阅读说明：本技术 β-D-(1,4)-甘露糖醛酸寡糖及其中间体的制备方法 (Preparation method of beta-D- (1,4) -mannuronic acid oligosaccharide and intermediate thereof ) 是由 傅东林 王世胜 张亚珍 肖中平 张真庆 于 2020-05-19 设计创作，主要内容包括：本发明公开了一种经济、高效地制备式IX所示的β-D-(1,4)-甘露糖醛酸寡糖的方法。特别地,本发明以经济易得的1,2,3,4,6-五-O-乙酰基-D-吡喃甘露糖(式X)为原料,制备关键中间体化合物I、化合物II和化合物III；将中间体化合物II和III偶联成寡糖受体化合物V,将中间体化合物I和II偶联成寡糖供体化合物VII；将化合物V和化合物VII偶联成寡糖化合物VIII,将寡糖化合物VIII脱除保护基得到β-D-(1,4)-甘露糖醛酸寡糖IX。(The invention discloses a method for economically and efficiently preparing beta-D- (1,4) -mannuronic acid oligosaccharide shown as a formula IX. In particular, the invention takes 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose (formula X) which is economic and easy to obtain as a raw material to prepare a key intermediate compound I, a compound II and a compound III; coupling the intermediate compounds II and III into an oligosaccharide acceptor compound V, and coupling the intermediate compounds I and II into an oligosaccharide donor compound VII; coupling the compound V and the compound VII into an oligosaccharide compound VIII, and removing a protecting group from the oligosaccharide compound VIII to obtain the beta-D- (1,4) -mannuronic acid oligosaccharide IX.)

1. a method for preparing beta-D- (1,4) -mannuronic acid oligosaccharide shown in formula VIII or formula IX,

the method comprises the following steps:

carrying out coupling reaction on the compound V and the compound VII and selectively removing the protecting group R³Generating a compound VIII;

and optionally, the compound VIII is subjected to one-time deprotection of the protecting group R²Generating beta-D- (1,4) -mannuronic acid oligosaccharide shown as the formula IX;

wherein m is an integer from 2 to 18; n and n' are each independently selected from integers from 0 to 8; r¹Is selected from C_1-8Alkyl, optionally substituted by C_1-8Alkyl substituted C_6-14An aryl group; r²A hydroxyl protecting group which can be removed through hydrogenation reaction catalyzed by palladium carbon or oxidation reaction catalyzed by palladium carbon; r³Is a hydroxyl protecting group which cannot be removed by palladium-carbon catalyzed hydrogenation or palladium-carbon catalyzed oxidation.

2. The process of claim 1, wherein the coupling reaction is carried out in the presence of a sterically hindered organic base, diphenyl sulfoxide and a sulfonic anhydride catalyst; then sequentially removing the hydroxyl protecting groups R at the 4-position³And hydroxy protecting groups R at other positions²To obtain the beta-D- (1,4) -mannuronic acid oligosaccharide compound shown as IX.

3. The process of claim 2 wherein the sulfonic anhydride catalyst is selected from methanesulfonic anhydride, trifluoromethanesulfonic anhydride, or p-toluenesulfonic anhydride.

4. The method of claim 1, further comprising:

carrying out coupling reaction on the compound II and the compound III to generate a 1, 4-glycosidic bond, and then selectively removing a 4-hydroxyl protecting group R³To obtain a compound IV;

then, carrying out coupling reaction on the compound IV and the compound II to generate a 1, 4-glycosidic bond, and then selectively removing a protecting group R of hydroxyl at the 4-position³(ii) a Optionally repeating the coupling reaction and deprotection of the group R³Until compound V is obtained;

wherein n and R²And R³As defined in claim 1; r⁴Selected from H, C_1-8Alkyl radical, C_6-14An aryl group; x is selected from fluorine, chlorine, bromine and iodine.

5. The process of claim 4, wherein the coupling reaction is carried out in the presence of a sulfonic acid catalyst; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate.

6. The method of claim 1, further comprising:

carrying out coupling reaction on the compound I and the compound II to generate a 1, 4-glycosidic bond, and then selectively removing a protecting group R of hydroxyl at the 4-position³To obtain a compound VI;

coupling reaction is carried out on the compound VI and the compound II to generate 1, 4-glycosidic bond, and then the protecting group R of the 4-hydroxyl is selectively removed³(ii) a Optionally repeating the coupling reaction and deprotection of the group R³Until compound VII is obtained;

wherein, n' and R¹、R²And R³As defined in claim 1; r⁴Selected from H, C_1-8Alkyl radical, C_6-14An aryl group; x is selected from fluorine, chlorine, bromine and iodine.

7. The process of claim 6, wherein the coupling reaction is carried out in the presence of a sulfonic acid catalyst; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate.

8. The method of any one of claims 4-7, wherein compound II is obtained by:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: selective removal of anomeric carbon protecting group-SR of Compound D¹To obtain a compound E;

and step 3: under alkaline conditions, the compound E is reacted with CX₃C(＝NR⁴) X or CX₃CN reacts to obtain a compound II;

wherein R is¹、R²And R³As defined in claim 1; r⁴Selected from H, C_1-8Alkyl radical, C_6-14An aryl group; x is selected from fluorine, chlorine, bromine and iodine.

9. The method of any one of claims 4-5, wherein compound III is obtainable by:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: under the alkaline condition, the compound D and the compound R²OH reaction to obtain a compound F;

and step 3: selective removal of the hydroxy-protecting group R at position 4 of Compound F³To obtain a compound III;

wherein R is¹、R²And R³As defined in claim 1.

10. The method of any of claims 6-9, further comprising:

step 1: 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose and anomeric carbon protective agent R¹SH reaction, and then hydrolysis reaction is carried out to obtain a compound A;

step 2: selectively protecting the hydroxyl groups at the 2-position and the 3-position of the compound A to obtain a compound B;

and step 3: oxidizing the hydroxyl group at the 6-position of the compound B into a carboxyl group, and then esterifying the carboxyl group to obtain a compound I;

wherein R is¹And R²As defined in claim 1.

11. The method of any one of claims 1-10,

R²is selected from C_6-14Arylmethyl or allyl, said C_6-14Arylmethyl optionally substituted by C_1-8Alkyl radical, C_1-8Alkoxy, halogen substitution;

R³is selected from C_1-8Alkyl acyl radical, C_1-8Alkoxyacyl group, C_6-14Arylacyl, tri (C)_1-8Alkyl) silyl, 9-fluorenylmethyloxycarbonyl, tri (C)_6-14Aryl) methyl; wherein C is_1-8Alkyl acyl and C_1-8C in alkoxyacyl_1-8Any carbon atom in the alkyl group may optionally be oxo.

12. The method of claim 11, wherein,

R²selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl;

R³selected from acetyl, levulinyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethyloxycarbonyl and trityl.

13. A compound of formula I:

or a salt thereof, wherein,

R¹is selected from C_1-8Alkyl, optionally substituted by C_1-8Alkyl substituted C_6-14An aryl group;

R²is selected from C_6-14Arylmethyl or allyl, said C_6-14Arylmethyl optionally substituted by C_1-8Alkyl, aryl, heteroaryl, and heteroaryl,C_1-8Alkoxy, halogen substitution.

14. A compound of formula I according to claim 13, or a salt thereof,

R¹selected from phenyl, o-tolyl, p-methylphenyl, 4-tert-butyl-2-methylphenyl, 2, 4-di-tert-butylphenyl, methyl or ethyl;

R²selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl.

15. A process for the preparation of a compound of formula I as claimed in claim 13 or 14 or a salt thereof, which comprises:

step 1: 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose and anomeric carbon protective agent R¹SH reaction, and then alkaline hydrolysis reaction is carried out to obtain a compound A;

step 2: selectively protecting the hydroxyl groups at the 2-position and the 3-position of the compound A to obtain a compound B;

and step 3: oxidizing the hydroxyl group at the 6-position of the compound B into a carboxyl group, and then esterifying the carboxyl group to obtain a compound I;

wherein R is¹And R²As defined in claim 13 or 14.

