDCl3):δ170.4,99.0,98.0,97.6,97.5,79.6,79.0,78.5,78.2,76.8,76.6,76.2,75.3,75.2,74.7,74.6,74.5,74.1,73.9,73.4,73.38,73.37,73.0,72.7,71.8,71.7,71.5,71.3,68.9,68.6,68.5,68.4,68.1,65.8,65.7,65.6,64.8,60.1,48.3,28.6,21.5,21.1.
(2) P-toluenesulfonyl 2,3,4, 6-tetra-oxo-benzyl-alpha-D-mannopyranosyl- (1 → 2) -3,4, 6-di-oxo-benzyl-alpha-D-mannopyranoside (H7)
Disaccharide H7 is a reported compound and can be prepared according to the methods of the references (Pure apple Chem,2016,89, 1011).1H NMR(600MHz,CDCl3):δ7.33–7.27(m,10H,ArH),7.27–7.23(m,14H,ArH),7.23–7.18(m,9H,ArH),7.17–7.13(m,2H,ArH),7.10–7.07(m,2H,ArH),6.96(m,2H,ArH),5.54(d,J＝1.8Hz,1H),5.16(d,J＝2.4Hz,1H),4.84(d,J＝10.8Hz,1H,-CH2Ph),4.80(d,J＝10.8Hz,1H,-CH2Ph),4.68-4.60(m,3H,-CH2Ph),4.58–4.48(m,4H,-CH2Ph),4.46–4.38(m,5H,-CH2Ph),4.29–4.24(m,2H),3.92–3.85(m,4H),3.84–3.78(m,3H),3.70(dd,J＝10.8,1.8Hz,1H),3.66(dd,J＝10.8,4.8Hz,1H),3.60(dd,J＝10.8,1.8Hz,1H),2.25(s,3H,-SPhCH3)；13C NMR(150MHz,CDCl3):δ99.7,87.6,80.3,79.7,76.0,75.2,75.0,74.97,74.90,74.8,73.2,73.0,72.8,72.5,72.3,72.2,72.1,69.3,69.0,21.1.
(3) 3-azido-1-propyl-2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) - [2,3,4, 6-tetra-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -3,4, 6-di-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) ] -2-hydroxy-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3, 4-tri-oxo-benzyl-alpha-D-galactopyranoside (H8-1)
Coupling reaction was carried out according to the synthesis method of A-2 in example 1 using disaccharide compound H7(39 mg, 0.036 mmol) prepared in step (2) as a glycosyl donor and intermediate H6-1(53 mg, 0.03 mmol) prepared in step (5) as a glycosyl acceptor to obtain intermediate H8-1(55 mg).1H NMR(600MHz,CDCl3):δ7.39–7.26(m,24H,ArH),7.25–7.14(m,52H,ArH),7.14–7.05(m,14H,ArH),5.25(d,J＝1.8Hz,1H),5.21(dd,J＝3.6,1.8Hz,1H,H-2),5.17(d,J＝3.6Hz,1H),5.05(d,J＝1.8Hz,1H),4.95–4.90(m,2H),4.85–4.71(m,8H,-CH2Ph),4.71–4.68(m,2H),4.67–4.59(m,8H),4.58(d,J＝1.8Hz,1H),4.57–4.53(m,2H),4.51(d,J＝3.00Hz,1H),4.49(d,J＝1.8Hz,1H),4.47–4.36(m,10H,-CH2Ph),4.34–4.25(m,3H,-CH2Ph),4.18(d,J＝11.4Hz,1H,-CH2Ph),4.15–4.12(m,1H),4.06(dd,J＝3.6,1.8Hz,1H),4.02–3.96(m,3H),3.94–3.89(m,4H),3.86–3.72(m,13H),3.71–3.43(m,13H),3.31–3.23(m,2H),3.23–3.14(m,2H),2.12(s,3H),1.74–1.60(m,2H)；13C NMR(150MHz,CDCl3):δ170.26,101.4,99.4,98.9,98.1,97.4,96.9,79.8,79.6,79.5,78.9,78.4,78.0,76.6,76.2,75.2,75.18,75.0,74.9,74.8,74.8,74.7,74.69,73.9,73.4,73.3,73.2,73.15,73.1,72.7,72.4,72.3,72.1,72.0,71.95,71.84,71.83,71.5,71.4,69.4,69.3,69.0,68.6,68.4,68.3,66.8,65.8,65.5,64.7,53.5,48.2,28.6,21.1.
