Preparation method of outer core octasaccharide of Helicobacter pylori lipopolysaccharide

Patent metadata

Field	Value
Publication number	US-10669353-B2
Application number	US-201816215414-A
Country	US
Kind code	B2
Filing date	Dec 10, 2018
Priority date	Dec 11, 2017
Publication date	Jun 2, 2020
Grant date	Jun 2, 2020

Abstract

The disclosure discloses a preparation method of outer core octasaccharide of Helicobacter pylori lipopolysaccharide, and belongs to the field of carbohydrate chemistry. The structure of the outer core octasaccharide of Helicobacter pylori is (α-D-Glc-(1-3)-α-D-Glc-(1-4)-β-D-Gal-(1-7)-D-α-D-Hep[α-D-Glc-(1-6)-α-D-Glc-(1-6)-α-D-Glc-(1-2)-D-α-D-Hep]-Linker. The structure consists of three monosaccharides: glucose, galactose and heptose. It contains five α-glycosidic bonds of glucose, one β-glycosidic bond of galactose and two α-glycosidic bonds of heptose. The disclosure prepares the octasaccharide by the synergistic action of remote neighboring group participation effect, solvent effect, temperature effect, additives and leaving groups. The reducing end of the octasaccharide may also be linked with a linker for future attachment to the protein to form a glycoconjugate for immunological studies.

First claim

What is claimed is:

1. An outer core octasaccharide of Helicobacter pylori or a derivative thereof, as shown in a Formula I:

wherein R is —(CH 2 ) n —N—Y 1 Y 2 or —(CH 2 ) n —N—Y 1 Y 2 (linker), n=1-10, N is nitrogen, Y 1 is H or benzyl (Bn), Y 2 is H or carbobenzoxy (Cbz); wherein R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 10 , R 12 , R 14 , R 15 , R 16 , R 18 , R 20 , R 21 , R 23 , R 24 , and R 26 are H or benzyl (Bn); wherein R 5 , R 11 , R 17 , R 19 , R 22 , and R 25 is H or 2-naphthylmethyl (Nap); and wherein R 4 , R 9 , R 13 , and R 27 are ester groups.

2. A method of synthesizing the outer core octasaccharide of Helicobacter pylori according to claim 1 , comprising the following steps:
step 1, synthesizing required monosaccharide building blocks A, B, C, and D:

wherein R is —(CH 2 ) n —N—Y 1 Y 2 or —(CH 2 ) n —N—Y 1 Y 2 (linker), n=1-10, N is nitrogen, Y 1 is benzyl (Bn), Y 2 is carbobenzoxy (Cbz); wherein R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 10 , R 12 , R 14 , R 15 , R 16 , R 18 , R 20 , R 21 , R 23 , R 24 , and R 26 are benzyl (Bn); wherein R 5 , R 11 , R 17 , R 19 , R 22 , and R 25 is 2-naphthylmethyl (Nap); and
wherein R 4 , R 9 , R 13 , and R 27 are ester groups;
step 2, assembling protected octasaccharide by a glycosylation reaction; and
step 3, deprotecting the octasaccharide.

3. The method according to claim 2 , wherein in step 1, when the monosaccharide building blocks A, B, C, and D are synthesized, the solvent used is one or more of dry dichloromethane, ethyl acetate, methanol, N,N-dimethylformamide, tetrahydrofuran, pyridine, chloroform, and 80% acetic acid aqueous solution; wherein a substrate concentration is 0.02-0.5 M; wherein an acid as a reagent or catalyst is hydrochloric acid, acetic acid or p-toluenesulfonic acid; wherein a reaction temperature is −78° C. to reflux temperature of the solvent; and wherein a reaction time is 1-48 h.