16. A compound of formula II:

or a salt thereof, wherein,

R²is selected from C_6-14Arylmethyl or allyl, said C_6-14Arylmethyl optionally substituted by C_1-8Alkyl radical, C_1-8Alkoxy, halogen substitution;

R³is selected from C_1-8Alkyl acyl radical, C_1-8Alkoxyacyl group, C_6-14Arylacyl, tri (C)_1-8Alkyl) silyl, 9-fluorenylmethyloxycarbonyl, tri (C)_6-14Aryl) methyl; wherein C is_1-8Alkyl acyl and C_1-8C in alkoxyacyl_1-8Any carbon atom in the alkyl group may optionally be oxo;

R⁴selected from hydrogen, C_1-8Alkyl radical, C_6-14An aryl group;

x is selected from fluorine, chlorine, bromine and iodine.

17. The compound of formula II or a salt thereof according to claim 16,

R²selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl;

R³selected from acetyl, levulinyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethyloxycarbonyl, trityl;

R⁴selected from hydrogen, C_1-4Alkyl radical, C_6-8Aryl radicals

X is selected from chlorine or bromine.

18. A process for the preparation of a compound of formula II, or a salt thereof, as claimed in claim 16 or 17, comprising:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: selective removal of anomeric carbon protecting group-SR of Compound D¹To obtain a compound E;

and step 3: reacting compound E with tri-CX under alkaline conditions₃C(＝NR⁴) X or CX₃CN nitrile reaction to obtain a compound II;

wherein R is¹As defined in claim 1; r²、R³、R⁴And X is as defined in claim 16 or 17.

19. A compound of formula III:

or a salt thereof, wherein,

R²is selected from C_6-14Arylmethyl or allyl, said C_6-14Arylmethyl optionally substituted by C_1-8Alkyl radical, C_1-8Alkoxy, halogen substitution.

20. The compound of formula III or a salt thereof according to claim 19,

R²selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl.

21. A process for the preparation of a compound of formula III, or a salt thereof, as claimed in claim 19 or 20, comprising:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: under the alkaline condition, the compound D and the compound R²OH reaction to obtain a compound F;

and step 3: selective removal of the hydroxy-protecting group R at position 4 of Compound F³To obtain a compound III;

wherein R is¹And R³As defined in claim 1, R²As defined in claim 19 or 20.

Technical Field

The invention belongs to the field of organic chemical synthesis, and relates to a preparation method of beta-D- (1,4) -mannuronic acid oligosaccharide (disaccharide to eicosaeose).

Background

The beta-D- (1,4) -mannuronic acid oligosaccharide widely exists in natural products, has the effects of promoting the growth of plant roots, inhibiting bacteria, promoting the generation of human keratinocytes and the like, can be combined with Toll-like receptors 2 and 4, shows the function of immunoregulation (CN103275133A), and has good curative effect on treating vascular dementia (CN 106344593A). 11/2/2019, the mannotena capsules approved by the national drug administration for treating mild to moderate alzheimer's disease are on the market, and the main components of the mannoprotein capsules are beta-D- (1,4) -mannuronic acid oligosaccharide and derivatives thereof.

At present, beta-D- (1,4) -mannuronic acid oligosaccharide is mainly obtained by degrading sodium alginate (CN100508985C), the degradation preparation process has great challenges for controlling the purity and impurities of the beta-D- (1,4) -mannuronic acid oligosaccharide, and the development of a preparation process which is efficient, simple to operate and capable of being accurately controlled is urgently needed. The preparation of derivatives of oligosaccharide of beta-D- (1,4) -Mannuronic Acid by solid phase Synthesis (WO2012138698), which is expensive, requires high equipment and is not easy to be industrially produced, or by liquid phase Synthesis (Cod ee, Jeronen D.C., van den Bos, Leendert J, de Jong, Ana-Rae, et al, the Stereodoring Effect of the Glycyl C5-Carboxylate Ester: Stereoselective Synthesis of beta-Mannuronic Acid Alginates [ J ]. Journal of Organic Chemistry,74(1): 38-47). In addition, the existing liquid phase synthesis method has the problems of low synthesis efficiency, complicated protective group removal and the like, and is not easy for industrial production. Therefore, the method for preparing the novel, high-efficiency and process-controllable beta-D- (1,4) -mannuronic acid oligosaccharide is developed and researched, and has important application value and economic value.

Disclosure of Invention

In a first aspect of the present invention, there is provided a process for producing beta-D- (1,4) -mannuronic acid oligosaccharide, which can economically and efficiently produce high-purity beta-D- (1,4) -mannuronic acid oligosaccharide. The method comprises the steps of carrying out coupling reaction on a compound V and a compound VII and selectively removing a protecting group R³Generating a compound VIII;

and optionally, the compound VIII is subjected to one-time deprotection of the protecting group R²Generating beta-D- (1,4) -mannuronic acid oligosaccharide shown as the formula IX;

wherein m is an integer from 2 to 18; n and n' are each independently selected from integers from 0 to 8; r¹Is selected from C_1-8Alkyl, optionally substituted by C_1-8Alkyl substituted C_6-14An aryl group; r²A hydroxyl protecting group which can be removed through hydrogenation reaction catalyzed by palladium carbon or oxidation reaction catalyzed by palladium carbon; r³Is a hydroxyl protecting group which cannot be removed by palladium-carbon catalyzed hydrogenation or palladium-carbon catalyzed oxidation.

In particular, the present invention can realize the preparation of intermediate compounds I, II and III starting from 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose (formula X), which is economically available; then coupling the intermediate compounds II and III into an oligosaccharide acceptor compound V, and coupling the intermediate compounds I and II into an oligosaccharide donor compound VII; and then coupling the compound V and the compound VII into an oligosaccharide compound VIII, and removing a protecting group to obtain a final product compound IX, namely the beta-D- (1,4) -mannuronic acid oligosaccharide. The invention provides an economic and efficient solution for synthesizing the beta-D- (1,4) -mannuronic acid oligosaccharide compound with the polymerization degree of 2 to 20, compared with the process for extracting and preparing the oligosaccharide mixture by degrading sodium alginate and the like, the scheme can obtain the oligosaccharide with single polymerization degree and high purity, and lays a solid foundation for further researching the pharmacological and biological activity of the oligosaccharide with single polymerization degree.

In a second aspect of the present invention, there are provided key intermediates compound I, compound II, compound III, compound V, compound VII and compound VIII for the synthesis of β -D- (1,4) -mannuronic acid oligosaccharide, having the structural formulae shown below:

wherein the content of the first and second substances,

R¹is selected from C_1-8Alkyl, optionally substituted by C_1-8Alkyl substituted C_6-14An aryl group; preferably, R¹Selected from phenyl, o-tolyl, p-methylphenyl, 4-tert-butyl-2-methylphenyl, 2, 4-di-tert-butylphenyl, methyl or ethyl;

R²is a hydroxyl protecting group which can be removed through palladium-carbon catalyzed hydrogenation reaction; preferably, R²Is selected from C_6-14Arylmethyl or allyl, said C_6-14Arylmethyl optionally substituted by C_1-8Alkyl radical, C_1-8Alkoxy, halogen substitution; more preferably, R²Selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl; most preferably, R²Is selected from benzyl;

R³is a hydroxyl protecting group which can not be removed through palladium-carbon catalyzed hydrogenation reaction; preferably, R³Is selected from C_1-8Alkyl acyl radical, C_1-8Alkoxyacyl group, C_6-14Arylacyl, tri (C)_1-8Alkyl) silyl, 9-fluorenylmethyloxycarbonyl, tri (C)_6-14Aryl) methyl; wherein C is_1-8Alkyl acyl and C_1-8C in alkoxyacyl_1-8Any carbon atom in the alkyl group may optionally be oxo; more preferably, R³Selected from acetyl, levulinyl, trimethylsilyl, t-butyldimethylsilyl, benzoyl, 9-fluorenylmethyloxycarbonyl, or trityl; most preferably, R³Selected from levulinyl;

R⁴selected from H, C_1-8Alkyl radical, C_6-14An aryl group;

x is selected from fluorine, chlorine, bromine and iodine;

m is an integer from 2 to 18; preferably, m is selected from 2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

n and n' are each independently selected from integers from 0 to 8; preferably, n and n' are each independently selected from 0, 1,2,3,4, 5, 6, 7 or 8.