(4) 3-azido-1-propyl-2-hydroxy-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) - [2,3,4, 6-tetra-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -3,4, 6-di-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) ] -2-hydroxy-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3, 4-tri-oxo-benzyl-alpha-D-galactopyranoside (H9-1)
Intermediate H8-1(30 mg, 0.01 mmol) from step (3) was deacetylated according to general method D in example 1 to give intermediate H9-1(25 mg, 86% yield).1H NMR(600MHz,CDCl3):δ7.38–7.31(m,8H,ArH),7.31–7.26(m,16H,ArH),7.25–7.08(m,66H,ArH),5.30(d,J＝1.8Hz,1H),5.11–5.08(m,2H),4.93(d,J＝1.8Hz,1H),4.89–4.82(m,3H),4.81–4.72(m,5H),4.72–4.68(m,5H),4.68–4.64(m,3H),4.63–4.56(m,6H),4.56–4.51(m,3H),4.51–4.48(m,2H),4.48–4.46(m,2H),4.46–4.43(m,3H),4.42–4.40(m,2H),4.40(d,J＝1.8Hz,2H),4.38–4.37(m,1H),4.35(s,1H),4.34–4.28(m,3H),4.18(dd,J＝2.4,1.8Hz,1H),4.11(dd,J＝2.4,1.8Hz,1H),4.05–3.98(m,2H),3.95–3.80(m,14H),3.79–3.70(m,8H),3.70–3.62(m,5H),3.60–3.54(m,4H),3.52–3.45(m,2H),3.35–3.25(m,2H),3.23–3.16(m,2H),2.53(d,J＝3.0Hz,1H),1.73–1.64(m,2H)；13C NMR(150MHz,CDCl3):δ101.0,99.4,99.0,98.8,97.3,96.9,79.9,79.7,79.6,78.8,78.7,76.6,76.4,75.3,75.1,75.06,75.0,74.9,74.87,74.86,74.8,74.7,74.6,74.4,74.1,73.4,73.2,73.18,73.1,73.09,72.9,72.3,72.28,72.2,72.0,71.95,71.8,71.4,71.2,69.3,69.3,69.0,68.6,68.3,68.1,66.5,65.9,65.6,64.7,53.5,48.2,28.6,21.5.
(5) 3-aminopropyl α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranosyl- (1 → 6) - [ α -D-mannopyranosyl- (1 → 2) - ] - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranoside (GM-3)
The hydrogenolysis removal reaction of benzyl group was performed according to general method E in example 1 using intermediate H9-1(20 mg, 0.005 mmol) prepared in step (4) to prepare the desired product, GM-3(4 mg, 90%).
