WO2020125307A1 - 一种绿脓假单胞菌o11血清型o抗原寡糖的化学合成方法 - Google Patents

一种绿脓假单胞菌o11血清型o抗原寡糖的化学合成方法 Download PDF

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WO2020125307A1
WO2020125307A1 PCT/CN2019/119584 CN2019119584W WO2020125307A1 WO 2020125307 A1 WO2020125307 A1 WO 2020125307A1 CN 2019119584 W CN2019119584 W CN 2019119584W WO 2020125307 A1 WO2020125307 A1 WO 2020125307A1
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compound
fucosamine
group
pseudomonas aeruginosa
glucose
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French (fr)
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尹健
胡静
刘中华
秦春君
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江南大学
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Priority to US17/017,896 priority patent/US20200405840A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to a chemical synthesis method of Pseudomonas aeruginosa O11 serotype O antigen oligosaccharide, which belongs to the field of chemistry.
  • Pseudomonas aeruginosa is a type of aerobic, rod-shaped Gram-negative bacteria that generally inhabits soil and water environments.
  • Pseudomonas aeruginosa is also a type of opportunistic pathogens widely present in the hospital environment, causing bacteremia, urinary tract infections and pneumonia and other diseases.
  • Susceptible populations of Pseudomonas aeruginosa include burn patients, patients in the ICU, patients after surgery, and patients with immune system defects such as cancer, diabetes, and congenital vesicular fibrosis.
  • VAP ventilator-associated pneumonia
  • HAP hospital-acquired pneumonia
  • aeruginosa isolated from VAP against gentamicin 51.1%, 95% CI 37.7–64.4%
  • amikacin 22.5%, 95% CI 14.3–33.6 %
  • Pseudomonas aeruginosa isolated from HAP is resistant to amikacin (22.2% (95% CI 13.8-33.6%)) and cefoperazone (50.0% (95 %CI 30.2–69.8%)) has high drug resistance.
  • P. aeruginosa has been clinically found to be resistant to antibiotics including polymyxin, fluoroquinolones, and carbapenems.
  • the World Health Organization listed Pseudomonas aeruginosa as one of the most urgently needed pathogens for research and development of new antibiotics or vaccines.
  • Vaccine research on Pseudomonas aeruginosa began in the 1980s, and the research field focused on the lipopolysaccharide, sugar-protein conjugate, flagella, inactivated or attenuated whole bacteria of the pathogenic bacteria and the DNA sequence of the bacteria aspect.
  • the lipopolysaccharide of Pseudomonas aeruginosa is composed of some simple sugars and glycosidic linkages that are not common in human body, and is an important virulence factor.
  • serotype antigens There are 20 different serotype antigens based on the types of monosaccharides and glycosidic linkages.
  • the specific structure is (-3)- ⁇ -LN-acetylfucosamine-(1-3)- ⁇ -DN-acetylfucosamine-(1-2)- ⁇ -D-glucose-(1- ] n 's O11 serotype antigen, which is a long-chain antigen formed by trisaccharide repeats, and the specific number of repeat units n is regulated by the gene of the pathogenic bacteria.
  • the compounds for this structure are all extracted from the wild disease-controlling bacteria containing the lipopolysaccharide structure or genetically engineered bacteria containing the structure, including the construction and cultivation of LPS high-expression strains, the degradation of proteinase k, and the thermal extraction of carbonic acid. Tricine-SDS-polyacrylamide gel electrophoresis (PAGE) analysis and Western immunoblot detection and other steps.
  • PAGE Tricine-SDS-polyacrylamide gel electrophoresis
  • the lipopolysaccharide obtained by the biological extraction method also has the problem of uneven chemical structure, and also has the problem of safety, because the lipopolysaccharide directly extracted from the bacterial body is likely to be doped with other bacterial virulence Factors not only do not provide immune protection to the human body, but also cause the human body to be directly infected by bacterial virulence factors and cause disease, which will cause serious side effects.
  • the synthesized lipopolysaccharide has a clear chemical structure And can ensure that only this kind of capsular polysaccharides participate in the composition of the vaccine, will not be doped with other substances.
  • the present invention synthesizes O11 serotype O antigen trisaccharide fragments of Pseudomonas aeruginosa through three sugar building block chemical methods, mainly including glucose building blocks, L-fucosamine building blocks, and D-rock Fucosamine building blocks and 1,2-cis-glycosidic bonds and 1,2- ⁇ -trans-glycosidic bonds.
  • the synthesis of D-fucosamine and the construction of 1,2- ⁇ -cis-glycosidic bond are the key steps of the target trisaccharide.
  • the present invention has developed a method for synthesizing D-fucosamine from D-glucose, and through the synergy of the solvent effect, the temperature effect and the additive effect, it has successfully solved the stereoscopic choice of constructing the 1,2- ⁇ -cis-glycosidic bond Sexual issues.
  • the three target sugars were synthesized using the three sugar-based building blocks, and the target three sugars obtained by removing the protection were as shown in Formula 1.
  • the reducing end of the trisaccharide is assembled with a connecting arm with an amino group to prepare for the next conjugated protein.
  • the first object of the present invention is to provide a method for synthesizing O11 serotype O antigen trisaccharide of Pseudomonas aeruginosa, the method comprising: using D-glucose blocks, L-fucosamine blocks, D-fucosamine building block to construct O antigen trisaccharide; wherein, D-glucose building block or L-fucosamine building block and D-fucose building block pass 1,2- ⁇ -cis-glycoside Bond connection, the D-glucose block and the L-fucosamine block are connected by a 1,2- ⁇ -trans glycosidic bond, and the construction of the 1,2- ⁇ -cis-glycoside bond is in a mixed solvent Carried out; the mixed solvent includes two or more of dichloromethane, ether, and thiophene.
  • the chemical structure of the D-glucose block is shown in Formula II, and the chemical structure of the L-fucosamine block is shown in Formula III.
  • the chemical structure of the block is shown in Formula IV,
  • Y 1 is hydrogen (H) or benzyl (Bn);
  • Y 2 is hydrogen (H) or benzylmethoxycarbonyl (Cbz);
  • R 1 includes but is not limited to hydrogen (H), ester, acetyl (Ac) , Benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), levulinyl (Lev), allylcarbonyl (Alloc);
  • R 2 , R 3 , R 4 is hydrogen (H) Or ester group, ether group including but not limited to acetyl (Ac), benzoyl (Bz), pivaloyl (Piv), chloroacetyl (C
  • the method includes synthesizing disaccharide fragments using D-glucose blocks and L-fucosamine blocks, the chemical structure of which is shown in Formula V.
  • the 1,2- ⁇ -trans glycosidic linkage between the D-glucose block and the L-fucosamine block is promoted by TMSOTf with dichloromethane as the Carried out in a solvent.
  • the method includes synthesizing trisaccharide fragments using disaccharide fragments and D-fucosamine building blocks, the chemical structural formula of which is shown in Formula I,
  • the method includes pre-synthesizing the O antigen trisaccharide precursor and reducing the remembering O antigen trisaccharide.
  • the chemical structural formula of the O antigen trisaccharide precursor includes the following general formula:
  • the terminal position of the D-glucose building block (Formula II) is trichloroacetimide ester, which is a leaving group, and the remaining substituent R n is shown in Formula II.
  • the method for synthesizing D-glucose blocks includes: starting with 1,2,3,4,6-penta-O-acetylglucose starting materials, using dry N, N- Dimethylformamide and hydrazine acetate remove the acetyl group at position 1 and leak -OH, and the bare -OH reacts with trichloroacetonitrile (Cl 3 CN) to obtain compound D-glucose block.
  • the terminal position of the L-fucose building block (Formula III) is protected by an allyl group (OAll), and the substituent R n at the remaining positions is shown in Formula III.
  • OAll allyl group
  • the L-fucose building block uses allyl 2-deoxy-2azido-L-fucose as the starting material, and dibutyltin oxide ( Bu 2 SnO) and 2-bromomethylnaphthalene selectively protect the hydroxyl group at position 4 to obtain 4-Nap fucose; then protect the remaining hydroxyl group at position 3 with benzyl group, and then dichlorodicyanobenzene Under the action of quinone, the Nap group at position 4 is removed to obtain the final sugar block B.
  • dibutyltin oxide Bu 2 SnO
  • 2-bromomethylnaphthalene selectively protect the hydroxyl group at position 4 to obtain 4-Nap fucose; then protect the remaining hydroxyl group at position 3 with benzyl group, and then dichlorodicyanobenzene Under the action of quinone, the Nap group at position 4 is removed to obtain the final sugar block B.
  • the terminal position of the D-fucosamine building block is a linker, as shown in Formula IV.
  • the method for synthesizing the D-fucosamine building block uses glucose to obtain D-fucosamine, including: 3,4,6-O-triacetyl mannose Enene is used as a raw material, and 1-selenbenzene-2 azide glucose is obtained through an azide and selenium reagent, acetyl group is removed, then 6-C is methylated, and finally D-fucosamine compounds are obtained.
  • the method specifically includes: taking 3,4,6-O-triacetylmannose as a raw material, under the action of iodobenzene diacetate (PhI(OAc) 2 ), and Azido trimethylsilane (TMS-N 3 ) and diphenyl diselenide (Ph 2 Se 2 ) generate 1-selenbenzene-2 azido-glucose, then remove the acetyl group under alkaline conditions, add 4-toluene Sulfonyl chloride achieves 6-C methylation to obtain D-fucosamine compounds.
  • iodobenzene diacetate PhI(OAc) 2
  • TMS-N 3 Azido trimethylsilane
  • Ph 2 Se 2 diphenyl diselenide
  • the method further includes protecting 3 and 4-C of the D-fucosamine compound, and then removing the 4-C Nap group and protecting it with an acetyl group.
  • N-butyl ammonium bromide (NBS) hydrolyzes the 1-position selenium phenyl of D-fucose, and fucosylose is made into glycosyl trichloroacetimide ester with Cl 3 CN and DBU, and then promoted by Lewis acid Assemble the amino linking arm, and finally remove the acetyl group at position 3 of D-fucose to obtain the D-fucose building block.