Drawings

FIG. 1 is a general synthetic route for compound VIII and compound IX.

FIG. 2 is a synthetic route to compound IX-1 and compound IX-2.

FIG. 3 is a synthetic route to compound IX-3 and compound IX-4.

FIG. 4 is a synthetic route to compound IX-5.

Detailed Description

Unless otherwise indicated, the term "alkyl" referred to herein may be a straight or branched chain saturated hydrocarbon group such as methyl, ethyl, propyl, butyl, octyl, isopropyl, tert-butyl, sec-pentyl and the like. The alkyl group may be unsubstituted or substituted with one or more substituents (e.g., halogen, alkoxy, aryl, aralkyl, aralkyloxy, and the like). C_1-nAlkyl (where n is an integer) refers to alkyl groups containing 1 to n carbon atoms, such as 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, and the like.

The term "aryl" refers to a monovalent unsaturated aromatic group having a single ring (such as phenyl) or fused rings (such as naphthyl or anthryl), optionally substituted with substituents such as halogen (including fluorine, chlorine, bromine, iodine), alkyl, aralkyl, alkoxy, aralkoxy, and the like. C_6-nAryl (where n is an integer) refers to an aryl group having 6 to n carbon atoms, such as phenyl, naphthyl, anthracenyl, or optionally substituted groups thereof.

The first aspect of the present invention relates to a process for the preparation of ss-D- (1,4) -mannuronic acid oligosaccharides represented by formula (VIII) or formula (IX),

the method comprises the following steps:

carrying out coupling reaction on the compound V and the compound VII and selectively removing the protecting group R³Generating a compound VIII;

and optionally, the compound VIII is subjected to one-time deprotection of the protecting group R²Generating beta-D- (1,4) -mannuronic acid oligosaccharide shown as the formula IX;

wherein m is an integer from 2 to 18; n and n' are each independently selected from integers from 0 to 8; r¹Is selected from C_1-8Alkyl, optionally substituted by C_1-8Alkyl substituted C_6-14An aryl group; r²Is a hydroxyl protecting group which can be removed through palladium-carbon catalyzed hydrogenation reaction; r³Is a hydroxyl protecting group which cannot be removed by palladium-carbon catalyzed hydrogenation.

The coupling reaction is carried out in the presence of organic base with large steric hindrance, diphenyl sulfoxide and a sulfonic anhydride catalyst; then sequentially removing the hydroxyl protecting groups R at the 4-position³And hydroxy protecting groups R at other positions²To obtain the beta-D- (1,4) -mannuronic acid oligosaccharide compound shown as IX. Preferably, the sulphonic anhydride catalyst is selected from the group consisting of methanesulphonic anhydride, trifluoromethanesulphonic anhydride or p-toluenesulphonic anhydride.

In one embodiment of the present invention, there is provided a method of synthesizing compound V, comprising:

dissolving the intermediate compound II and the intermediate compound III in a proper organic solvent, adding a dry molecular sieve, adding a sulfonic acid catalyst under the protection of nitrogen or argon, performing coupling reaction at a proper temperature to generate a 1, 4-glycosidic bond, and then selectively removing a 4-hydroxyl protecting group to obtain a compound IV; repeating the above coupling reaction and selectivity between compound IV and compound IIRemoval of the protecting group R for the hydroxyl group at position 4³Optionally repeating the above steps of coupling and deprotecting the compound obtained until compound V is obtained.

In a preferred embodiment of the present invention, the organic solvent in the synthesis of compound V is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N, N-dimethylformamide, anhydrous N, N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate, preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the protecting group of the hydroxyl group at the 4-position is preferably hydrazine acetate, and the molar equivalent of the hydrazine acetate is 3 to 8eq, preferably 3 to 5.5eq, for example 4.3 eq.

In one embodiment of the present invention, there is provided a method of synthesizing compound VII, comprising:

dissolving compound I and compound II in proper organic solvent, adding dried molecular sieve, protecting with nitrogen or argon, adding sulfonic acid catalyst at proper temperature, coupling reaction to generate 1, 4-glycosidic bond, and selectively removing 4-hydroxy protecting group R³To obtain a compound VI; repeatedly carrying out the coupling reaction and selectively removing the protecting group R at the 4-position on the compound VI and the compound II³A step (2); optionally, the obtained compound is further subjected to the above steps of coupling reaction and deprotection, until the compound VII is obtained.

In a preferred embodiment of the present invention, the organic solvent in the synthesis of compound VII is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N, N-dimethylformamide, anhydrous N, N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate; the sulfonic acid catalyst is preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the 4-position hydroxyl protecting group is preferably hydrazine acetate.

In one embodiment of the present invention, there is provided a method of synthesizing compound IX, comprising:

dissolving a compound V and a compound VII in a proper organic solvent, adding a dry molecular sieve, adding a large steric hindrance organic base, diphenyl sulfoxide and sulfonic anhydride for catalysis under the protection of nitrogen or argon at a proper temperature, carrying out a coupling reaction to generate a 1, 4-glycosidic bond, and selectively removing a protecting group R of a 4-hydroxyl group³Obtaining a compound VIII; adding palladium carbon (palladium content is 5% -10%) into compound VIII, hydrogenating and removing all R of compound VIII²Protecting groups to obtain the beta-D- (1,4) -mannuronic acid oligosaccharide compound IX.

In a preferred embodiment of the present invention, the molar ratio of compound V to compound VII in the synthesis of compound IX is 1: 0.9; the solvent of the coupling reaction is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N, N-dimethylformamide, anhydrous N, N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sterically hindered organic base is selected from: 1, 8-diazabicycloundecen-7-ene, 2, 6-di-tert-butylpyridine or 2,4, 6-tri-tert-butylpyrimidine, preferably 2, 6-di-tert-butylpyridine; the sulfonic anhydride catalyst is selected from: methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride, preferably trifluoromethanesulfonic anhydride; the molar equivalent of the sulfonic anhydride is preferably 0.05 eq; the reaction temperature is preferably-60 ℃; the reagent for removing the 4-position hydroxyl protecting group is preferably hydrazine acetate, and the molar equivalent is preferably 4.3 eq; the temperature for removing the protecting group is preferably 25 ℃.

The starting compounds used above, formula I and formula II, respectively, can be obtained from compounds of formula X.

The compound of formula X is 1-5 substituted mannopyranose, wherein the substituent may be C₁-C₆Acyl, for example formyl, acetyl, propionyl. An example of a compound of formula X is 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose.

In a preferred embodiment of the present invention, exemplified by the simple and readily available starting material 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose (compound of formula X), the general reaction scheme is as follows:

wherein R is⁵Is C_1-8The acyl group of (2) is preferably an acetyl group.

In one embodiment of the present invention, there is provided a process for the synthesis of intermediate compound I comprising:

step 1: 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose and anomeric carbon protective agent R¹SH reaction, and then alkaline hydrolysis reaction is carried out to obtain a compound A;

step 2: selectively protecting the hydroxyl groups at the 2-position and the 3-position of the compound A to obtain a compound B;

and step 3: the hydroxyl group at the 6-position of compound B is oxidized to a carboxyl group, and the carboxyl group is esterified to give compound I.

In a preferred embodiment, the method comprises:

step 1, in a dry organic solvent, taking 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose as a raw material, stirring at a proper temperature in the presence of an acid catalyst, under the protection of nitrogen or argon, adding a proper anomeric carbon protective agent R¹SH reacts, and then alkali hydrolysis is carried out to obtain a compound A;

step 2, under the catalysis of Lewis acid, selecting proper hydroxyl protecting groups, selectively protecting the hydroxyl at the 4-position and the 6-position of the compound A, then selectively protecting the hydroxyl at the 2-position and the 3-position, and finally selectively removing the protecting groups at the 4-position and the 6-position to obtain a compound B;

and 3, adding an oxidant into the compound B in an organic solvent, selectively oxidizing 6-hydroxyl of the compound B into carboxyl, and reacting with an alkylating reagent at a proper temperature and in a solvent under an alkaline condition to obtain the compound I.