Example 4: 3-aminopropyl α -D-mannopyranosyl- (1 → 3) - α -D-mannopyranosyl- (1 → 2) - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranosyl- (1 → 6) - [ α -D-mannopyranosyl- (1 → 2) - ] - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranoside (GM-4)
Coupling reaction of 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate and p-tolyl 2-oxo-benzoyl-4, 6, -di-oxo-benzyl-1-thio-alpha-D-mannopyranoside at-15 deg.C under the catalysis of trimethyl trifluoromethanesulfonate (TMSOTf) to produce disaccharide H10 (yield 70%); next, at-15 deg.C, disaccharide donor H10 and hexasaccharide acceptor H9-1 were dissolved in ether with N-iodosuccinimide(NIS) and AgOTf catalyze the coupling reaction to obtain the octasaccharide H11-1, and each J in H11-1C-1,H-1Coupling constant values are all larger than 170 Hz, and the glycosidic bonds are all determined to be alpha bonds; finally, H11-1 was selectively freed from acetyl and benzoyl protecting groups with 1M sodium methoxide in methanol solution (yielding H12-1), and then benzyl and azido protecting groups were catalytically hydrogenolyzed with palladium on carbon under hydrogen atmosphere to produce the octasaccharide target product GM-4 (82% yield in two steps). The synthetic route of the antrodia camphorata galactomannan octaglucoside derivative GM-4 is as follows:
Figure BDA0002061221750000181
(1) p-toluenesulfonyl 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl- (1 → 3) -2-oxo-benzoyl-4, 6-di-oxo-benzyl-alpha-D-mannopyranoside (H10)
Coupling was performed according to the synthesis method of A-1 in example 1 using 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate (730 mg, 1.15 mmol) as a glycosyl donor and p-tolyl 2-oxo-benzoyl-4, 6-di-oxo-benzyl-1-thio-alpha-D-mannopyranoside (547 mg, 0.96 mmol) as a glycosyl acceptor to obtain intermediate H10(702 mg, 70% yield).1H NMR(600MHz,CDCl3):δ7.99-7.96(dd,J＝7.8,1.2Hz,2H,ArH),7.52(t,J＝7.8Hz,1H,ArH),7.36-7.28(m,13H,ArH),7.27-7.23(m,5H,ArH),7.23-7.20(m,4H,ArH),7.18-7.15(m,5H,ArH),7.06-7.01(m,4H,ArH),5.68(dd,J＝3.0,1.8Hz,1H,H-2),5.57(d,J＝1.8Hz,1H,H-1),5.37(dd,J＝3.2,1.8Hz,1H,H-2′),5.20(d,J＝1.8Hz,1H,H-1′),4.74(dd,J＝10.8,5.4Hz,2H),4.68(t,J＝12.4Hz,2H,-CH2Ph),4.55(d,J＝10.8,1H,-CH2Ph),4.50-4.38(m,4H),4.35(m,1H),4.32(d,J＝11.4Hz,1H,-CH2Ph),4.27(dd,J＝9.6,3.0Hz,1H),4.19(t,J＝9.6Hz,1H),3.93-3.87(m,2H),3.84-3.80(m,2H),3.72(dd,J＝11.4,1.8Hz,1H),3.63(dd,J＝10.8,3.0Hz,1H),3.58(dd,J＝10.8,1.2Hz,1H),2.27(s,3H,-SPhCH3),2.07(s,3H,-OCOCH3)；13C NMR(150MHz,CDCl3):δ170.3,165.5,99.7,86.2,77.8,77.5,75.4,75.0,74.6,73.9,73.8,73.4,73.3,72.5,72.4,71.9,68.9,68.8,68.3,21.13,21.07.
(2) 3-azido-1-propyl-2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 3) -2-oxo-benzoyl-4, 6-di-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -2-hydroxy-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) - [2,3,4, 6-tetra-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -3,4, 6-di-O-benzyl-alpha-D-mannopyranosyl- (1 → 2) ] -2-hydroxy-3, 4-di-O-benzyl-6-hydroxy-alpha-D-mannopyranosyl- (1 → 6) -2,3,4, -tri-O-benzyl-alpha-D-galactopyranoside (H11-1)
Intermediate H11-1(45 mg) was prepared by coupling the intermediate H9-1(54 mg, 0.02 mmol) prepared in example 3 as a glycosyl acceptor using intermediate H10(25 mg, 0.024 mmol) prepared in step (1) as a glycosyl donor and intermediate H11-1 (54 mg) prepared in example 1 as the synthetic method of A-2.1H NMR(600MHz,CDCl3):δ8.07–8.02(m,2H,ArH),7.58–7.53(m,1H,ArH),7.39–7.26(m,30H,ArH),7.25–7.15(m,71H,ArH),7.13–7.04(m,16H,ArH),5.60(dd,J＝3.0,2.4Hz,1H,H-2),5.40(dd,J＝3.6,1.8Hz,1H,H-2′),5.26(d,J＝1.8Hz,1H),5.19–5.17(m,2H),5.16(d,J＝2.4Hz,1H),5.04(d,J＝1.8Hz,1H),4.92(d,J＝1.8Hz,1H),4.88(d,J＝1.8Hz,1H),4.87–4.79(m,4H),4.78–4.70(m,8H),4.70–4.58(m,10H),4.58–4.49(m,8H),4.48–4.29(m,15H),4.24(m,2H),4.21–4.12(m,3H),4.06(dd,J＝3.6,1.6Hz,1H),4.03–3.95(m,5H),3.95–3.47(m,37H),3.30–3.24(m,2H),3.22–3.14(m,2H),2.05(s,3H),1.72–1.61(m,2H)；13C NMR(150MHz,CDCl3):δ170.1,165.5,101.4,99.6,99.5,99.13,99.12,98.9,97.4,96.9,79.8,79.63,79.62,79.1,78.9,78.3,78.0,76.6,76.3,76.26,75.4,75.3,75.2,75.18,75.17,75.05,75.04,75.02,74.96,74.90,74.87,74.82,74.77,74.73,74.72,74.68,74.66,74.57,74.56,73.8,73.3,73.26,73.25,73.23,73.2,73.16,73.09,73.08,73.0,72.7,72.4,72.3,72.2,72.03,72.02,72.01,71.99,71.97,71.96,71.86,71.82,71.8,71.4,69.4,69.3,69.1,68.9,68.7,68.65,68.2,66.8,65.6,65.4,64.7,48.2,48.1,28.6,28.5,21.0.