  • the 3-C protective reagent includes methoxybenzyl chloride (PMBCl) and dibutyltin oxide (Bu2SnO).
  • the 4-C protecting agent includes benzyl.
  • the removal of the 4-C Nap group is achieved using DDQ.
  • the Lewis acid includes trimethylsilyl triflate.
  • the second object of the present invention is to provide an O11 serotype O antigen trisaccharide of Pseudomonas aeruginosa assembled with an amino linking arm, which is prepared by the above method.
  • the third object of the present invention is to provide a method for preparing a sugar-protein conjugate using the aforementioned O11 serotype O antigen trisaccharide of Pseudomonas aeruginosa assembled with an amino linking arm.
  • the fourth object of the present invention is to provide a method for preparing a sugar-protein conjugate, the method comprising O11 serotype O antigen trisaccharide of Pseudomonas aeruginosa, the Pseudomonas aeruginosa
  • the preparation method of O11 serotype O antigen trisaccharide is the above method.
  • the fifth object of the present invention is to apply the above-mentioned O11 serotype O antigen trisaccharide of Pseudomonas aeruginosa assembled with an amino linking arm in the development or preparation of a Pseudomonas aeruginosa vaccine or Pseudomonas aeruginosa Application of medicine for diseases caused by bacterial infection.
  • the present invention synthesizes O-antigen trisaccharide fragments of Pseudomonas aeruginosa O11 serotype by chemical synthesis.
  • a method for converting D-mannose into D-fucose is developed. The method is simple and efficient, except that the 6-C methylation is about 50, and the rest can be more than 80%;
  • the block is used to synthesize O-antigen trisaccharide fragments of P. aeruginosa O11 serotype.
  • the commonly used glycosidic bond is constructed by using the neighboring group assisted by the acyl group at the C-2 position of the sugar ring to efficiently construct the 1,2-trans glycosidic bond, and the 2-position acetyl group is the easiest in the sugar chemical protection strategy.
  • the most effective protective agent is the construction of trans-glycosidic bonds in the present invention.
  • each reaction condition has a great influence on the reaction. Among them, when the sugar block is designed, it is selectively protected with a large steric hindrance in C-6.
  • the method of the present invention uses a suitable mixed solvent to achieve the uniform construction of the cis-glycosidic bond.
  • the stereo selectivity can reach 100%.
  • the present invention successfully completes the preparation of O11 trisaccharide repeat units through the selection and optimization of protecting groups and the strategy of glycosylation reaction.
  • the Pseudomonas aeruginosa O11 serotype O-antigen trisaccharide fragment synthesized by the method of the present invention is assembled with an amino linking arm and can be connected to an antigen protein to form a glycoconjugate, which is useful for the development of treatment and prevention of Pseudomonas aeruginosa vaccine Has an important role.
  • FIG. 1 Synthetic route diagram of monosaccharide building block 3 (D-glucose building block);
  • FIG. 1 Synthetic route diagram of monosaccharide building block 7 (L-fucose building block);
  • FIG. 1 Synthesis route of monosaccharide building block 17 (D-fucosamine building block);
  • Figure 4 Synthetic roadmap of construction method 1 of monosaccharide block 17;
  • Figure 5 Synthetic roadmap of construction method 2 of monosaccharide block 17;
  • Figure 8 Design diagram of the synthetic route of trisaccharide S23.
  • the method for calculating the yield of the present invention is "product (mol)/reaction substrate (mol)*100%".
  • the methods for identifying the structure of compounds in the present invention are the determination of nuclear magnetic resonance spectrum (400M), the determination of high-resolution mass spectrometry, and the determination of the optical rotation value. The results are listed in the specific synthesis of each compound.
  • sugar block 7 L-fucose block
  • the acetyl group at positions 3 and 4 is first removed in an alkaline environment containing sodium methoxide
  • the resulting dihydroxy compound is selectively protected with a Nap group at the 4 position under the action of dibutyltin oxide and bromomethylnaphthalene to obtain compound 5, and the hydroxyl group of 3-Nap compound 5 is protected with a benzyl group to produce compound 6 .
  • the Nap group of 6 was removed by DDQ to obtain compound 3-OH compound 7.
  • the synthetic route of the sugar block 17 is shown in FIG. 3.
  • the Ts group of 6-Ts glucose 10 was iodinated with sodium iodide, followed by sodium cyanoborohydride (NaCNBH 3 ) to reduce the 6th position to a methyl group to obtain D-fucose compound 11.
  • Selective protection of position 3 of the D-fucose compound with p-methoxybenzyl chloride (PMBCl) and dibutyltin oxide (Bu 2 SnO) yields compound 12, followed by protection of the hydroxyl group at position 4 with benzyl to yield compound 13.13
  • the Nap at position 4 was removed with DDQ and then protected with an acetyl group to give compound 14.
  • Tetrabutylammonium bromide was used to hydrolyze the 1-position selenophenyl of compound 14, followed by Cl 3 CN and DBU to make 1-OH fucose into glycosyl trichloroacetimide ester donor 15. Subsequently, under the promotion of trimethylsilyl triflate, compound 15 reacts with a three-carbon connecting arm to obtain a fucose compound 16 assembled with the connecting arm. Finally, the acetyl group of compound 16 is removed in an alkaline environment of methanol and sodium methoxide to obtain sugar block 17.
  • reaction solution was washed with saturated NaHCO 3 (20 ml) three times, the organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried and purified through a silica gel column (petroleum ether/ethyl acetate, 6:1) to obtain compound 9 ( 2.4g, 5.15mmol, 70%).
  • Compound 10 Compound 9 (0.5 g, 1.1 mmol) was dissolved in methanol (2.2 ml), and sodium methoxide (29 mg, 0.5 mmol) was added. The reaction solution was stirred at room temperature for 2h, and TLC detection showed that the raw material reaction was complete. Neutralize the reaction solution to ⁇ 7 with hydrogen type Amberlite IR120 resin. Filter the resin and spin the solvent.
  • Compound 12 Compound 11 (3.0 g, 9.1 mmol) was dissolved in anhydrous toluene (54 ml), and Bu 2 SnO (3.5 g, 13.7 mmol) was added under the protection of argon. The reaction solution was heated to 118°C and refluxed for 1.5h with stirring. Subsequently, the reaction liquid was cooled to 60°C, PMBCl (1.9 ml, 13.7 mmol) and TBAI (5.1 g, 13.7 mmol) were added, and stirring was continued for 2 h.
  • Compound 14 Compound 13 (900 mg, 1.67 mmol) was dissolved in DCM (84 ml), and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (564 mg, 2.5 mmol) and water (4.2 ml). The reaction was stirred at room temperature for 7h, and the reaction was detected to be complete by TLC. DCM (50 ml) was added for dilution, and the reaction solution was washed twice with 10% sodium thiosulfate (100 ml).
  • Compound 15 Compound 14 (580 mg/1.3 mmol) was dissolved in a mixed solution of tetrahydrofuran (6 ml) and water (6 ml), and cooled to 0°C. Tetrabutylammonium bromide (538mmol, 3mmol) was added, and the reaction solution was returned to room temperature and stirred for 5h. TLC detection showed that the reaction of the raw materials was complete.
  • the new compound (417 mmol, 1.3 mmol) was dissolved in anhydrous dichloromethane (14 ml) and cooled to 0°C. Trichloroacetonitrile (0.4 ml, 3.78 mmol) and DBU (23 ⁇ L, 0.13 mmol) were added in sequence, and the reaction solution was continuously stirred for 5 h. TLC detected that the raw material was completely reacted. The reaction solution was directly concentrated at room temperature, and purified through a silica gel column (petroleum ether/ethyl acetate, 10:1) to obtain compound 15 (518 mg, 1.1 mmol, 88%).
  • the method 1 for synthesizing D-fucose blocks is summarized in the synthesis methods of similar compounds reported in the existing literature, as shown in FIG. 4.
  • galactenose S1 as the starting material, under the combined action of diphenyl diselenide, trimethylsilyl azide and diacetyl iodobenzene, selenium phenyl and The azido group obtains the important intermediate S2.
  • the acetyl group at the 3rd, 4th and 6th positions is removed to obtain the compound S3, and then the benzylidene group is introduced at the 4th and 6th positions to obtain the compound S4.
  • the inventors tried different conditions, including: acetonitrile and TBAI were reacted under the conditions of 100°C and 80°C and methyl ethyl ketone and sodium iodide at 80°C, but they failed to introduce iodine at the sixth position The product S8, and then unable to continue to synthesize the target synthetic D-fucose blocks along the route.
  • disaccharide 18 relies on the glucose compound 3 previously synthesized from the route of FIG. 1 and the L-fucose compound 7 synthesized from the route of FIG. 2 under the catalysis of Lewis acid TMSOTf. Subsequently, the allyl group at the reducing end of the disaccharide 18 was dissolved in palladium chloride water in methanol solution to obtain a 1-OH disaccharide compound 19. Finally, 2,2,2-trifluoro-N-phenyliminoacetyl chloride was used to make 19 into a disaccharide donor 20 containing N-phenyl-trifluoroacetimide ester.
  • Compound 18 Dissolve monosaccharide donor 3 (500 mg, 1.0 mmol) and glycosyl acceptor 7 (165 mg, 0.52 mmol) in anhydrous DCM (10 ml) under nitrogen, add Molecular sieve. The reaction solution was stirred at room temperature for 30 min, then cooled to 0°C, and TMSOTf (36 ⁇ L, 0.2 mmol) was added. The reaction solution was slowly returned to room temperature and stirring was continued for 5 hours. TLC detection showed that the raw material reaction was complete.
  • Compound 20 Compound 19 (86 mg, 0.14 mmol) was dissolved in anhydrous DCM and cooled to 0°C. Under the protection of argon, 2,2,2-trifluoro-N-phenyliminoacetyl chloride (0.1 ml) and DBU (30 ⁇ L) were added. The reaction solution was returned to room temperature and stirred for 20h. After concentration at room temperature, purification through a silica gel column (petroleum ether/ethyl acetate, 2.5:1) gave compound 20 (67 mg).