In a more preferred embodiment, the synthesis of intermediate compound I employs a one-pot two-step reaction, and the intermediate does not need to be isolated and purified. Wherein the organic solvent in step 1 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous toluene, anhydrous N, N-dimethylformamide, or anhydrous N, N-dimethylacetamide; the appropriate temperature is 0-25 ℃, preferably 0-5 ℃; the acidic catalyst is selected from: boron trifluoride diethyl etherate, acetyl chloride or hydrogen chloride gas, preferably boron trifluoride diethyl etherate; the molar equivalent of boron trifluoride diethyl etherate is preferably 0.1 eq; the base is selected from: sodium ethoxide, potassium ethoxide, magnesium ethoxide, sodium methoxide, potassium methoxide or magnesium methoxide, preferably sodium methoxide; the molar equivalent of sodium methoxide is preferably 0.1 eq; the solvent for the alkaline hydrolysis is selected from: methanol, ethanol or tetrahydrofuran, preferably methanol; the anomeric carbon protective agent added in the reaction is selected from: thiophenol, o-tolylthiophenol, p-methylphenylthiol, 4-tert-butyl-2-methylphenylthiol, 2, 4-di-tert-butylthiophenol, methanethiol or ethanethiol, preferably p-methylphenylthiol; the molar equivalent of anomeric carbon protectors is preferably 1.05 eq.

Wherein the protecting agent for protecting the 4-and 6-positions of compound a in step 2 is selected from: xylylene acetal, p-methoxyphenyldiacetal or 2, 2-dimethoxypropane (acetonylidene), preferably xylylene acetal; the molar equivalent of xylylene acetal is preferably 0.9 eq; the Lewis acid catalyst is selected from: p-toluenesulfonic acid, trifluoromethanesulfonic acid, aluminum trichloride and ferric trichloride, preferably ferric trichloride; the protecting agent for protecting the hydroxyl at the 2-position and the 3-position of the compound A is selected from benzyl bromide, benzyl chloride, p-methoxy benzyl bromide, p-methoxy benzyl chloride or allyl bromide, preferably benzyl bromide; the equivalent of benzyl bromide is preferably 1.9 eq.

Wherein, the oxidant in the step 3 is selected from: tetramethyl piperidine nitrogen oxide, sodium hypochlorite, potassium bromide, hydrogen peroxide, tert-butyl hydroperoxide or iodobenzene diacetate, preferably tetramethyl piperidine nitrogen oxide or iodobenzene diacetate; the molar equivalent of the tetramethylpiperidine nitroxide is preferably 0.2eq, or the molar equivalent of the iodobenzene diacetate is preferably 2.5 eq; the organic solvent is selected from: any one or more of dichloromethane, acetonitrile, water, tetrahydrofuran, N-dimethylformamide or N, N-dimethylacetamide, preferably selected from: an acetonitrile-water mixed solvent (acetonitrile to water volume ratio of about 5:1 to 1:5), a tetrahydrofuran-water mixed solvent (tetrahydrofuran to water volume ratio of about 5:1 to 1:5), or a dichloromethane-water mixed solvent (dichloromethane to water volume ratio of about 5:1 to 1:5), more preferably a dichloromethane-water mixed solvent (dichloromethane to water volume ratio of about 1: 2); the temperature of the oxidation reaction is 0 ℃ to 25 ℃, and the preferred temperature is 25 ℃; the base is selected from: potassium carbonate, cesium carbonate, sodium carbonate, calcium carbonate, silver carbonate, triethylamine or diisopropylethylamine, preferably potassium carbonate or triethylamine; the molar equivalent of the base is preferably 0.5eq to 3eq, more preferably 1.5eq of potassium carbonate or 1eq of triethylamine; the alkylating agent is selected from: bromobenzyl, chlorobenzyl, p-methoxybenzyl bromide, p-methoxybenzyl chloride or allyl bromide, preferably bromobenzyl; the solvent for the alkylation reaction is selected from: tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide, acetone or acetonitrile, preferably acetone; the alkylation reaction temperature is 25 ℃ to 100 ℃, preferably 30 ℃.

In one embodiment of the present invention, there is provided a process for the synthesis of intermediate compound II comprising:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: selective removal of anomeric carbon protecting group-SR of Compound D¹To obtain a compound E;

and step 3: under alkaline conditions, the compound E is reacted with CX₃CN or CX₃C(＝NR⁴) X reacts to obtain a compoundII。

In a preferred embodiment, the method comprises:

step 1: protecting the 4-hydroxyl of the compound I in a dry organic solvent under the alkaline condition by using a proper protective agent to obtain a compound D;

step 2: selective removal of anomeric carbon protecting group-SR of Compound D¹To obtain a compound E;

and step 3: reacting compound E with CX in dry organic solvent under alkaline condition₃CN or CX₃C(＝NR⁴) Reaction of X (e.g., trichloroacetonitrile) affords compound II.

In a more preferred embodiment, the synthesis of intermediate compound II employs a one-pot three-step reaction, and the intermediate does not need to be isolated and purified. Wherein the suitable protecting agent in step 1 is selected from acetyl chloride, acetic anhydride, trimethylchlorosilane, tert-butyldimethylchlorosilane, benzoyl chloride, 9-fluorenylmethyl chloroformate, triphenylchloromethane, acetylpropionyl chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protecting agent is preferably 1.5 eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N, N-dimethylformamide or anhydrous N, N-dimethylacetamide, preferably anhydrous dichloromethane; the preferred reaction temperature is 25 ℃; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N-p-dimethylaminopyridine or pyridine, preferably N, N-p-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5 eq.

Wherein, the reagent for removing the anomeric carbon protecting group in the step 2 is selected from N-chlorosuccinimide or N-bromosuccinimide, preferably N-bromosuccinimide; the reaction temperature is preferably 25 ℃.

Wherein the organic solvent in step 3 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N, N-dimethylformamide or anhydrous N, N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably 0 ℃ to 10 ℃; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N-p-dimethylaminopyridine or pyridine, preferably 1, 8-diazabicyclo [5.4.0] undec-7-ene; the molar equivalent of the base is preferably 0.5 eq.

In one embodiment of the present invention, there is provided a process for the synthesis of intermediate compound III comprising:

step 1: protecting the 4-hydroxy of the compound I to obtain a compound D;

step 2: under the alkaline condition, the compound D and the compound R²OH reaction to obtain a compound F;

and step 3: selective removal of the hydroxy-protecting group R at position 4 of Compound F³To obtain the compound III.

In a preferred embodiment, the method comprises:

step 1, protecting 4-hydroxy of a compound I by using a proper protective agent in a dry organic solvent under an alkaline condition to obtain a compound D;

step 2. adding R to Compound D in a Dry organic solvent in the Presence of a sterically hindered base²OH, diphenyl thiophenol and a sulfonic anhydride catalyst react to obtain a compound F;

and 3, selectively removing the 4-hydroxyl protecting group of the compound F in an organic solvent to obtain a compound III.

In a more preferred embodiment of the present invention, the synthesis of intermediate compound III employs a one-pot three-step process. Wherein the suitable protecting agent in step 1 is selected from: acetyl chloride, acetic anhydride, trimethylchlorosilane, tert-butyldimethylchlorosilane, 9-fluorenylmethyl chloroformate, benzoyl chloride, triphenylchloromethane, levulinic acid chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protective agent is preferably 1.5 eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N, N-dimethylformamide or anhydrous N, N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably 25 ℃; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N-p-dimethylaminopyridine or pyridine, preferably N, N-p-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5 eq.

Wherein the organic solvent in step 2 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N, N-dimethylformamide, anhydrous N, N-dimethylacetamide, anhydrous toluene or anhydrous dimethylsulfoxide, preferably anhydrous dichloromethane; the sterically hindered organic base is selected from: 1, 8-diazabicycloundecen-7-ene, 2, 6-di-tert-butylpyridine or 2,4, 6-tri-tert-butylpyrimidine, preferably 2, 6-di-tert-butylpyridine; the molar equivalent of the base is preferably 2.2 eq; the sulfonic anhydride catalyst is selected from: methanesulfonic anhydride, trichloromethanesulfonic anhydride, tribromomethanesulfonic anhydride, trifluoromethanesulfonic anhydride or toluenesulfonic anhydride, preferably trifluoromethanesulfonic anhydride; the molar equivalent of trifluoromethanesulfonic anhydride is preferably 0.05 eq; the reaction temperature is preferably-60 ℃.