(3) 3-azido-1-propyl-2-hydroxy-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 3) -2-hydroxy-4, 6-di-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -2-hydroxy-3, 4, 6-tri-oxo-benzyl- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranosyl- (1 → 6) - [2,3,4, 6-tetra-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) -3,4, 6-di-oxo-benzyl- α -D-mannopyranosyl- (1 → 2) ] -2-hydroxy-3, 4-di-oxo-benzyl-6-hydroxy- α -D-mannopyranosyl- (1 → 6) -2,3,4, -tri-oxo-benzyl- α -D-galactopyranoside (H12-1)
Intermediate H11-1(50 mg, 0.01 mmol) from step (2) was taken for deacetylation and benzoyl removal according to general procedure D in example 1 to give intermediate H12-1(45 mg, 94% yield).1H NMR(600MHz,CDCl3):δ7.39–7.31(m,9H,ArH),7.30–7.04(m,106H,ArH),5.26(s,1H),5.17(d,J＝3.6Hz,1H),5.04(dd,J＝4.2,1.8Hz,2H),5.00(d,J＝1.8Hz,1H),4.92(s,1H),4.86–4.76(m,7H),4.76–4.70(m,6H),4.69–4.61(m,8H),4.61–4.50(m,12H),4.49–4.38(m,12H),4.38–4.23(m,9H),4.19–4.12(m,2H),4.06(s,1H),4.04–3.62(m,30H),3.62–3.43(m,10H),3.31–3.24(m,2H),3.22–3.13(m,2H),2.28(d,J＝1.8Hz,1H)1.73–1.62(m,2H)；13C NMR(150MHz,CDCl3):δ101.9,101.4,99.8,99.5,99.3,98.9,97.4,96.9,82.6,80.1,79.79,79.78,79.6,79.6,79.5,78.9,78.3,76.6,76.2,75.3,75.2,75.1,75.0,74.97,74.9,74.86,74.85,74.8,74.77,74.76,74.7,74.68,74.66,74.6,74.56,74.55,74.3,73.6,73.4,73.28,73.23,73.22,73.20,73.19,73.17,73.1,73.0,72.7,72.4,72.3,72.2,72.0,71.96,71.95,71.9,71.7,71.5,71.4,69.36,69.34,69.1,68.9,68.8,68.77,68.75,66.76,65.5,65.4,64.7,48.2,28.6.