  • the disaccharide donor 20 and the monosaccharide acceptor 17 are catalyzed by Lewis acid TMSOTf under the combined action of dichloromethane, diethyl ether, and thiophene to obtain 1,2-cis-linked trisaccharide 21.
  • Thioacetic acid and pyridine were used to reduce the azido group of 21 to acetamido compound 22 at zero degrees.
  • the 4 acetyl groups on the 22 glucosyl group were removed under basic conditions to obtain compound 23.
  • palladium carbon and hydrogen are used to remove all aromatic groups to obtain the P. aeruginosa O11 serotype O antigen trisaccharide assembled with amino linking arms.
  • Disaccharide donor 20 35 mg
  • monosaccharide acceptor 17 45 mg
  • the molecular sieve was stirred at room temperature for 30 min, and then cooled to 0°C.
  • TMSOTf was added and stirring was continued for 8h. TLC detection showed that all the glycosyl donors had reacted.
  • Compound 24 Compound 23 (12 mg) was dissolved in a mixed solvent of methanol (3 ml), dichloromethane (1 ml) and water (1 ml), and two drops of glacial acetic acid were added, followed by palladium on carbon (10 mg). The reaction was carried out in a BLT-2000 medium-pressure hydrogenation instrument with a hydrogen pressure of 0.4 MPa and a reaction time of 12 hours. After the reaction was completed, the reaction solution was filtered through celite, concentrated, and purified by CHROMAFIX C 18 ec to obtain compound 24 (7 mg).
  • Example 7 Investigating the effect of solvents on the construction of cis-glycosidic bonds in trisaccharide 24
  • the acetamido group is assembled at the monosaccharide stage to obtain the acetamidobiose donor S21 and the acetamido acceptor S22. In this way, the glycosylation reaction failed to successfully complete the synthesis of the target trisaccharide S23.
  • the reason for the analysis was that the acetylamino group at the reducing position of the donor disaccharide was activated to form a stable oxazoline ring intermediate, which limited the glycosylation reaction. get on.
  • the above results indicate that using non-participating azido groups as acetamido precursors will help to improve the reactivity and stereoselectivity of target oligosaccharide synthesis.

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Abstract

一种利用D-葡萄糖砌块、L-岩藻糖胺砌块、D-岩藻糖胺砌块构建绿脓假单胞菌O11血清型O抗原寡糖的方法；其中，D-葡萄糖砌块或L-岩藻糖胺砌块与D-岩藻糖胺砌块通过1,2-α-顺式-糖苷键连接，D-葡萄糖砌块与L-岩藻糖胺砌块通过1,2-β-反式糖苷键连接，其中，1,2-α-顺式-糖苷键的构建是在混合溶剂中进行的；其中，混合溶剂包括二氯甲烷、乙醚、噻吩中的两种或两种以上。

Description

一种绿脓假单胞菌O11血清型O抗原寡糖的化学合成方法 技术领域
本发明涉及一种绿脓假单胞菌O11血清型O抗原寡糖的化学合成方法，属于化学领域。
背景技术
绿脓假单胞菌是一类需氧的、杆状的革兰氏阴性菌，一般栖息于土壤和水环境中。特别的，绿脓假单胞菌也是一类广泛存在于医院环境中的机会主义致病菌，引发菌血症，尿路感染以及肺炎等疾病。绿脓假单胞菌的易感人群包括烧伤病人、ICU中的病人、手术后病人以及免疫系统缺陷病人例如癌症、糖尿病和先天性囊泡纤维症患者。在我国大陆地区的呼吸机相关性肺炎(VAP)和医院获得性肺炎(HAP)中绿脓假单胞菌的流行性研究发现，在VAP中，绿脓假单胞菌占所有分离株的19.4％((95％置信区间(CI)17.6-21.2％))，HAP的比例比较相似，为17.8％(95％CI 14.6-21.6％)。此外，该研究还发现从VAP中分离到的绿脓假单胞菌对庆大霉素(51.1％,95％CI 37.7–64.4％)和阿米卡星(22.5％,95％CI 14.3–33.6％)具有较高的耐药性，而从HAP中分离得到的绿脓假单胞菌对阿米卡星(22.2％(95％CI 13.8–33.6％))和头孢哌酮(50.0％(95％CI 30.2–69.8％))具有较高的耐药性。
在全球范围内，临床上发现绿脓假单胞菌对包括多粘菌素、氟喹诺酮类、碳青霉烯类等抗生具有一定的耐药性。2016年，世界卫生组织将绿脓假单胞菌列为目前最急需研究和发展新型抗生素或者疫苗的致病菌之一。截至目前，市场上还没有针对该致病菌的疫苗问世。有关绿脓假单胞菌的疫苗研究始于1980年代，研究领域集中于该致病菌的脂多糖、糖-蛋白缀合物、鞭毛、灭活或者减毒全菌以及该菌的DNA序列等方面。绿脓假单胞菌的脂多糖由一些在人体上不常见的单糖以及糖苷键连接方式构成，是一种重要的毒力因素。根据单糖种类的和糖苷键连接方式的不同，绿脓假单胞菌共有20种不同的血清型抗原。其中具体结构为[-3)-α-L-N-乙酰岩藻糖胺-(1-3)-α-D-N-乙酰岩藻糖胺-(1-2)-β-D-葡萄糖-(1-] n的O11血清型抗原，这是一个由三糖重复片段收尾相接而成的一个长链抗原，具体的重复单元数n受该致病菌的基因所调控。
目前，针对该结构化合物都是从含有该脂多糖结构的野生治病菌或者含有编码该结构的基因工程菌中提取得，包括LPS高表达菌株的构建和培养，蛋白酶k的降解，石炭酸热萃取，Tricine-SDS-polyacrylamide gel electrophoresis(PAGE)分析和Western immunoblot检测等一系列步骤。此外，使用生物提取方法得到的脂多糖还存在化学结构不均一的问题，同时还存在着安全性的问题，因为是从菌体中直接提取的脂多糖，很有可能会掺杂其他细菌毒力因子，不仅不能够对人体产生免疫保护，反而会使人体受到菌体毒力因子的直接感染而致病，这样会产生严重的副作用。为了避免这样的问题，我们决定利用化学合成的方法来直接合成该脂多糖多糖，这样就避免了化学结构不均一以及掺杂其他未知毒力因子的问题，合成出的脂多糖具有明确的化学结构，并且可以保证仅有此种荚膜多糖参与疫苗的组成，不会掺杂其他物质。
然而，在复杂寡糖合成过程中，糖苷键的构建是寡糖合成中最基本但是也最为棘手和关键的问题，由于糖类化合物结构的多样性，立体化学的复杂性，因此与其他结构的有机化合物不同，寡糖的合成的方法学依然是不成熟和不完善的，被认为是有机化学领域中唯一存在众多方法(数十种之多)但又有没有一个具有普适性的方法为大家公认的领域。因为糖模块的结构复杂、顺式糖苷键选择性低，导致难以该结构合成构建，目前还没有报道利用化学合成的方法来构建该化合物，从而制约了该结构化解无的化学合成方法的研究。
发明内容
为了解决上述问题，本发明通过三种糖砌块化学方法合成绿脓假单胞菌的O11血清型O抗原三糖片段，主要包括葡萄糖砌块、L-岩藻糖胺砌块、D-岩藻糖胺砌块以及1,2- -顺式-糖苷键和1,2-β-反式糖苷键。其中D-岩藻糖胺的合成和1,2-α-顺式-糖苷键的构建是该目标三 糖的关键步骤。本发明开发了一条由D-葡萄糖合成D-岩藻糖胺的方法，并且通过溶剂效应，温度效应和添加剂效应的协同作用成功解决了构建1,2-α-顺式-糖苷键的立体选择性问题。利用合成的三个糖基砌块合成了保护目标三糖，最后脱去保护得到的目标三糖如式1。同时，三糖的还原端组装有带有氨基的连接臂，为下一步的缀合蛋白作准备。
本发明的第一个目的是提供一种绿脓假单胞菌的O11血清型O抗原三糖的合成方法，所述方法包括：利用D-葡萄糖砌块、L-岩藻糖胺砌块、D-岩藻糖胺砌块构建O抗原三糖；其中，D-葡萄糖砌块或L-岩藻糖胺砌块与D-岩藻糖胺砌块通过1,2-α-顺式-糖苷键连接，D-葡萄糖砌块与L-岩藻糖胺砌块通过1,2-β-反式糖苷键连接，所述1,2-α-顺式-糖苷键的构建是在混合溶剂中进行的；所述混合溶剂包括二氯甲烷、乙醚、噻吩中的两种或两种以上。