Wherein, the organic solvent in the step 3 is dichloromethane and/or pyridine, preferably dichloromethane: pyridine ═ 5:1 (V/V); the reagent for selectively removing the hydroxyl protecting group at the 4-position of the compound F is preferably hydrazine acetate, and the equivalent thereof is preferably 4.3 eq.

In a preferred embodiment of the present invention, the compound I, the compound II and the compound III are respectively a compound I-1, a compound II-1 and a compound III-1 represented by the following structural formulae:

the invention has the advantages that:

the method for preparing beta-D- (1,4) -mannuronic acid oligosaccharide (compound IX) adopts a convergent synthesis strategy, assembles an oligosaccharide donor (compound VII) and an oligosaccharide acceptor (compound V) into an oligosaccharide compound VIII with higher glycosyl number by a brand-new oligosaccharide intermediate compound I, a compound II and a compound III, and removes a protecting group R in the compound VIII once again²Synthesis of beta-D- (1,4) -oligomannuronateSugar (compound IX). Compared with the prior art, the preparation method is simpler, more economical and more efficient.

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

The following examples provide, in part, a complete synthesis scheme for the disaccharides (. beta. -D- (1,4) -mannuronic acid (compound IX-1), tetrasaccharides (compound IX-2), trisaccharides (compound IX-3), pentasaccharides (compound IX-4) and heptasaccharides (compound IX-5) which further illustrate the invention, but are not intended to be limiting thereof.

The starting materials and reagents in the examples are all commercially available except where specifically noted.

The room temperature in the examples means 20 to 30 ℃.

The atmospheric pressure in the examples means 1 atmosphere.

The water in the examples refers to deionized water.

Meanings of abbreviations

Abbreviations	Means of
		TLC	Thin layer chromatography
EA	Ethyl acetate
		Hex	Hexane (C)
DCM	Methylene dichloride
		TEMPO	Tetramethyl piperidine nitroxide
DIC	Diisopropylcarbodiimide
		DMAP	Dimethylaminopyridine
NBS	Bromosuccinimide
		DBU	1, 8-diazabicycloundec-7-enes
TTBP	2,4, 6-Tri-tert-butylpyridines
		Tf2O	Trifluoromethanesulfonic anhydride
LevOH	Levulinic acid
		TMSOTf	Trimethylsilyl trifluoromethanesulfonate
CNCCl3	Trichloroacetonitrile
		min	Minute (min)
mL	Milliliter (ml)
		mmol	Millimole

Example 1: synthesis of Compound I-1

The first step is as follows: 1,2,3,4, 6-penta-O-acetyl-D-mannopyranose (19.5g,50.0mmol) was weighed out and placed in a 500mL eggplant-shaped flask, and anhydrous dichloromethane (150mL) was added to give a colorless transparent solution, and p-tolylthiophenol (6.2g,50.0mmol) and boron trifluoride ether solution (12.7mL, 100mmol) were added thereto under ice bath. After stirring in an ice bath for half an hour, stirring was continued at room temperature for 24 hours and the solution changed from light yellow to pink. TLC (EA/Hex. 1/2) for disappearance of starting material and saturated NaHCO₃The solution is quenched to react, a dichloromethane layer is separated by a separating funnel, the solvent is evaporated under reduced pressure, 200mL of anhydrous methanol is added for redissolving, sodium methoxide (256mg, 4.7mmol) is added, the mixture is stirred at room temperature for 12 hours, TLC (EA/Hex ═ 1/2) detects that raw materials are completely reacted, the pH value is adjusted to be neutral by dilute hydrochloric acid, the solvent is evaporated under reduced pressure, dichloromethane (100mL) and water (100mL) are added, the mixture is stirred at room temperature for half an hour, a water layer is separated by the separating funnel, water is evaporated under reduced pressure, and the crude compound A-1 is directly used for the next step without purification.

The second step is that: crude Compound A-1 (ca 47.3mmol) was dissolved in 200mL anhydrous DMF and PhCH (OMe) was added₂(7.9g,52.03mmol,1.1eq), p-toluenesulfonic acid (899mg, 4.7mmol, 0.1eq), reaction temperature controlled at 50 deg.C, reaction under reduced pressure for 2 hours, TLC (CH)₃OH/DCM ═ 1/9) indicated complete reaction of the starting materials. The reaction was cooled to room temperature, NaH (5.68g,141.9mmol,3.0eq) was added under ice-bath for 20min, then BnBr (20.2 g,118.3mmol,2.5eq) was added and after 20min the ice-bath was removed and stirred at room temperature for 12 h, TLC (EA/Hex ═ 1/4) indicated complete conversion of the starting material. The NaH was quenched with methanol (10mL) while cooling on ice, ethyl acetate (200mL) was added to dilute the reaction,the reaction mixture was washed three times with saturated brine (200mL), concentrated under reduced pressure to remove the solvent, and then dissolved in 300mL of methanol, followed by addition of p-toluenesulfonic acid (899mg, 10%) and reaction at room temperature for 12 hours, and TLC (EA/Hex. 1/9) indicated that the starting material was completely reacted. Adjusting pH of the reaction solution to 8 with saturated sodium bicarbonate solution, separating organic layer with separating funnel, removing solvent under reduced pressure, separating and purifying by column chromatography, and eluting with eluent at a ratio (EA/Hex: 1/12-CH)₃OH/DCM ═ 1/9), the organic phase was dried and evacuated to constant weight to give compound B-1 as a white solid (11.8g, 25.5mmol) in 51% yield (yield based on acetyl mannose). The nuclear magnetic data are shown below:

¹H NMR(600MHz,CDCl₃)δ7.37–7.21(m,12H),7.06(d,J＝7.9Hz, 2H),5.45(d,J＝1.3Hz,1H),4.60(d,J＝12.2Hz,1H),4.57–4.46(m,3H), 4.17–4.00(m,2H),3.94(dd,J＝3.0,1.5Hz,1H),3.85–3.73(m,2H),3.68 (dd,J＝9.1,3.1Hz,1H),3.35(s,1H),2.70(d,J＝3.7Hz,1H),2.28(s,3H).

the third step: compound B-1(6.3g, 13.6mmol) was weighed into a 250mL eggplant-shaped bottle, 87mL of a mixed solution of water and methylene chloride (v/v. 1/2) was added, colorless and transparent, TEMPO (424mg, 2.7mmol, 0.2eq) was added to turn the solution red brown, and PhI (OAc) was added₂(10.9g, 33.9mmol, 2.5eq) and stirred vigorously at room temperature for 3 hours, the solution turned brown-yellow and TLC (EA/Hex-1/1) indicated complete reaction of starting material. Adding saturated Na into the reaction solution₂S₂O₃Aqueous solution (10mL), pH 3 adjusted with dilute hydrochloric acid, stirred for 10 minutes, dichloromethane 100mL extracted and separated, the solvent was removed under reduced pressure, 180mL acetone was added to redissolve, BnBr (4.6g,27.2mmol,2eq), K were added₂CO₃(2.8g,20.4mmol,1.5eq) under nitrogen at room temperature for 3 hours with stirring, the solution is reddish brown, TLC (CH)₃OH/DCM ═ 1/9) indicated complete disappearance of starting material, the reaction was adjusted to neutral pH with dilute hydrochloric acid, the fractions were extracted with dichloromethane (100mL), the organic phase was concentrated under reduced pressure and purified by column chromatography (EA/Hex ═ 1/10-1/1) to give compound I-1(4.3g, 7.5mmol) as a reddish brown oil in 55% yield (based on compound B-1). The nmr data are as follows:

¹H NMR(600MHz,CDCl₃)δ7.70–7.28(m,17H),6.97(d,J＝7.9Hz, 2H),5.23(dd,J＝44.0,12.3Hz,2H),5.00(d,J＝11.4Hz,1H),4.86(d,J＝ 11.4Hz,1H),4.75(q,J＝12.0Hz,2H),4.68(d,J＝0.9Hz,1H),4.45(td,J ＝9.5,2.5Hz,1H),4.10(t,J＝6.4Hz,1H),3.76(d,J＝9.6Hz,1H),3.58– 3.41(m,1H),3.08(d,J＝2.5Hz,1H),2.30(s,3H).

example 2: synthesis of Compound II-1

The first step is as follows: compound I-1(15.0g,26.3mmol) was weighed into a 1000mL eggplant-shaped bottle, 400mL of anhydrous dichloromethane was added, the solution was reddish brown, LevOH (7.6g,65.8 mmol,2.5eq), DIC (8.3g,10.2mL,65.8mmol,2.5eq), and DMAP (8.1g, 65.8mmol,2.5eq) were added, and the solution was stirred at room temperature for 3 hours, becoming cloudy yellow. TLC (EA/Hex-1/2) indicated complete conversion of starting material. Saturated brine (500mL) was added, the organic phase was separated by extraction and concentrated under reduced pressure to give crude compound D-1, which was used directly in the next step.