(4) 3-azido-1-propyl α -D-mannopyranosyl- (1 → 3) - α -D-mannopyranosyl- (1 → 2) - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranosyl- (1 → 6) - [ α -D-mannopyranosyl- (1 → 2) - α -D-mannopyranosyl- (1 → 2) ] - α -D-mannopyranosyl- (1 → 6) - α -D-galactopyranoside (GM-4)
The intermediate product H12-1(20 mg, 0.005 mmol) prepared in step (3) was subjected to hydrogenolysis removal reaction of benzyl group according to general method E to prepare the target product GM-4(4 mg, 85%), of the target product GM-41The H NMR spectrum is shown in figure 1,13the C NMR spectrum is shown in FIG. 2.1H NMR(600MHz,D2O):δ5.08(s,1H),4.93–4.90(m,2H),4.89–4.86(s,1H),4.83–4.80(m,2H),4.78(d,J＝1.8Hz,1H),4.73(s,1H),4.01(d,J＝2.4Hz,1H),3.92–3.82(m,5H),3.80–3.34(m,46H),3.00–2.87(m,2H),1.85–1.74(m,2H)；13C NMR(150MHz,D2O):δ102.10,102.09,102.04,100.56,98.27,98.08,97.88,97.49,78.73,78.52,78.33,77.68,73.16,73.15,73.14,73.09,72.57,70.90,70.35,70.11,70.10,70.01,69.84,69.73,69.38,69.26,69.22(2C),69.20,69.13,68.90,68.84,68.19,67.84,66.84,66.72,66.70,66.61,66.60,66.46,66.45,66.16,65.79,64.93,60.94,60.92,60.91(2C),60.90,60.75,37.77,26.27.

Claims (8)

1. The preparation method of the antrodia camphorata galactomannan oligosaccharide derivative comprises the following steps:
Figure DEST_PATH_IMAGE002
wherein n is any one integer from 0 to 4, characterized by the following steps:
(1) taking 2-oxy-chloroacetyl-3, 4-di-oxy-benzyl-6-oxy-tert-butyl dimethyl silicon-based-alpha-D-mannopyranosyl trichloroacetimidate as a glycosyl donor, taking azido alkyl 2,3, 4-tri-oxy-benzyl-alpha-D-galactopyranoside as a glycosyl acceptor, and carrying out coupling reaction a to obtain an intermediate product H1; wherein n is any one integer of 0 to 4;
Figure DEST_PATH_IMAGE004
(2) taking the intermediate product H1 prepared in the step (1) to carry out silane protecting group removing reaction to prepare an intermediate product H2;
Figure DEST_PATH_IMAGE006
(3) taking 2-oxo-acetyl-3, 4, 6-tri-oxo-benzyl-alpha-D-mannopyranosyl trichloroacetimidate as a glycosyl donor, taking p-tolyl 2,3, 4-tri-oxo-benzyl-1-thio-alpha-D-galactopyranoside as a glycosyl acceptor, and carrying out coupling reaction a to obtain an intermediate product H3;
Figure DEST_PATH_IMAGE008
(4) taking the intermediate product H3 prepared in the step (3) as a glycosyl donor, taking the intermediate product H2 prepared in the step (2) as a glycosyl acceptor, and carrying out coupling reaction b to prepare an intermediate product H4;
Figure DEST_PATH_IMAGE010
(5) taking the intermediate product H4 prepared in the step (4) to carry out chloroacetyl and acetyl removal reaction to prepare an intermediate product H5;
Figure DEST_PATH_IMAGE012
(6) carrying out hydrogenolysis removal reaction on the intermediate product H5 prepared in the step (5) to prepare the antrodia camphorata galactomannan oligosaccharide glycoside derivative I,
Figure DEST_PATH_IMAGE002A
。
2. the method of claim 1, wherein the reaction comprises one or more of the following:
(a) the method for the coupling reaction a in the step (1) and the step (3) comprises the following steps: dissolving a glycosyl donor and a glycosyl acceptor with the mass ratio of (1.1-1.2) 1 in dry dichloromethane, adding a 4A molecular sieve, stirring at room temperature for 20-40 minutes in a nitrogen atmosphere, cooling the reaction solution to-20-0 ℃, adding trimethyl trifluoromethanesulfonate, wherein the addition of the trimethyl trifluoromethanesulfonate is 10-30% of the mass of the glycosyl acceptor, stirring for 20-40 minutes, slowly heating the reaction solution to room temperature, neutralizing with triethylamine after the reaction is complete, and separating to obtain a target product;
(b) the method for the silane protecting group removing reaction in the step (2) comprises the following steps: dissolving a raw material in dichloromethane, adding a trifluoroacetic acid solution with the volume percentage of 90% under the stirring condition, wherein the adding amount ratio of the raw material to the trifluoroacetic acid solution is 1:5, the unit is g/ml, stirring and reacting for 20-40 minutes at room temperature, removing a solvent, and separating to obtain a target product;