在本发明的一种实施方式中，所述D-葡萄糖砌块的化学结构如式II所示，L-岩藻糖胺砌块的化学结构式如式III所示，D-岩藻糖胺砌块的化学结构式如式IV所示，
Figure PCTCN2019119584-appb-000001
其中，linker包括-(CH 2) n-N-Y 1Y 2，或者O-(CH 2) n-SY 1(Y 2)，或者O-(CH 2) n-N 3，n＝1～10；Y 1为氢(H)或者苄基(Bn)；Y 2为氢(H)或者苄甲氧羰基(Cbz)；R 1包括但不限定于氢(H)、酯基、乙酰基(Ac)、苯甲酰基(Bz)、新戊酰基(Piv)、氯乙酰基(ClAc)、乙酰丙酰基(Lev)、烯丙羰酰基(Alloc)；R 2，R 3，R 4为氢(H)或者酯基、醚基包括但不限于乙酰基(Ac)、苯甲酰基(Bz)、新戊酰基(Piv)、氯乙酰基(ClAc)、乙酰丙酰基(Lev)、烯丙羰酰基(Alloc)以及苄基(Bn)、对甲氧基苄基(pMBn)、烯丙基(All)、三苯甲基(Tr)、单甲氧基三苯甲基(Mmt)和硅醚类基团基团；R 5,R 6包括但不限于氢(H)、醚类基团、苄基(Bn)、对甲氧基苄基(pMBn)、烯丙基(All)、三苯甲基(Tr)、单甲氧基三苯甲基(Mmt)和硅醚类基团基团；R 7，R 8，R 9，R 10包括氢(H)、氮(N)或者乙酰基(Ac)。
在本发明的一种实施方式中，所述方法包括利用D-葡萄糖砌块和L-岩藻糖胺砌块合成二糖片段，其化学结构如式V所示，
Figure PCTCN2019119584-appb-000002
在本发明的一种实施方式中，所述D-葡萄糖砌块与L-岩藻糖胺砌块的1,2-β-反式糖苷键连接是在TMSOTf促进作用下，以二氯甲烷作为溶剂中进行的。
在本发明的一种实施方式中，所述方法包括利用二糖片段与D-岩藻糖胺砌块合成三糖片段，其化学结构式如式I所示，
Figure PCTCN2019119584-appb-000003
在本发明的一种实施方式中，所述方法包括预先合成O抗原三糖前体，还原记得O抗原三糖，所述O抗原三糖前体的化学结构式包括如下通式：
Figure PCTCN2019119584-appb-000004
在本发明的一种实施方式中，所述D-葡萄糖砌块(式II)的端基位是三氯乙酰亚胺酯，为离去基团，其余取代基R n参见式II。
在本发明的一种实施方式中，所述D-葡萄糖砌块的合成方法包括：以为1,2,3,4,6-五-O-乙酰基葡萄糖起始原料，利用干燥N、N-二甲基甲酰胺和乙酸肼脱去1号位乙酰基裸漏出-OH，裸露的-OH与三氯乙腈(Cl 3CN)反应得到化合物D-葡萄糖砌块。
在本发明的一种实施方式中，所述L-岩藻糖砌块(式III)端基位为烯丙基(OAll)保护，其余位置取代基R n参见式III。
在本发明的一种实施方式中，所述L-岩藻糖砌块是以烯丙基2-脱氧-2叠氮基-L-岩藻糖苷为起始原料，用二丁基氧化锡(Bu 2SnO)和2-溴甲基萘的作用下选择性保护4号位的羟基得到4-Nap岩藻糖；然后用苄基保护剩余的3号位羟基，再在二氯二氰基苯醌的作用下脱去4号位的Nap基团得到最终糖砌块B。
在本发明的一种实施方式中，所述D-岩藻糖胺砌块的端基位为linker，如式IV所示。
在本发明的一种实施方式中，所述D-岩藻糖胺砌块的合成方法利用葡萄糖构建得到D-岩藻糖胺，包括：以3,4,6-O-三乙酰基甘露糖烯为原料，经过叠氮化物、硒试剂得到1-硒苯-2叠氮葡萄糖，脱除乙酰基，然后6-C进行甲基化，最终得到D-岩藻糖胺类化合物。
在本发明的一种实施方式中，所述方法具体包括：以3,4,6-O-三乙酰基甘露糖烯为原料，在二乙酸碘苯(PhI(OAc) 2)作用下，与叠氮基三甲基硅烷(TMS-N 3)和二苯基二硒(Ph 2Se 2)生成1-硒苯-2叠氮葡萄糖，然后碱性条件下脱除乙酰基，加入4-甲苯磺酰氯实现6-C甲基化，得到D-岩藻糖胺化合物。
在本发明的一种实施方式中，所述方法还包括将D-岩藻糖胺化合物的3、4-C进行保护，然后脱去4-C的Nap基团，用乙酰基保护起来，四丁基溴化铵(NBS)水解D-岩藻糖的1号位硒苯基，用Cl 3CN和DBU将岩藻糖做成糖基三氯乙酰亚胺酯，随后在路易斯酸的促进下组装上氨基连接臂，最后脱去D-岩藻糖3号位上的乙酰基得到D-岩藻糖胺砌块。
在本发明的一种实施方式中，所述3-C的保护试剂包括甲氧基苄氯(PMBCl)和氧化二丁基锡(Bu2SnO)。
在本发明的一种实施方式中，所述4-C的保护试剂包括苄基。
在本发明的一种实施方式中，所述4-C的Nap基团的脱除去利用DDQ实现的。
在本发明的一种实施方式中，所述路易斯酸包括三氟甲磺酸三甲基硅脂。
本发明的第二个目的是提供一种组装有氨基连接臂的绿脓假单胞菌的O11血清型O抗原三糖，所述抗原三糖是利用上述方法制备得到的。
本发明的第三个目的是提供一种糖-蛋白缀合物的制备方法，所述方法是利用上述的组装有氨基连接臂的绿脓假单胞菌的O11血清型O抗原三糖。
本发明的第四个目的是提供一种糖-蛋白缀合物的制备方法，所述方法是包含绿脓假单胞菌的O11血清型O抗原三糖，所述绿脓假单胞菌的O11血清型O抗原三糖的制备方法为上述方法。
本发明的第五个目的是将上述的组装有氨基连接臂的绿脓假单胞菌的O11血清型O抗原三糖应用于在开发或制备绿脓假单胞菌疫苗或者绿脓假单胞菌感染导致的疾病的药物中的应用。
本发明有益效果：
1、本发明是通过化学方法合成得到绿脓假单胞菌O11血清型O-抗原三糖片段。本发明中开发了一种由D-甘露糖转变为D-岩藻糖的方法，方法简便高效，除了6-C甲基化约50左右，其余都可在80％以上；然后所得糖基砌块用于合成绿脓假单胞菌O11血清型O-抗原三糖片段。
2、常用糖苷键的构建是利用糖环C-2位酰基辅助下的邻基参与效应，高效率的构建1,2-反式糖苷键，2-位乙酰基作为糖化学保护策略中最易得、最有效的保护剂，如本发明中反式糖苷键的构建。然而对于1,2-顺式反应，由于反应方法是没有普适性的，各反应条件对反应影响较大，其中有设计糖砌块的时候选择性在C-6用大空间位阻的保护基保护和在特别的针对半乳糖的在C-4位引入酰基形成类似长程邻基辅助效应的机制的报道，本发明方法采用一种适宜的混合溶剂，实现了顺式糖苷键的均一构建，立体选择性可达100％。
3、本发明通过保护基团的选择和优化以及糖苷化反应的策略，成功完成了O11三糖重复单元的制备。本发明方法合成的绿脓假单胞菌O11血清型O-抗原三糖片段组装有氨基连接臂，可以和抗原蛋白相连制成糖缀合物，对发展治疗和预防绿脓假单胞菌疫苗具有重要作用。
附图说明
图1：单糖砌块3(D-葡萄糖砌块)的合成路线图；
图2：单糖砌块7(L-岩藻糖砌块)的合成路线图；
图3：单糖砌块17(D-岩藻糖胺砌块)的合成路线图；
图4：单糖砌块17构建方法一的合成路线图；
图5：单糖砌块17构建方法二的合成路线图；
图6：二糖20的合成路线图；
图7：三糖24的合成路线图。
图8：三糖S23的合成路线设计图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或者制造商建议的条件进行，所用的试剂或者仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明的产率计算方法为“产物(mol)/反应底物(mol)*100％”。本发明中化合物结构鉴定的方法为核磁共振图谱的测定(400M)，高分辨质谱测定，旋光值测定，结果在每个化合物的具体合成中列出。
实施例1
糖砌块3(D-葡萄糖砌块)的合成如图1：
如图1所示，以全乙酰葡萄糖1作为起始原料，在干燥的N，N-二甲基甲酰胺中利用乙酸肼(NH 2-NH 2-AcOH)选择性的水解掉1号位上的乙酰基。得到的1-OH葡萄糖2在然后无水二氯甲烷中利用三氯乙腈(Cl 3CN)和1,8-二氮杂二环十一碳-7-烯(DBU)生成葡萄糖基三氯乙酰亚胺酯3。
具体试验操作和步骤：
化合物2：全乙酰葡萄糖1(5.0g，12.8mmol)溶解在无水DMF(65ml)中，加入醋酸肼(1.45g，15.6mmol)。反应在40℃下搅拌6h，用TLC监测反应物消失。反应液冷却到室温，用饱和NaCI溶液洗涤，分离有机相，用无水Na 2SO 4干燥，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，2:1)得化合物2(3.9g，11.2mmol)。该化合物的鉴定数据已有相关文献报道。
化合物3：化合物2(1.1g，3.16mmol)溶解在无水DCM(30ml)并且冷却到0℃。依次往反应液里面加入Cl 3CN(3.2ml，31.6mmol)和DBU(0.45ml，0.3mmol)，该反应在冰 浴下搅拌4h，用TLC监测反应原料消失。反应液恢复到室温，用饱和NaHCO 3萃取，分离有机相，用无水Na 2SO 4干燥，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得化合物3(1.3g，2.8mmol)。该化合物的鉴定数据已有相关文献报道。
实施例2：
糖砌块7(L-岩藻糖砌块)的合成路线为图2。
如图2所示，以烯丙基2-脱氧-2叠氮基-L-岩藻糖苷4为起始原料，先在含有甲醇钠的碱性环境下脱去3,4号位的乙酰基，得到的二羟基化合物在二丁基氧化锡和溴甲基萘的作用下在4号位选择性的用Nap基保护起来得到化合物5，3-Nap化合物5的羟基用苄基保护生成化合物6。最后用DDQ脱去6的Nap基得到化合物3-OH化合物7。
具体试验操作和步骤：
化合物5：烯丙基2-脱氧-2叠氮基-L-岩藻糖苷4(500mg，2.2mmol)容易无水甲苯(22ml)，然后加入二丁基氧化锡(814mg，3.3mmol)。将反应液加热到110℃搅拌1h。随后反应液冷却到40℃，加入溴甲基萘(814mg，3.3mmol)和四丁基溴化铵(1.05g，3.3mmol)，继续反应20h。反应完全后加入乙酸乙酯(30ml)稀释，用饱和食盐水洗涤。分离有机相，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，5:1)得化合物5(527mg，1.7mmol，79％)。[α] D 20＝34.0°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ7.85(dt,J＝10.5,7.7Hz,4H,Ar-H),7.59–7.45(m,3H,Ar-H),5.94(ddt,J＝16.8,10.9,5.6Hz,1H,Ally-H),5.33(dd,J＝17.2,1.7Hz,1H,Ally-CH 2a),5.21(dd,J＝10.5,1.5Hz,1H,Ally-CH 2b),4.88(s,2H,2Nap-H),4.12(dd,J＝12.9,6.2Hz,1H,Ally-H),4.23(d,J＝8.1Hz,1H,1-H),4.12(dd,J＝12.9,6.2Hz,1H,Ally-OCH 2b),3.74(d,J＝3.3Hz,1H,4-H),3.66(dd,J＝10.0,8.0Hz,1H,2-H),3.47(q,J＝6.5Hz,1H,5-H),3.35(dd,J＝10.0,3.3Hz,1H,3-H),2.32(s,1H,4-OH),1.34(d,J＝6.4Hz,6-CH 3). 13C NMR(101MHz,CDCl 3)δ134.65,133.65,133.20,128.55,127.93,127.77,126.98,126.33,126.22,125.75,117.61,100.75,79.41,77.33,77.22,77.01,76.70,72.19,70.19,69.97,68.34,62.57,16.33.HRMS(ESI)m/z calcd for C 20H 23N 3O 4Na[M+Na] +391.1581,found 391.1564。