The second step is that: the crude compound D-1 was dissolved in 320mL acetone/water (v/v ═ 15/1), NBS (16.8g,4eq) was added as a light yellow solution, which turned reddish brown after several minutes, and the solution was stirred at room temperature for 1 hour, becoming pale yellow. TLC (EA/Hex-1/2) indicated complete conversion of starting material. With saturated Na₂S₂O₃The reaction was quenched with a solution (10mL), extracted with additional dichloromethane (500mL), the organic layer was washed once with saturated brine, and the solvent was removed by concentration under reduced pressure to give compound E-1 as a yellow oil which was used directly in the next reaction.

The third step: dissolving the crude compound E-1 in 90mL of anhydrous dichloromethane, and adding CNCCl₃(6.3g,4.3mL,5eq) and DBU (663mg,0.7mL,4.4mmol,0.5eq), the solution was dark brown and was stirred for 3 hours under ice bath. TLC (EA/Hex-1/2) indicated complete conversion of starting material. The reaction mixture was concentrated under reduced pressure and purified by column chromatography to give an eluent (EA/Hex: 1/6-1/1) in 73% yield (based on compound I-1) of compound II-1 as a pale yellow oil (13.5g, 19.2 mmol). The nuclear magnetic data are shown below:

¹H NMR(400MHz,CDCl₃)δ7.43–7.16(m,15H),6.53(d,J＝3.3 Hz,1H),5.60(t,J＝7.5Hz,1H),5.12(t,J＝12.9Hz,1H),5.03(d,J＝12.2 Hz,1H),4.88(d,J＝46.7Hz,2H),4.65(ddd,J＝34.3,16.0,6.9Hz,1H), 4.51(d,J＝7.6Hz,1H),4.49–4.38(m,1H),3.89(dd,J＝7.8,2.9Hz,1H), 3.85–3.76(m,1H),2.72–2.44(m,3H),2.39(d,J＝7.2Hz,1H),2.15(d,J ＝11.7Hz,3H).

example 3: synthesis of Compound III-1

Compound D-1(300mg,0.45mmol) was weighed out and dissolved in 20mL of anhydrous DCM, TTBP (334.8mg,1.35mmol,3eq), diphenylsulfoxide (109mg,0.54mmol, 1.2eq), and dried 4A molecular sieve (500mg) were added under nitrogen protection, and the solution was stirred at-78 deg.C for 10 min. Adding Tf₂O (141mg,0.5mmol,1.1 eq). The solution was stirred for 10min, benzyl alcohol (54mg,0.5 mmol,1.1eq) was added and the reaction was carried out at-78 ℃ for 1 h, TLC (EA/Hex ═ 1/2) indicated complete conversion of the starting material. Saturated sodium bicarbonate was added to adjust pH to neutral, the molecular sieve was removed, the organic phase was concentrated under reduced pressure to give crude compound F-1, which was redissolved in 20mL of DCM/pyridine (v/v ═ 4/1), hydrazine acetate (180mg,1.95mmol,4.33eq) was added, and the mixture was stirred at room temperature for 2 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. The reaction mixture was quenched by addition of acetone, diluted with dichloromethane, and then diluted with dilute hydrochloric acid to neutral pH, washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the organic phase was concentrated and purified by column chromatography at an eluent ratio (EA/Hex ═ 1/4-1/1) to give compound III-1(224mg, 0.41mmol) as a colorless oil in 90% yield (based on compound D-1). The nuclear magnetic data are shown below:

¹H NMR(600MHz,CDCl₃)δ7.49–7.06(m,20H),5.24(q,J＝12.3 Hz,2H),5.03(d,J＝1.6Hz,1H),4.74(d,J＝11.9Hz,1H),4.70–4.54(m, 3H),4.50(d,J＝11.9Hz,1H),4.35(td,J＝9.2,2.4Hz,1H),4.20(d,J＝9.3 Hz,1H),3.80(dd,J＝9.2,3.0Hz,1H),3.78–3.71(m,1H),2.82(d,J＝2.4 Hz,1H).

example 4: synthesis of Compound IV-1

Compound II-1(2.0g,2.84mmol) and compound III-1(1.7g,3.12mmol, 1.1eq) were weighed out and dissolved in 80mL of anhydrous DCM to give a reddish brown solution, dried 4 Angstrom molecular sieve (1.8g) was added, stirring was carried out at-40 ℃ for 10min, and TMSOTf (126mg,0.57mmol, 0.2eq), N, was added₂The reaction was stirred for 1 hour under protection and TLC (EA/Hex ═ 1/2) indicated disappearance of compound II-1. Adding triethylamine to adjust pH to neutrality, removingThe organic phase was redissolved in 20mL of DCM/pyridine (v/v. 4/1) and hydrazine acetate (1130mg,12.3mmol,4.33 eq) was added and stirred at room temperature for 5 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. The reaction was quenched by addition of acetone, diluted with dichloromethane, then diluted with dilute hydrochloric acid to neutral pH, washed once with saturated brine, dried over anhydrous sodium sulfate for the organic phase, filtered, concentrated for purification by column chromatography, and the eluent ratio (EA/Hex ═ 1/8-1/1) gave compound IV-1 as a pale yellow oil (1813mg, 1.8mmol) in 64% yield. The nuclear magnetic data are shown below:

¹H NMR(600MHz,CDCl₃)δ7.35–6.92(m,35H),5.19(s,1H),5.08 (d,J＝12.2Hz,1H),5.00(d,J＝12.2Hz,1H),4.94(d,J＝12.2Hz,1H), 4.80(dd,J＝27.2,12.1Hz,2H),4.71(d,J＝12.2Hz,1H),4.57(dd,J＝12.1, 4.8Hz,2H),4.52–4.35(m,8H),4.35–4.28(m,1H),4.18(t,J＝9.5Hz, 1H),4.03–3.93(m,1H),3.72(t,J＝4.2Hz,1H),3.64–3.56(m,2H),3.15 (dd,J＝9.5,2.8Hz,1H),2.94(s,1H).

example 5: synthesis of beta-D- (1,4) -mannuronic acid disaccharide (Compound IX-1)

Compound IV-1(500mg, 0.5mmol) was dissolved in 22mL of THF/H₂To O/t-BuOH (v/v/v ═ 1/1/0.2), palladium on carbon (palladium content 10%) (50mg) was added, the reaction was stirred at 25 ℃ under hydrogen for 48 hours, TLC (EA/Hex ═ 1/2) indicated that the raw material disappeared, the palladium on carbon was filtered off, the palladium on carbon layer was washed three times with water (50mL × 3), the aqueous phases were combined, extracted once with EA (100mL), water was concentrated under reduced pressure to 10mL, and freeze-dried to give compound IX-1(160mg, 0.4mmol) as a white solid with a yield of 86%. The nuclear magnetic data are shown below:

¹H NMR(600MHz,D2O)δ5.23–5.12(m,1H),4.75(d,J＝9.4Hz, 1H),4.67–4.57(m,1H),4.40(t,J＝6.8Hz,1H),4.08(dt,J＝45.1,20.4Hz, 1H),4.02–3.85(m,2H),3.85–3.67(m,2H),3.67–3.48(m,1H).