(c) the method of the coupling reaction b in the step (4) is as follows: dissolving a glycosyl donor and a glycosyl acceptor with the mass ratio of (1.1-1.3):1 in dry dichloromethane, adding a 4A molecular sieve, stirring at room temperature for 20-40 minutes under a nitrogen atmosphere, cooling the reaction solution to-40 to-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of (1-2.5):1, wherein the mass ratio of the N-iodosuccinimide to the glycosyl acceptor is (1.0-1.2):1, stirring for 50-70 minutes, slowly raising the reaction solution to the room temperature, neutralizing with triethylamine after the reaction is completed, and separating to obtain a target product;
(d) the method for removing chloroacetyl and acetyl in the step (5) comprises the following steps: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strong acid cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain a target product;
(e) the method for the hydrogenolysis removal reaction of benzyl group in the step (6) is: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
3. The preparation method of the antrodia camphorata galactomannan-oligoglycoside derivative comprises the following steps:
Figure DEST_PATH_IMAGE015
wherein R is1Is alpha-D-mannopyranosyl or alpha-D-mannopyranosyl- (1 → 2) -alpha-D-mannopyranosyl, n is any integer from 0 to 4, characterized in that the steps are as follows:
1) the process for preparing galactomannan oligosaccharide derivative according to claim 1, wherein the intermediate H4 is obtained from steps (1) - (4);
2) taking the prepared intermediate product H4 to carry out chloroacetyl removal reaction to prepare an intermediate product H6;
Figure DEST_PATH_IMAGE017
3) taking a compound H7 as a glycosyl donor, taking an intermediate product H6 prepared in the step 2) as a glycosyl acceptor, and preparing an intermediate product H8 through a coupling reaction b;
Figure DEST_PATH_IMAGE019
wherein, H7 is:
Figure DEST_PATH_IMAGE021
or
Figure DEST_PATH_IMAGE023
R3Comprises the following steps:
Figure DEST_PATH_IMAGE025
or
Figure DEST_PATH_IMAGE027
4) Taking the intermediate product H8 prepared in the step 3) to perform acetyl removal reaction to prepare an intermediate product H9;
Figure DEST_PATH_IMAGE029
5) carrying out hydrogenolysis removal reaction on the intermediate product H9 prepared in the step 4) to prepare an antrodia camphorata galactomannan oligosaccharide glycoside derivative II;
Figure DEST_PATH_IMAGE015A
wherein R is1Comprises the following steps:
Figure DEST_PATH_IMAGE032
or
Figure DEST_PATH_IMAGE034
。
4. The method of claim 3, wherein the method comprises one or more of the following:
a) the method for the chloroacetyl group removal reaction in the step 2) comprises the following steps: dissolving raw materials in a mixed solvent of dichloromethane and anhydrous methanol, wherein the volume ratio of the dichloromethane to the anhydrous methanol is 1 (3-5), thiourea and 2, 6-dimethylpyridine are added according to the mass ratio of (4-6) to 0.1, the addition of the thiourea is 4-6 times of the mass of the raw materials, the reflux reaction is carried out at the temperature of 60-70 ℃, the reaction is complete, and the target product is obtained by concentration and separation;
b) the method of the coupling reaction b in step 3) is: taking a glycosyl donor and a glycosyl acceptor with the mass ratio of (1.1-1.3) 1 to dissolve in dry dichloromethane, adding a 4 angstrom molecular sieve, stirring at room temperature for 20-40 minutes under a nitrogen atmosphere, cooling the reaction liquid to-40 to-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of (1-2.5) 1 to the mass ratio of the addition amount of the N-iodosuccinimide to the addition amount of the glycosyl acceptor of (1.0-1.2) 1, stirring for 50-70 minutes, slowly raising the reaction liquid to the room temperature, neutralizing with triethylamine after the reaction is completed, and separating to obtain a target product;