化合物6：将化合物5(527mg，1.7mmol)溶于无水DMF(17ml)中，冷却至0℃，冰浴30min。往反应瓶中加入NaH(140mg，3.4mmol)，继续在0℃下搅拌30min，加入溴化苄(0.5ml，3.4mmol)。反应液恢复到室温，继续搅拌4h，待反应原料全部消失之后加入DCM(20ml)稀释，用冰水淬灭反应。用饱和NaHCO 3洗涤三次，合并有机相，浓缩后经硅胶柱纯化(石油醚/乙酸乙酯，25:1)得到化合物6(531mg，1.2mmol，68％)[α] D 20＝50.3°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.90–7.77(m,4H,Ar-H),7.57–7.45(m,3H,Ar-H),7.38–7.21(m,6H,Ar-H),5.94(dddd,J＝16.9,10.8,6.1,5.0Hz,1H,Ally-H),5.32(dt,J＝17.2,1.7Hz,1H,Ally-H),5.19(dq,J＝10.5,1.5Hz,1H,Ally-H),4.97(d,J＝11.7Hz,1H,PhCH),4.87(s,2H,2Nap-H)4.71(d,J＝11.7Hz,1H,PhCH-H),4.39(ddt,J＝12.9,5.0,1.6Hz,1H,Ally-H),4.22(d,J＝8.0Hz,1H,1-H),4.10(ddt,J＝12.9,6.1,1.4Hz,1H,Ally-H),3.87(dd,J＝10.4,8.0Hz,1H,2-H),3.55(d,J＝2.8Hz,1H,4-H),3.42-3.38(m,1H,5-H),3.36(dd,J＝10.4,2.8Hz,1H,3-H),1.20(d,J＝6.4Hz,3H,6-CH 3).
13C NMR(101MHz,Chloroform-d)δ138.38,135.34,133.93,133.38,133.23,128.55,128.48,128.36,128.07,127.89,127.83,126.79,126.38,126.21,125.90,117.58,101.11,81.07(C-1),75.17,74.85,72.87,70.74,70.07,63.31,17.02.HRMS(ESI)m/z calcd for C 27H 29N 3O 4Na[M+Na] +482.2050,found 482.2041。
化合物7：将化合物6(531mg，1.16mmol)溶于二氯甲烷(50ml)和水(5ml)的混合溶剂中，反应液冷却至0℃，加入DDQ(540mg，2.32mmol)。反应液在室温下搅拌2h，待TLC监测原料全部反应完全后，用10％Na 2S 2O 3洗涤反应液，分离有机相，用无水硫酸钠干燥后浓缩，经硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物7(325mg，0.96mmol，85％)。[α] D 20＝-3.6°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.42–7.28(m,5H,Ar-H),5.94(dddd,J＝16.7,10.4,6.1,5.1Hz,1H,Ally-H),5.33(dq,J＝17.2,1.6Hz,1H,Ally-H), 5.21(dq,J＝10.5,1.4Hz,1H,Ally-H),4.81(d,J＝11.6Hz,1H,Bn-H),4.72(d,J＝11.6Hz,1H,Bn-H),4.41(ddt,J＝12.9,5.1,1.6Hz,1H,Ally-H),4.26(d,J＝7.7Hz,1H,1-H),4.11(ddt,J＝12.9,6.1,1.4Hz,1H,Ally-H),3.58–3.49(m,3H,2-H,4-H,5-H),3.45(dd,J＝10.4,3.3Hz,1H,3-H),2.14(s,1H,-OH),1.31(d,J＝6.5Hz,3H,6-Me)。 13C NMR(101MHz,Chloroform-d)δ138.02,133.75,128.68,128.30,128.18,117.63,101.11,78.60,76.04,73.12,70.95,70.17,64.73,17.01。HRMS(ESI)m/z calcd for C 16H 21N 3O 4Na[M+Na] +342.1424,found 342.1430。
实施例3：
糖砌块17的合成路线为图3。
如图3所示，以3,4,6-O-三乙酰基甘露糖糖烯8为起始原料，与二乙酸碘苯(PhI(OAc) 2)、叠氮基三甲基硅烷(TMS-N 3)和二苯基二硒(Ph 2Se 2)加成反应得到1-硒苯基-2-叠氮基化合物9。用甲醇和甲醇钠脱去9的三个乙酰基，然后利用接着用4-甲苯磺酰氯(TsCl)选择性保护6号位羟基得到化合物10。接下来，6-Ts葡萄糖10的Ts基团用碘化钠碘代，紧接着用氰基硼氢化钠(NaCNBH 3)把6号位还原成甲基，得到D-岩藻糖化合物11。用对甲氧基苄氯(PMBCl)和氧化二丁基锡(Bu 2SnO)选择性保护D-岩藻糖化合物的3号位得到化合物12，随后用苄基保护4号位羟基得到化合物13。13的4号位Nap用DDQ脱去，随后用乙酰基保护起来得到化合物14。用四丁基溴化铵(NBS)水解化合物14的1号位硒苯基，紧接着用Cl 3CN和DBU将1-OH岩藻糖做成糖基三氯乙酰亚胺酯供体15。随后在三氟甲磺酸三甲基硅脂的促进下化合物15与一个三碳连接臂反应，得到一个组装有连接臂的岩藻糖化合物16。最后在甲醇和甲醇钠的碱性环境下脱去化合物16的乙酰基得到糖砌块17。
具体试验操作和步骤：
化合物9：将半乳糖烯化合物8(2.0g，7.35mmol)溶于无水二氯甲烷(35ml)中，然后冷却到-30℃。在氩气保护下加入二乙酸碘苯(2.4g，7.35mmol)和叠氮三甲基硅烷(1.8ml，14.7mmol)，继续在-30℃下搅拌0.5h，然后缓慢恢复到室温，继续搅拌3h之后经TLC监测原料反应完全能。反应液用饱和NaHCO 3洗涤(20ml)3次，分离有机相用无水硫酸钠干燥，过滤，滤液旋干后经由硅胶柱纯化(石油醚/乙酸乙酯，6:1)，得到化合物9(2.4g，5.15mmol，70％)。[α] D 20＝931.7°(c＝1.00,CHCl3)。 1H NMR(400MHz,Chloroform-d)δ7.78-7.11(m,5H,Ar-H),6.01(d,J＝5.4Hz,1H,H-1),5.47(dd,J＝3.4,1.4Hz,1H,4-H),5.12(dd,J＝10.9,3.3Hz,1H,3-H),4.67(ddd,J＝7.1,5.8,1.3Hz,1H,5-H),4.26(dd,J＝10.9,5.4Hz,1H,2-H),4.08(dd,J＝11.5,5.9Hz,1H,6a-H),4.02(dd,J＝11.4,7.1Hz,1H,6b-H),2.15(s,3H,COCH 3),2.07(s,3H,COCH 3),1.98(s,3H,COCH 3). 13C NMR(101MHz,Chloroform-d):δ169.93,137.08,135.12,129.91,129.19,128.56,128.23,128.04,127.97,84.44,77.22,75.34,73.68,73.44,70.09,67.05,58.99,21.67,20.83。HRMS(ESI)m/z calcd for C 18H 21N 3NaO 7Se[M+Na] +494.0442。
化合物10：将化合物9(0.5g，1.1mmol)溶解在甲醇(2.2ml)里，加入甲醇钠(29mg，0.5mmol)。反应液在室温下搅拌2h，TLC检测显示原料反应完全。用氢型Amberlite IR120树脂中和反应液至～7。过滤树脂，将溶剂旋干。
将上一步的粗产物溶解在无水吡啶(8ml)里，在氩气保护下加入TsCl(253mg，1.3mmol)。反应液在室温下搅拌12h，TLC检测原料反应完全。用DCM稀释反应液，用1M盐酸洗涤，然后用饱和碳酸氢钠萃取。合并有机相，用无水硫酸钠干燥，浓缩后用硅胶柱纯化(二氯甲烷/甲醇，200:1)得到化合物10(384mg，077mmol，70％)。[α] D 20＝133.6°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.72(d,J＝8.3Hz,2H,Ar-H),7.58(dt,J＝6.7,1.6Hz,2H,Ar-H),7.36-7.23(m,6H,Ar-H),5.86(d,J＝5.3Hz,1H,H-1),4.57-4.50(m,1H,5-H),4.29(dd,J＝10.6,6.0Hz,1H,6 a),4.10–3.93(m,3H,H-2,H-4,6 b-H),3.78(ddd,J＝9.8,6.0,3.3Hz,1H,H-3),2.68(dd,J＝6.5,4.8Hz,2H,3-OH,4-OH),2.44(s,3H,PhCH 3)。 13C NMR(101MHz,CDCl 3)δ145.20,135.04,132.36,132.11,132.01,129.94,129.24,128.78,128.66,128.24,128.00,127.67,84.62,77.34,77.22,77.02,76.70,70.62,69.97,67.87,67.55,61.57,21.68。HRMS(ESI)m/z calcd for C 19H 21N 3NaO 5SSe[M+Na] +506.0265。