¹³C NMR(151MHz,D₂O)δ172.85,172.73,101.35,101.06,99.82, 95.47,94.08,93.28,92.97,79.52,77.87,76.14,75.36,75.22,72.32,72.27, 71.82,71.16,70.11,69.96,69.84,69.80,69.75,69.64,69.59,69.53,69.43, 69.21,69.04,67.91,67.79,67.75,67.66.

example 6: synthesis of Compound VI-1

Weighing compound II-1(2.0g,2.84mmol) and compound I-1(1.78g,3.13mmol,1.1 eq) and dissolving in 80mL of anhydrous DCM to give a reddish brown color, adding dried 4 Angstrom molecular sieve (2.0g), stirring at-40 deg.C for 10min, adding TMSOTf (126mg,0.57mmol, 0.2eq), N₂Stirring was continued for 1 hour under protection and TLC (EA/Hex ═ 1/2) indicated complete reaction of compound II-1. Adding saturated sodium bicarbonate to adjust pH to neutral, filtering to remove molecular sieve, extracting, drying, concentrating, purifying by column chromatography to obtain the final product with eluent ratio (EA/Hex: 1/8-1/1) to obtain pale yellow solid compound VI-1(2.7 g, 2.42mmol), and yield is 86%. The nuclear magnetic data are shown below:

¹H NMR(600MHz,CDCl₃)δ7.39–7.15(m,34H),6.90(d,J＝8.0Hz, 2H),5.68(d,J＝6.8Hz,1H),5.56–5.46(m,1H),5.13–5.02(m,2H),4.92 (dd,J＝36.4,12.2Hz,2H),4.75–4.53(m,3H),4.53–4.38(m,7H),4.38– 4.26(m,1H),4.16(s,1H),3.83(dt,J＝9.6,3.2Hz,2H),3.73(dd,J＝8.1, 2.7Hz,1H),3.40(dd,J＝9.6,2.8Hz,1H),2.57(dt,J＝19.8,7.8Hz,1H), 2.52–2.37(m,2H),2.36–2.25(m,1H),2.22(s,3H),2.15–2.05(m,3H)

example 7: synthesis of beta-D- (1,4) -mannuronic acid tetrasaccharide (Compound IX-2)

The first step is as follows: VI-1(500mg,0.45mmol) was weighed out and dissolved in 20mL of anhydrous DCM, TTBP (334.8mg,1.35mmol,3eq), diphenyl sulfoxide (109mg,0.54mmol, 1.2eq), dried 4A molecular sieve 500mg were added under nitrogen protection, and the solution was stirred at-78 deg.C for 10 min. Adding Tf₂O (141mg,0.5mmol,1.1 eq). The solution was stirred for 10min, compound IV-1(500mg, 0.5mmol,1.1eq) was added and the reaction was allowed to react at-78 ℃ for 1 h, TLC (EA/Hex ═ 1/2) indicated complete conversion of the starting material. Saturated sodium bicarbonate was added to adjust pH to neutral, the molecular sieve was removed, the organic phase was concentrated under reduced pressure, redissolved in 20mL of DCM/pyridine (v/v-4/1), hydrazine acetate (180mg,1.95mmol,4.33eq) was added, and stirred at room temperature for 2 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. Adding acetone to quench reaction, diluting with dichloromethane, adding diluted hydrochloric acid to adjust pH to neutral, washing with saturated sodium chloride solution, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating the organic phaseThe phases were purified by column chromatography, and the ratio of the eluates (EA/Hex: 1/4-1/1) gave compound VIII-1(766mg, 0.405mmol) as a pale yellow solid in 90% yield (based on compound VI-1).

The second step is that: compound VIII-1(766mg, 0.405mmol) was dissolved in 22mL of THF/H₂To O/t-BuOH (v/v/v ═ 1/1/0.2), palladium on carbon (palladium content 10%) (300mg) was added, the reaction was stirred at 25 ℃ under hydrogen for 48 hours, TLC (EA/Hex ═ 1/2) indicated that the raw material disappeared, the palladium on carbon was filtered off, the palladium on carbon layer was washed three times with water (50mL × 3), the aqueous phases were combined, extracted once with EA (100mL), water was concentrated under reduced pressure to 10mL, and freeze-dried to give compound IX-2 (263mg, 0.365mmol) as a white solid with a yield of 90%.

¹H NMR(600MHz,D₂O)δ5.22(d,J＝4.4Hz,1H),4.98–4.80(m, 2H),4.82–4.76(m,2H),4.46(d,J＝6.6Hz,1H),4.17(t,J＝6.9Hz,1H), 4.13–3.87(m,8H),3.87–3.65(m,3H),3.65–3.48(m,2H).

¹³C NMR(150MHz,D₂O)δ172.68,172.11,171.66,100.40,100.37, 99.86,95.47,93.99,92.93,77.94,77.84,77.66,76.54,75.23,74.38,73.03, 72.93,72.31,71.81,71.16,70.97,69.97,69.60,69.53,69.46,69.19,69.04, 67.63.

Example 8: synthesis of beta-D- (1,4) -mannuronic acid trisaccharide (Compound IX-3)

The first step is as follows: weighing compound II-1(2.0g,2.84mmol) and compound IV-1(3.12g,3.13 mmol,1.1eq) and dissolving in 100mL of anhydrous DCM to give a reddish brown solution, adding dried 4 Angstrom molecular sieve (1.8g), stirring at-40 deg.C for 10min, adding TMSOTf (126mg,0.57mmol, 0.2eq), N₂The reaction was stirred for 1 hour with protection. TLC (EA/Hex ═ 1/2) indicated the disappearance of compound II-1. Triethylamine was added to adjust the pH to neutral, the molecular sieve was removed, the organic phase was concentrated under reduced pressure, redissolved in 50mL DCM/pyridine (v/v-4/1), hydrazine acetate (1130mg,12.3mmol,4.33 eq) was added and stirred at room temperature for 5 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. Adding acetone to quench reaction, diluting with dichloromethane, adding diluted hydrochloric acid to adjust pH to neutral, washing with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the organic phase, purifying by column chromatography, eluting with an eluent at a ratio (EA/Hex: 1/8-1/1),compound V-1 was obtained as a pale yellow oil (2547mg, 1.76mmol), 62% yield. The nuclear magnetic data are shown below:

¹H NMR(600MHz,CDCl3)δ7.26(ddd,J＝48.8,27.9,19.6Hz,50H), 5.25(s,1H),5.14–4.97(m,4H),4.88(dd,J＝27.9,12.0Hz,3H),4.73(t,J ＝11.9Hz,2H),4.62(ddd,J＝33.3,22.0,11.5Hz,6H),4.54–4.39(m,8H), 4.39–4.27(m,2H),4.17(t,J＝8.7Hz,1H),4.04(s,1H),3.79(d,J＝8.3Hz, 1H),3.70(d,J＝14.7Hz,3H),3.48(d,J＝9.3Hz,1H),3.45–3.34(m,1H), 3.11(d,J＝8.3Hz,1H),2.82(s,1H).

the second step is that: compound V-1(500mg, 0.34mmol) was dissolved in 20mL of THF/H₂To O/t-BuOH (v/v/v ═ 1/1/0.2), palladium on carbon (palladium content 10%) (50mg) was added, the reaction was stirred at 25 ℃ under hydrogen for 48 hours, TLC (EA/Hex ═ 1/2) indicated that the raw material disappeared, the palladium on carbon was filtered off, the palladium on carbon layer was washed three times with water (50mL × 3), the aqueous phases were combined, extracted once with EA (100mL), water was concentrated under reduced pressure to 10mL, and freeze-dried to give compound IX-3 (167mg, 0.31mmol) as a white solid with a yield of 90%. The nuclear magnetic data are shown below:

¹H NMR(600MHz,D₂O)δ5.17(d,J＝4.3Hz,1H),4.77(s,1H),4.41 (d,J＝6.6Hz,1H),4.11(d,J＝6.7Hz,1H),4.05–3.94(m,3H),3.94–3.89(m,2H),3.88(d,J＝9.9Hz,1H),3.82–3.77(m,1H),3.77–3.65(m, 3H),3.58(dd,J＝9.5,3.0Hz,1H).