4. The method according to claim 2 , wherein step 2 comprises (1) assembly of a trisaccharide donor, (2) assembly of a pentasaccharide acceptor, and (3) assembly of a fully protected octasaccharide;
wherein,
the assembly conditions of the trisaccharide donor are that a substrate is sugar building blocks A and B, and a reaction solvent is Et 2 O/DCM; adding thiophenein an amount of 10 equivalents of donor, and using a desiccant; wherein a reaction temperature is 0° C. to room temperature, and reaction process is under inert gas protection; wherein after reaction materials disappear, quenching reaction with pyridine or triethylamine, filtering, washing, extracting and drying are carried out, and purifying a product by a silica gel column;
wherein the assembly conditions of the pentasaccharide acceptor are that the substrate is sugar building blocks A, C, and D, and the reaction solvent is Et 2 O/DCM or DCM; adding thiophene in an amount of 10 equivalents of donor, and using a desiccant; wherein a reaction temperature is 0° C. to room temperature, and a reaction process is under inert gas protection;
wherein after reaction materials disappear, quenching reaction with pyridine or triethylamine, filtering, washing, extracting and drying are carried out, and purifying a product by a silica gel column; and
wherein the assembly conditions of the octasaccharide is that the substrate is the above trisaccharide donor and the pentasaccharide acceptor, and organic solvent for glycosidation is dry dichloromethane or dry diethyl either or both; wherein glycosylation reaction is carried out under the action of Lewis acid to obtain the protected octasaccharide; wherein after reaction materials disappear, quenching with pyridine or triethylamine, filtering, washing, extracting and drying are carried out, and purifying product by a silica gel column.

5. The method according to claim 4 , wherein,
the desiccant is one or more of 3 Å molecular sieve, 4 Å molecular sieve, 5 Å molecular sieve, anhydrous sodium sulfate, anhydrous magnesium sulfate, and anhydrous calcium sulfate;
wherein mass ratio of the desiccant to a reactant is 1.0 to 4.0;
wherein the inert gas is nitrogen or argon;
wherein the Lewis acid is TMSOTf, TfOH, or AgOTf, and for the reaction of thioglycoside, NIS is additionally added as a promoter together with Lewis acid.

6. The method according to claim 4 , wherein substrate concentration during the glycosylation reaction is 0.01 M to 0.1 M, the reaction temperature is −50° C. to 0° C. to room temperature; wherein the room temperature is 20-30° C.; and reaction time is 1 h to 7 h.

7. The method according to claim 5 , wherein substrate concentration during the glycosylation reaction is 0.01 M to 0.1 M, the reaction temperature is −50° C. to 0° C. to room temperature; wherein the room temperature is 20-30° C.; and reaction time is 1 h to 7 h.

8. The method according to claim 2 , wherein in step 3, the acyl of the protected octasaccharide is removed under alkaline conditions, and solvent used is one or more of methanol, tetrahydrofuran, and dichloromethane; and wherein after completion of reaction, the reaction is neutralized with H + resin, reaction temperature is room temperature, and the reaction time is 2 h to 12 h to obtain a semi-deprotected octasaccharide.

9. The method according to claim 8 , wherein in step 3, the semi-deprotected octasaccharide is completely deprotected by using palladium carbon and hydrogen, and solvent used is one or more of tetrahydrofuran, dichloromethane, ethyl acetate, tert-butanol, water, methanol, and acetic acid; wherein the palladium carbon is 10% palladium carbon; wherein mass ratio of palladium carbon to the reactant is 0.1:1-0.5:1; wherein hydrogen pressure for debenzylation and 2-naphthylmethyl removal is 1-100 atm; and wherein the reaction temperature is normal temperature, and reaction time is 1-48 h.

10. A Helicobacter Pylori vaccine, comprising the outer core octasaccharide of Helicobacter Pylori according to claim 1 , which services as the epitope.

11. A method for producing a α-glycosidic bond of glucose, comprising co-distilling glycosyl donor and acceptor for 2 to 3 times in toluene, adding dry DCM or DCM/Et 2 O in a volume ratio of 1:2; wherein substrate concentration is 0.01-0.1 M, and the activated 3 Å or 4 Å molecular sieve is used as a desiccant; adding thiophene in an amount of 10 equivalents of glycosyl donor; stirring reaction at room temperature for 20-30 min, then cooling to −50-0° C.; adding TMSOTf as a promotor, and carrying reaction time for 1-7 h; and terming reaction with pyridine.

Assignees

• Univ Jiangnan
• Max Planck Inst Of Colloids And Interfaces

Inventors

• Yin Jian
• Hu Jing
• Seeberger Peter
• Zou Xiaopeng

Classifications

• C08B37/006 Primary

  Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof · CPC title

• A61K39/00

  Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) · CPC title