化合物11：在氩气保护下，将化合物10(380mg，0.76mmol)溶于2-丁酮(9.6ml)中，加入碘化钠(568mg，3.8mmol)。反应液在80℃下搅拌12h，TLC检测原料反应完全。反应液恢复到室温，加入乙酸乙酯(10ml)稀释，用10％硫代硫酸钠洗涤两次，水洗涤两次，合并有机相，用无水硫酸钠干燥。浓缩后经由硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到新化合物(259mg，0.57mmol，75％)。[α] D 20＝317.6°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.61–7.43(m,4H,Ar-H),7.39–7.12(m,6H,Ar-H),5.96(d,J＝5.3Hz,1H,1-H),4.44–4.33(m,1H,5-H),4.22(d,J＝3.7Hz,1H,4-H),4.09(dd,J＝10.2,5.3Hz,1H,2-H),3.79(ddd,J＝9.9,6.1,3.3Hz,1H,3-H),3.17(dd,J＝12.7,8.2Hz,1H,6-H a),3.06(dd,J＝12.7,6.0Hz,1H,6-H b),2.75(d,J＝6.2Hz,1H,3-OH),2.45(d,J＝3.9Hz,1H,4-OH)。 13C NMR(101MHz,CDCl 3)δ134.58,132.92,132.87,132.81,129.49,129.32,129.28,129.18,128.19,127.97,127.46,85.04,84.62,77.36,77.25,77.04,76.73,72.32,71.32,68.93,61.71,26.94。HRMS(ESI)m/z calcd for C 12H 14IN 3NaO 3Se[M+Na] +477.9143。
在氩气的保护下将新化合物溶解在DMF(2ml)中，加入氰基硼氢化钠(48mg，0.75mmol)。反应在95℃下搅拌12h，TLC检测原料反应完全。反应液恢复到室温，用水和乙酸乙酯洗涤反应液，合并有机相，用无水硫酸钠干燥，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯2:1)得到化合物11(35mg，0.1mmol，70％)。[α] D 20＝259.4°(c＝1.00,CHCl 3)。 1HNMR(400MHz,Chloroform-d)δ7.73-7.45(m,2H,Ar-H),7.40-6.93(m,3H,Ar-H),5.92(d,J＝5.3Hz,1H,H-1),4.39(dt,J＝7.4,6.0Hz,1H,5-H),4.05(dd,J＝10.0,5.3Hz,1H,2-H),3.91–3.74(m,2H,H-3.4-H),2.60(d,J＝6.3Hz,1H,3-OH),2.26(d,J＝4.0Hz,1H,4-OH),1.27(d,J＝6.6Hz,3H,6-CH 3). 13C NMR(101MHz,Chloroform-d)δ134.48,129.15,127.90,85.00,77.22,71.46,71.29,68.69,61.88,15.98。HRMS(ESI)m/z calcd for C 12H 15N 3NaO 3Se[M+Na] +346.0230,found346.0226。
化合物12：将化合物11(3.0g，9.1mmol)溶于无水甲苯(54ml)中，在氩气保护下加入Bu 2SnO(3.5g，13.7mmol)。反应液加热到118℃搅拌下回流1.5h。随后将反应液冷却至60℃，加入PMBCl(1.9ml，13.7mmol)和TBAI(5.1g，13.7mmol)，继续搅拌2h。反应液冷却到室温，加入乙酸乙酯(80ml)稀释，用水(100ml)洗涤2次，分离有机相，用无水硫酸钠干燥，滤液浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，6:1)得到化合物12(2.7g，6mmol，66％)。[α] D 20＝137.9°(c＝1.00,CHCl3)。1H NMR(400MHz,Chloroform-d)δ7.26(s,11H,Ar-H),5.88(d,J＝5.4Hz,1H,H-1),4.70(d,J＝11.0Hz,1H,Bn-H),4.63(d,J＝11.0Hz,1H,Bn-H),4.36-4.24(m,1H,5-H),4.15(dd,J＝10.2,5.3Hz,1H,2-H),3.86(dt,J＝3.2,1.5Hz,1H,4-H),3.82(s,3H,Me-H),3.69(dd,J＝10.2,3.2Hz,1H,3-H),2.35(t,J＝1.6Hz,1H,4-OH),1.26(d,J＝6.6Hz,3H,6-Me). 13C NMR(101MHz,Chloroform-d)δ159.74,134.40,129.75,129.10,129.08,127.75,114.14,85.28,78.85,71.84,68.61,68.55,60.18,55.31,16.05。HRMS(ESI)m/z calcd for C 20H 23N 3O 4SeNa[M+Na] +472.0751,found 472.0725。
化合物13：用将化合物12(2.2g，4.9mmol)溶于DMF(30ml)中，0℃下搅拌30min，在氩气保护下缓慢划入氢化那(396mg，9.8mmol)。冰浴下继续搅拌30min，往反应液中逐滴加入溴化苄(1.2ml，9.8mmol)。继续搅拌反应2h，TLC检测原料全部反应完全，加入冰水淬灭反应，用DCM稀释反应液，萃取后由无水硫酸钠干燥，浓缩液使用硅胶柱纯化(石油醚/乙酸乙酯，30:1)得到化合物13(1.9g，3.6mmol，74％)。[α] D 20＝71.5°(c＝1.00,CHCl 3)。 1HNMR(400MHz,Chloroform-d)δ7.57-7.55(m,2H,Ar-H),7.36-7.25(m,10H,Ar-H),6.94-6.91(m,2H,Ar-H),5.92(d,J＝5.3Hz,1H,H-1),4.94(d,J＝11.4Hz,1H,PhCH),4.77-4.66(m,2H,PhCH2),4.60(d,J＝11.5Hz,1H,PhCH),4.33(dd,J＝10.2,5.3Hz,1H,H-2),4.21(q,J＝6.5Hz,1H,H-5),3.83(s,3H,OCH 3),3.71(dd,J＝10.3,2.7Hz,1H,H-3),3.68(dd,J＝2.8,1.1Hz,1H,H-4),1.12(d,J＝6.4Hz,3H,6-CH3)。 13C NMR(101MHz,CDCl3)δ159.63,138.33,134.48,129.72,129.16,128.90,128.44,128.32,127.90,127.77,114.14,85.79,80.47,77.16,76.01,75.13,72.40,69.56,61.03,55.46,16.69。HRMS(ESI)m/z calcd for C 27H 29N 3NaO 4Se[M+Na] +562.1221, found 562.1209。
化合物14：将化合物13(900mg，1.67mmol)溶解于DCM(84ml)中，在氩气保护下加入2,3-二氯-5,6-二氰基-1,4-苯醌(564mg，2.5mmol)和水(4.2ml)。反应在室温下搅拌7h，用TLC检测反应完全。加入DCM(50ml)稀释，用10％硫代硫酸钠(100ml)洗涤反应液两次。分离有机相，经由无水硫酸钠，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，10:1)得到新产物(573mg，1.4mmol，82％)。把新产物溶解在干燥的吡啶(3.2ml)中，冷却至0℃，30min后缓慢加入乙酸酐(0.5ml，5.12mmol)，反应液恢复到室温继续搅拌5h，TLC检测原料反应完全。用1M的盐酸洗涤反应液，接着用饱和碳酸氢钠和饱和食盐水萃取。分离有机相，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯＝20:1)得到化合物14(598mg，1.3mmol，quant)。 1H NMR(400MHz,Chloroform-d)δ7.68-7.21(m,9H,Ar-H),5.91(d,J＝5.3Hz,1H,H-1),5.46(dd,J＝3.3,1.2Hz,1H,H-4),4.75(d,J＝10.7Hz,1H,PhCH 2),4.53(d,J＝10.7Hz,1H,PhCH),4.48-4.37(m,1H,H-5),4.12(dd,J＝10.3,5.4Hz,1H,H-2),3.78(dd,J＝10.4,3.3Hz,1H,H-3),2.15(s,3H,COCH 3),1.12(d,J＝6.5Hz,3H,6-CH 3)。 13C NMR(101MHz,Chloroform-d)δ170.52,136.90,134.53,129.12,128.50,128.34,128.07,127.88,85.08,71.68,68.89,67.77,60.36,20.76,16.07。HRMS(ESI)m/z calcd for C 21H 23N 3NaO 4Se[M+Na] +484.0751,found 484.0749。
化合物15：将化合物14(580mg/1.3mmol)溶于四氢呋喃(6ml)和水(6ml)的混合溶液，并且冷却至0℃。加入四丁基溴化铵(538mmol，3mmol)，反应液回复到室温继续搅拌5h，TLC检测显示原料反应完全。反应液用10％硫代硫酸钠洗涤，用DCM和水萃取，分离有机相，用无水硫酸钠干燥，浓缩后用硅胶柱纯化(石油醚/乙酸乙酯，4:1)得到新化合物(417mmol，1.3mmol，quant.)[α] D 20＝54.9°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.43–7.30(m,8H,Ar-H),5.47(dd,J＝3.3,1.3Hz,1H,1a-H),5.34(dd,J＝3.4,1.1Hz,0.8H,4b-H),5.32(d,J＝3.6Hz,1H,4a-H),4.77(d,J＝10.8Hz,1H,PhCH),4.75(d,J＝11.1Hz,0.8H,PhCH),4.54(d,J＝7.9Hz,1H,1b-H),4.53(d,J＝10.8Hz,1H,PhCH),4.53(d,J＝11.1Hz,0.8H,PhCH),4.32(qd,J＝6.6,1.3Hz,1H,5-H),4.03(dd,J＝10.5,3.2Hz,1H,2a-H),3.74(dd,J＝10.5,3.5Hz,1H,3a-H),3.69(td,J＝6.4,1.1Hz,0.8H,5b-H),3.58(dd,J＝10.2,7.9Hz,0.8H,2b-H),3.45(dd,J＝10.3,3.4Hz,0.8H,3b-H),2.19(s,2.4H,COCH 3),2.18(s,3H,COCH 3),1.25(d,J＝6.5Hz,2.4H,6b-CH 3),1.19(d,J＝6.5Hz,3H,6a-CH 3).。 13C NMR(101MHz,CDCl3)δ170.90,170.88,137.15,137.04,128.63,128.52,128.40,128.22,128.17,96.20,92.46,77.91,77.48,77.36,77.16,77.16,76.84,74.36,71.91,71.67,69.69,69.46,68.39,65.21,64.03,59.82,20.95,20.93,16.55,16.42。HRMS(ESI)m/z calcd for C 15H 19N 3O 5Na[M+Na] +344.1217,found 344.1208。