¹³C NMR(151MHz,D2O)δ185.70,172.66,172.09,171.67,100.34, 99.85,93.97,92.93,77.93,77.84,77.73,77.64,74.36,73.03,72.30,71.79, 71.12,70.95,70.56,70.08,69.98,69.55,69.48,69.18,69.02,67.72,67.61.

example 9: synthesis of beta-D- (1,4) -mannuronic acid pentasaccharide (Compound IX-4)

The first step is as follows: VI-1(500mg,0.45mmol) was weighed out and dissolved in 20mL of anhydrous DCM, TTBP (334.8mg,1.35mmol,3eq), diphenyl sulfoxide (109mg,0.54mmol, 1.2eq), dried 4 Angstrom molecular sieves 500mg were added under nitrogen protection, the solution was stirred at-78 deg.C for 10min, Tf was added₂O (141mg,0.5mmol,1.1eq), the solution was stirred for 10min, compound V-1 (723mg,0.5mmol,1.1eq) was added and the reaction was allowed to react at-78 ℃ for 1 h, TLC (EA/Hex ═ 1/2) indicated complete conversion of the starting material. Saturated sodium bicarbonate was added to adjust pH to neutral, the molecular sieve was removed, the organic phase was concentrated under reduced pressure, redissolved in 20mL of DCM/pyridine (v/v-4/1), hydrazine acetate (180mg,1.95mmol,4.33eq) was added, and stirred at room temperature for 2 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. Adding acetone to quench the reaction, diluting with dichloromethane, adding diluted hydrochloric acid to adjust the pH value to be neutral, washing with saturated saline solution once, drying an organic phase with anhydrous sodium sulfate, filtering, concentrating the organic phase, and purifying by column chromatography, wherein the ratio of an eluent (EA/Hex is 1/4-1/1) to obtain a pale yellow solid compound VIII-2(935mg, 0.40mmol) and the yield is 88% (calculated on the compound VI-1).

The second step is that: compound VIII-2(935mg, 0.40mmol) was dissolved in 22mL of THF/H₂To O/t-BuOH (v/v/v ═ 1/1/0.2), palladium on carbon (palladium content 10%) (300mg) was added, the reaction was stirred at 25 ℃ under hydrogen for 48 hours, TLC (EA/Hex ═ 1/2) indicated that the raw material disappeared, the palladium on carbon was filtered off, the palladium on carbon layer was washed three times with water (50mL × 3), the aqueous phases were combined, extracted once with EA (100mL), water was concentrated under reduced pressure to 10mL, and freeze-dried to give compound IX-4 (323mg, 0.36mmol) as a white solid with a yield of 90%. The nuclear magnetic data are shown below:

¹H NMR(600MHz,D₂O)δ5.17(s,1H),4.86(s,1H),4.63–4.51(m, 3H),4.29(d,J＝5.8Hz,1H),4.05(s,1H),3.97(s,3H),3.91(t,J＝10.4Hz, 2H),3.84(d,J＝9.3Hz,6H),3.76(dd,J＝14.9,7.5Hz,2H),3.72(d,J＝18.2Hz,3H),3.58(d,J＝7.0Hz,1H),3.53(s,1H).

13C NMR(151MHz,D₂O)δ174.35,174.12,173.65,173.37,172.69, 100.23,100.15,99.78,97.86,93.81,93.18,78.07,78.00,77.86,77.79,74.50, 72.39,71.24,70.38,70.15,69.80,69.71,69.51,69.01,67.99,67.45,61.48.

example 10: synthesis of beta-D- (1,4) -mannuronic acid heptasaccharide (Compound IX-5)

The first step is as follows: compound VI-1(500mg,0.45mmol) was weighed out and dissolved in 20mL of DCM/pyridine (v/v ═ 4/1), hydrazine acetate (180mg,1.95mmol,4.33eq) was added, and stirred at room temperature for 2 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. Adding acetone to quench reaction, diluting with dichloromethane, adding diluted hydrochloric acid to adjust pH to neutral, washing with saturated saline solution, and collecting organic solutionThe phases were dried over anhydrous sodium sulfate and filtered, the organic phase was concentrated, dried 4 angstrom molecular sieve (2.0g) was added, redissolved in 20mL of anhydrous DCM, and compound II-1(317m g,0.45mmol) was added and the solution was reddish brown, N₂Under protection, the temperature was controlled below-40 ℃ and TMSOTf (20mg,0.09mmol, 0.2eq) was added after stirring for 10min and stirred for 1 h. TLC (EA/Hex ═ 1/2) indicated complete reaction of compound II-1. Adding saturated sodium bicarbonate to adjust the pH value to be neutral, filtering to remove a molecular sieve, extracting, drying, concentrating, carrying out silica gel column chromatography separation, and obtaining the compound VII-1(560m g, 0.36mmol) as a light yellow solid with the eluent ratio (EA/Hex ═ 1/8-1/1) and the yield of 80% (calculated by the compound VI-1).

The second step is that: VII-1(500mg,0.32mmol) was weighed and dissolved in 20mL of anhydrous DCM, TTBP (238mg,0.96mmol,3eq), diphenyl sulfoxide (78mg,0.38mmol, 1.2eq), dried 4A molecular sieve (500mg) were added under nitrogen protection, the solution was stirred at-78 deg.C for 10min, Tf was added₂O (100mg,0.35mmol,1.1eq), the solution was stirred for 10min, compound VIII-1 (663mg,0.35mmol,1.1eq) was added and the reaction was reacted at-78 ℃ for 1 h, TLC (EA/Hex ═ 1/2) indicated complete conversion of the starting material. Saturated sodium bicarbonate was added to adjust pH to neutral, the molecular sieve was removed, the organic phase was concentrated under reduced pressure, redissolved in 20mL of DCM/pyridine (v/v-4/1), hydrazine acetate (128mg,1.39mmol,4.33eq) was added, and stirred at room temperature for 2 hours. TLC (EA/Hex 1/2) indicated disappearance of starting material. The reaction mixture was quenched with acetone, diluted with dichloromethane, diluted with dilute hydrochloric acid to neutral pH, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography at an eluent ratio (EA/Hex ═ 1/4-1/1) to give compound VIII-3(880mg, 0.27mmol) as a pale yellow solid in 85% yield (based on compound VII-1).

The third step: compound VIII-3(880mg, 0.27mmol) was dissolved in 22mL of THF/H₂O/t-BuOH (v/v/v ═ 1/1/0.2), palladium on carbon (palladium content 10%) (300mg) was added, the reaction was stirred at 25 ℃ under hydrogen for 48 hours, TLC (EA/Hex ═ 1/2) indicated that the raw material disappeared, the palladium on carbon was filtered off, the palladium on carbon layer was washed three times with water (50mL × 3), the aqueous phases were combined, extracted once with EA (100mL), water was concentrated under reduced pressure to 10mL, and freeze-dried to give compound IX-5 (300mg, 0.24mmol) as a white solid with a yield of 8, and yield of 89 percent. The nuclear magnetic data are shown below:

¹H NMR(600MHz,D₂O)δ5.96(d,J＝3.9Hz,1H),5.15(s,1H),4.84 (s,2H),4.65(d,J＝3.9Hz,3H),4.60(s,3H),4.16(s,1H),4.10–4.01(m, 1H),3.96(s,3H),3.91(s,2H),3.84(d,J＝16.4Hz,5H),3.70(s,6H),3.66 (d,J＝6.7Hz,4H),3.56(dd,J＝12.2,6.7Hz,3H).

¹³C NMR(151MHz,D₂O)δ174.37,174.05,174.02,174.01,173.94, 173.84,173.60,100.23,100.15,99.78,97.86,93.81,93.19,78.08,78.00, 77.87,77.79,74.64,72.39,71.25,70.38,70.16,69.80,69.71,69.51,69.01, 68.00,67.45,61.49.