c) the method for the acetyl group removal reaction in the step 4) comprises: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strong acid cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain a target product;
d) the method for the hydrogenolysis removal reaction of benzyl in the step 5) comprises the following steps: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
5. The preparation method of the antrodia camphorata galactomannan oligosaccharide derivative comprises the following steps:
Figure DEST_PATH_IMAGE036
wherein R is1Is alpha-D-mannopyranosyl or alpha-D-mannopyranosyl- (1 → 2) -alpha-D-mannopyranosyl, R2Is alpha-D-mannopyranosyl or alpha-D-mannopyranosyl- (1 → 3) -alpha-D-mannopyranosyl, n is any integer from 0 to 4, characterized in that it comprises the following steps:
(i) the process for preparing the galactomannan oligosaccharide derivative of antrodia according to claim 3, wherein the process comprises steps 1) -4) to obtain intermediate H9;
(ii) taking a compound H10 as a glycosyl donor, taking the intermediate product H9 prepared in the step (i) as a glycosyl acceptor, and performing coupling reaction b to prepare an intermediate product H11;
Figure DEST_PATH_IMAGE038
wherein, H10 is:
Figure DEST_PATH_IMAGE040
or
Figure DEST_PATH_IMAGE042
R4Comprises the following steps:
Figure DEST_PATH_IMAGE044
or
Figure DEST_PATH_IMAGE046
(iii) (iii) taking the intermediate product H11 prepared in the step (ii) to carry out acetyl and benzoyl removal reaction to prepare an intermediate product H12;
Figure DEST_PATH_IMAGE048
wherein R is5Comprises the following steps:
Figure DEST_PATH_IMAGE050
or
Figure DEST_PATH_IMAGE052
(iv) (iv) carrying out hydrogenolysis removal reaction on the intermediate product H12 prepared in the step (iii) to obtain the antrodia camphorate galactomannan oligosaccharide glycoside derivative III,
Figure DEST_PATH_IMAGE036A
wherein R is1Comprises the following steps:
Figure DEST_PATH_IMAGE055
or
Figure DEST_PATH_IMAGE057
Wherein R is2Comprises the following steps:
Figure DEST_PATH_IMAGE059
or
Figure DEST_PATH_IMAGE061
。
6. The process for preparing the galactomannan oligosaccharide derivative of antrodia camphorata according to claim 5, wherein the coupling reaction b in step (ii) is performed by: taking a glycosyl donor and a glycosyl acceptor with the mass ratio of (1.1-1.3):1, dissolving in dry dichloromethane, adding a 4A molecular sieve, stirring at room temperature for 20-40 minutes under a nitrogen atmosphere, cooling the reaction liquid to-40 to-15 ℃, adding N-iodosuccinimide and silver trifluoromethanesulfonate with the mass ratio of (1-2.5):1, wherein the mass ratio of the addition amount of the N-iodosuccinimide to the addition amount of the glycosyl acceptor is (1.0-1.2):1, stirring for 50-70 minutes, slowly raising the reaction liquid to the room temperature, neutralizing with triethylamine after the reaction is completed, and separating to obtain the target product.
7. The process for preparing the galactomannan-oligoglycoside derivative of antrodia according to claim 5, wherein the acetyl and benzoyl removal reaction in step (iii) is carried out by: dissolving the raw materials in anhydrous methanol, adding 1M sodium methoxide-methanol solution to adjust pH to 9.5-10.5, reacting completely, and adding strongly acidic cation exchange resin (H)+) Neutralizing, filtering, concentrating and separating to obtain the target product.
8. The method for preparing the galactomannan oligosaccharide derivative of antrodia camphorata according to claim 5 wherein the hydrogenolysis reaction to remove benzyl group in step (iv) comprises: dissolving 8-12 parts by mass of raw materials in deionized water, adding 1-2 parts by mass of catalytic palladium carbon under the protection of nitrogen, introducing hydrogen into a reaction bottle, displacing the nitrogen, stirring the reaction solution at room temperature for 24-30 hours under the atmosphere of the hydrogen, filtering to remove the palladium carbon, distilling under reduced pressure to remove the solvent, separating and freeze-drying to obtain the target product.
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