将新化合物(417mmol，1.3mmol)溶于无水二氯甲烷(14ml)，冷却至0℃。依次加入三氯乙腈(0.4ml，3.78mmol)和DBU(23μL，0.13mmol)，反应液继续搅拌5h，TLC检测原料反应完全。直接将反应液在室温下浓缩，经由硅胶柱纯化(石油醚/乙酸乙酯，10:1)得到化合物15(518mg，1.1mmol，88％)。
化合物16：糖基三氯乙酰亚胺酯15(518mg，1.1mmol)和氮-苄基-氮-甲酸苄酯-丙醇(500mg，1.7mmol)溶于无水DCM中，加入
Figure PCTCN2019119584-appb-000005
分子筛，室温下搅拌半小时。然后将反应液冷却至-40℃，在氩气下加入缓缓加入TMSOTf(0.3ml，1.3mmol)。反应液在-40℃下继续搅拌4h，用TLC监测原料反应完全，用三乙胺(1ml)淬灭反应。反应液用饱和NaHCO 3萃取，分离有机相，用无水Na 2SO 4干燥后经由硅胶柱纯化得到化合物16(396mg，0.44mmol，60％)。[α] D 20＝-10.8°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.45–7.16(m,15H,Ar-H),5.23–5.12(m,2H,PhCH),4.74–4.59(m,3H,3-H,2PhCH),4.61–4.38(m,2H,2NPhCH),4.21(dd,J＝33.3,8.0Hz,1H,1-H),4.02–3.87(m,1H,linker-OCH),3.79(t,J＝9.5Hz,1H 2-H),3.67(d,J＝3.0Hz,1H,4-H),3.63–3.27(m,4H,5-H linker-OCH,linker-NCH 2),2.07(s,3H,COCH 3),2.01–1.76(m,2H,linker-CH 2),1.24(d,J＝6.4Hz,3H,6-CH3). 13C NMR(101MHz,CDCl 3)δ 170.38,137.95,137.70,128.52,128.46,128.40,128.32,127.97,127.92,127.87,127.27,101.99,77.37,77.25,77.05,76.74,76.19,75.59,74.23,70.48,67.40,67.19,61.11,50.90,44.67,43.62,28.48,28.03,20.89,16.57.HRMS(ESI)m/z calcd for C 33H 38N 4O 7Na[M+Na] +625.2633,found625.2650。
化合物17：将化合物16(262mg，0.42mmol)溶于甲醇(4ml)中，加入甲醇钠(40mg)。反应液在室温下搅拌2h，待原料全部反应完全之后，用甲醇稀释反应液，用氢型Amberlite IR120树脂中和反应液至pH～7。滤去树脂，滤液浓缩后经由硅胶柱纯化(石油醚/乙酸乙酯，3:1)得到化合物17(244mg，0.42mmol，quant.)。[α] D 20＝-24.1°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.76–6.87(m,15H,Ar-H),5.22(d,J＝9.4Hz,2H,PhCH),4.83(d,J＝11.6Hz,1H,PhCH),4.78(d,J＝11.6Hz,1H,PhCH),4.58(d,J＝14.0Hz,2H,PhCH),4.25–4.08(m,1H,1-H),4.05–3.86(m,1H,linker-OCH),3.62–3.34(m,7H,2-H,3-H,4-H,5-H,linker-OCH,linker-NCH2),2.46(s,1H,-OH),2.02–1.80(m,2H,linker-CH 2),1.31(d,J＝6.4Hz,3H,6-CH 3). 13C NMR(101MHz,Chloroform-d)δ138.00,128.58,128.56,128.50,128.26,128.08,127.95,127.89,102.12(1-C),78.48,77.36,75.94,72.96,70.84,67.24,64.70,50.94,43.74,28.58,16.91。HRMS(ESI)m/z calcd for C 31H 36N 4NaO 6[M+Na] +583.2527,found 583.2531。
实施例4：D-岩藻糖砌块合成方法的考察
方法一：
根据已有文献报道的类似化合物合成方法中总结得到合成D-岩藻糖砌块的方法1，如图4所示。以半乳糖烯糖S1作为起始原料，在二苯基二硒醚、三甲基硅烷基叠氮和二乙酰基碘苯的共同作用下，在其一号二号位分别引入硒苯基和叠氮基，得到重要的中间体S2，在甲醇甲醇钠作用下，脱去三四六号位的乙酰基得到化合S3，接着在四六位引入苄叉基得到化合物S4，再将三号位用乙酰基暂时保护起来得到化合物S5，然后在三甲基硅三氟甲磺酸和硼烷的共同作用下，将苄叉基选择性打开，将苄基引入到四号位，得到化合物S6，然后在裸露的六号位在对甲苯化酰氯和吡啶的作用下引入对甲苯磺酰基，得到化合物S7，然而在将六号位的对甲苯磺酰基用碘原子取代时，反应无法顺利进行。
发明人尝试了不同条件，包括：乙腈和TBAI在分别在100℃，80℃条件下以及丁酮和碘化钠在80℃的条件下进行反应，均未能在六号位引入碘，得到相应的产物S8，进而无法继续沿路线合成目标合成D-岩藻糖砌块。
方法二：
对方法一进行改进，改进之后的合成路线如图5所示。以全乙酰葡萄糖烯糖S9为起始原料，利用自由基加成反应，在一号二号位分别引入硒苯基和叠氮基，得到化合物S10，然后脱乙酰后得到化合物S11，紧接着四六位用苄叉基同时保护得到化合物S12，然后将三号位暂时用乙酰基保护后得到化合物S13，接下来在80％醋酸溶液中打开苄叉基得到化合物S14，在六号位在对甲苯磺酸和吡啶的条件下引入对甲苯磺酰基，得到化合物S15，然后在2-丁酮的回流溶液中与碘化钠反应，六号位引入碘，得到化合物S16。用氰基硼氢化钠还原的方法还原化合物S16的六号位羟基，使之还原成甲基，得化合物S17，然后在四号位先引入三氟甲磺酰基，得到化合物S18，再用亚硝酸钾还原，得到四号位竖直羟基的化合物S19，即成功将四号位平伏的羟基转变为竖直键的羟基，从而转变成D-岩藻糖构型，而后在氧化银的中性条件下，在四号位的羟基上引入苄基，得到化合物S20。
这种方法尽管能够得到所需的化合物S20，但是实用性不高，主要体现在路线步骤太长，共计有11个反应模块，尤其是在4-OH转位和最后一步利用Ag 2O上OBn基团两步反应产率都不足50％上，以至于该路线总产率不足2％。
实施例5：
二糖20的合成路线为图6。
如图6所示，二糖18的合成依靠先前由图1路线合成的葡萄糖化合物3和由图2路线合成的L-岩藻糖化合物7在路易斯酸TMSOTf的催化下完成。随后在甲醇溶液中利用氯化钯水 解掉二糖18还原端的烯丙基得到1-OH二糖化合物19。最后用2,2,2-三氟-N-苯基亚氨代乙酰氯将19做成含有N-苯基-三氟乙酰亚胺酯的二糖供体20。
具体试验操作和步骤：
化合物18：在氮气保护下将单糖供体3(500mg，1.0mmol)和糖基受体7(165mg，0.52mmol)溶于无水DCM(10ml)，加入
Figure PCTCN2019119584-appb-000006
分子筛。反应液在室温下搅拌30min，然后冷却至0℃，加入TMSOTf(36μL，0.2mmol)。反应液缓慢回复到室温并继续搅拌5h，TLC检测发现原料反应完全。反应液用三乙胺淬灭，用饱和碳酸氢钠溶液萃取，浓缩液经硅胶柱纯化(石油醚/乙酸乙酯，2:1)得到化合物18(300mg,0.46mmol,88％)。[α] D 20＝2.3°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.52–6.73(m,6H，Ar-H),5.84(ddt,J＝16.4,10.8,5.6Hz,1H，Ally-H),5.23(d,J＝17.3Hz,1H,Ally-H),5.17–5.05(m,3H,Ally-H,3’-H,4’-H),5.03(t,J＝8.2Hz,1H,2’-H),4.80(d,J＝11.3Hz,1H,Bn-H),4.77(d,J＝7.7Hz,1H,1’-H),4.46(d,J＝11.4Hz,1H,Bn-H),4.29(dd,J＝13.0,5.0Hz,1H,Ally-H),4.20(dd,J＝12.3,4.4Hz,1H,6’-Ha),4.17–4.08(m,2H,6’-Hb,1-H),4.00(dd,J＝13.0,6.2Hz,1H,Ally-H),3.66(qd,J＝10.3,4.9Hz,3H,5’-H,2-H,3-H),3.43(d,J＝2.5Hz,1H,4-H),3.35(d,J＝6.4Hz,1H,5-H),1.99(s,3H,CH 3CO),1.95(s,3H,CH 3CO),1.92(s,3H,CH3CO),1.79(s,3H,CH 3CO),1.10(d,J＝6.3Hz,3H,6-CH 3)。 13C NMR(101MHz,Chloroform-d)δ170.38,170.08,169.04,168.88,137.76,133.47,128.04,127.91,127.52,117.24,100.54,98.46,79.37,77.03,75.44,74.68,72.99,71.87,71.34,70.06,69.76,68.00,61.87,61.62,20.46,20.32,16.50。HRMS(ESI)m/z calcd for C 30H 39N 3NaO 13[M+Na] +672.2375,found 672.2377。
化合物19：将化合物18(300mg，0.46mmol)溶于无水甲醇(23ml)，随后将反应液加热到40℃。加入氯化钯(16mg)，继续搅拌1h，TLC检测原料反应完全。反应液用DCM稀释并用饱和碳酸氢钠萃取，分离有机相，浓缩后经硅胶柱(石油醚/乙酸乙酯，1:1)纯化得到化合物19(256mg，0.40mmol，88％)。[α] D 20＝-7.1°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)。-anomerδ7.35–7.22(m,5H,Ar-H),5.23(d,J＝3.5Hz,1H,1-H),5.22–5.00(m,3H,2’-H,3’-H,4’-H),4.86–4.72(m,2H,1’-H,Bn-H),4.43(d,J＝11.0Hz,1H,Bn-H),4.38(d,J＝7.8Hz,1H,1’-H),4.28(dd,J＝10.6,2.7Hz,1H,3-H),4.25–4.11(m,2H,6’-Hab),4.11–4.02(m,1H,5-H),3.75–3.65(m,2H,2-H,5’-H),3.61(d,J＝2.8Hz,1H,4-H),2.01(s,3H,OAc-Me),1.97(s,3H,OAc-Me),1.93(s,3H,OAc-Me),1.72(s,3H,OAc-Me),1.10(d,J＝6.4Hz,3H,6-Me)。-anomerδ7.35–7.22(m,5H,Ar-H),5.22–5.00(m,3H,2’-H,3’-H,4’-H),4.80(d,J＝11.5Hz,1H,Bn-H),4.47(d,J＝11.3Hz,1H,Bn-H),4.38(d,J＝7.8Hz,1H,1’-H),4.22–4.03(m,4H,6ab-H,1’-H,3-H),3.74–3.58(m,3H,2-H,4-H,5’-H),3.51–3.41(m,1H,5-H),2.01(s,3H,OAc-Me),1.97(s,3H,OAc-Me),1.94(s,3H,OAc-Me),1.72(s,3H,OAc-Me),1.10(d,J＝6.4Hz,3H,6-Me)。 13C NMR(101MHz,CDCl 3)δ170.79,170.69,170.43,170.38,169.33,169.29,169.15,169.05,137.87,128.44,128.39,128.36,128.32,128.24,128.14,128.12,128.00,127.93,102.18,101.52,98.74,98.67,98.65,96.22,92.72,79.54,79.42,79.37,77.37,77.25,77.14,77.05,76.87,76.73,76.48,75.73,75.52,75.17,75.03,75.00,74.96,74.06,73.33,73.23,73.20,72.13,72.10,71.97,71.82,71.56,70.69,70.62,70.40,68.25,68.20,66.62,63.71,63.53,62.05,61.99,61.78,58.93,43.75,29.68,20.71,20.69,20.66,20.58,20.56,20.54,16.78,16.67,16.61,16.57。HRMS(ESI)m/z calcd for C 27H 35N 3NaO 13[M+Na] +632.2062,found 632.2039。
化合物20：将化合物19(86mg，0.14mmol)溶于无水DCM中并冷却到0℃。在氩气保护下加入2,2,2-三氟-N-苯基亚氨代乙酰氯(0.1ml)和DBU(30μL)。反应液恢复到室温继续搅拌20h。在室温下浓缩后经由硅胶柱纯化(石油醚/乙酸乙酯，2.5:1)得到化合物20(67mg)。
实施例6：
三糖24的合成路线为图7。
如图7所示，二糖供体20和单糖受体17在二氯甲烷，乙醚和噻吩等溶剂的共同作用下由路易斯酸TMSOTf催化得到1,2-cis连接的三糖21。用硫代乙酸和吡啶在零度下将21的叠氮基还原成乙酰氨基化合物22。22葡萄糖基上的4个乙酰基在碱性条件下脱除得到化合物23。最后，用钯碳和氢气脱去所有的芳香基团得到组装有氨基连接臂的绿脓假单胞菌O11血清型O抗原三糖。
具体试验操作和步骤：
化合物21：在氩气保护下将二糖供体20(35mg)和单糖受体17(45mg)溶于无水DCM(0.9ml)、乙醚(0.9ml)和噻吩(0.9ml)的混合溶剂中，加入
Figure PCTCN2019119584-appb-000007
分子筛，在室温下搅拌30min，随后冷却至0℃。加入TMSOTf，继续搅拌8h，TLC检测显示糖基供体全部反应完毕。用三乙胺淬灭反应，用饱和碳酸氢钠萃取，有机相浓缩后经硅胶柱(石油醚/乙酸乙酯，3:1)和Sephadex LH-20凝胶柱(DCM/MeOH，1:1)纯化得到化合物21(37mg)。[α] D 20＝-44.5°(c＝1.00,CHCl 3)。 1H NMR(400MHz,Chloroform-d)δ7.41–7.19(m,20H,Ar-H),5.30–5.04(m,6H,2PhCH,1’-H,2”-H,3”-H,4”-H),4.83(d,J＝11.1Hz,1H,PhCH),4.77-4.75(m,3H,2PhCH,1”-H),4.55-4.50(m,2H,NPhCH),4.42(d,J＝11.0Hz,1H,PhCH),4.33–4.21(m,1H,6”-H),4.18(d,J＝7.0Hz,1H,1-H),4.07(m,2H,3’-H,6”-H),4.00–3.88(m,1H,linker-OCH),3.81(t,J＝9.0Hz,1H,2-H),3.79–3.67(m,2H,5’-H,5”-H),3.62(dd,J＝10.7,3.7Hz,1H,2’-H),3.53-3.26(m,6H,linker-OCH,linker-NCH 2,3-H,4H,4’-H),2.04(s,1H,COCH 3),2.00(s,1H,COCH 3),1.96–1.81(m,2H,linker-CH 2),1.80(s,1H,COCH 3),1.30(d,J＝6.7,3H,6-CH 3),1.10(d,J＝6.4Hz,3H,6’-CH 3)。 13C NMR(101MHz,CDCl 3)δ170.70,170.41,169.22,168.95,138.47,137.94,137.78,128.50,128.44,128.38,127.99,127.96,127.89,127.86,127.77,127.62,127.25,102.44,100.52,99.60,79.20,79.11,77.34,77.22,77.02,76.70,75.31,75.26,73.29,72.29,71.96,70.71,68.03,67.50,67.18,63.61,61.76,58.26,50.90,49.78,44.76,43.68,29.69,28.52,28.09,23.42,20.66,20.59,16.95,16.54。HRMS(ESI)m/z calcd for C 58H 69N 7NaO 18[M+Na] +1174.4591,found1174.4626。
化合物22：将化合物21(30mg)溶于干燥吡啶(0.5ml)和硫代乙酸(0.5ml)里。0℃下搅拌12h，用TLC检测原料反应完全，用甲苯与反应液共沸三次后用硅胶柱纯化(二氯甲烷/甲醇，50:1)得到化合物21(30mg)。[α] D 20＝49.5°(c＝1.00,CHCl 3). 1H NMR(400MHz,Chloroform-d)δ7.80–6.90(m,20H,Ar-H),6.83(d,J＝8.1Hz,1H,NHAc-H),6.18(d,J＝9.2Hz,1H,NHAc-H),5.27–5.13(m,3H,3”-H,2Cbz-H),5.10–4.99(m,2H,1’-H,4”-H),4.96(t,J＝8.7Hz,1H,2”-H),4.88(d,J＝11.1Hz,1H,Bn-H),4.84–4.79(m,1H,Bn-H),4.74(d,J＝15.5Hz,2H,Bn-H),4.63(d,J＝8.0Hz,3H,1”-H,3-H,2’-H),4.41(d,J＝10.9Hz,1H,Bn-H),4.34–4.25(m,2H,6”-H,1-H),4.25–4.13(m,1H,2-H),4.09(t,J＝9.0Hz,1H,6”-H),3.86(dtt,J＝34.7,10.8,6.0Hz,3H,5’-H,4-H,3’-H),3.66(dd,J＝9.7,6.0Hz,1H,5”-H),3.60–3.52(m,1H,3-H),3.44(d,J＝7.3Hz,2H,5-H,4’-H),3.34–3.08(m,1H,linker-OCH 2-H),2.87(dt,J＝14.3,5.0Hz,1H,linker-NH 2-H),2.14(s,3H,OAc-Me),2.09(s,3H,NHAc-Me),2.06(s,3H,OAc-Me),2.00(s,3H,OAc-Me),1.89(s,3H,NHAc-Me),1.80(s,3H,OAc-Me),1.68(d,J＝9.6Hz,1H),1.32(d,J＝6.3Hz,3H,6’-Me),1.17(d,J＝6.5Hz,3H,6-Me). 13C NMR(101MHz,Chloroform-d)δ171.62,171.05,170.77,170.42,169.28,168.83,156.56,138.81,138.17,137.98,137.35,136.86,128.66,128.55,128.51,128.47,128.36,128.17,128.09,127.96,127.91,127.80,127.76,127.71,127.65,127.50,127.28,127.19,126.35,101.24,100.77,99.67,80.00,79.66,78.94,77.83,77.23,75.05(d,J＝13.2Hz),73.18,72.20,72.07,70.73,68.21,67.52,67.33,65.76,61.95,52.22,49.70,47.60,42.47,31.92,29.69,26.87,23.33,20.84,20.58,20.56,17.24,16.73。
HRMS(ESI)m/z calcd for C 58H 69N 7NaO 18[M+Na] +1206.4998。
化合物23：将化合物22(22mg)溶解于甲醇中(5ml)并加入甲醇钠(10mg)。反应液在室温下搅拌2h，用TLC检测原料反应完全，用氢型Amberlite IR120树脂中和反应和至～7。过滤树脂，反应液浓缩后经硅胶柱纯化(二氯甲烷：甲醇，10:1)得到化合物23(18mg)。 [α] D 20＝-16.3°。 1H NMR(400MHz,Chloroform-d)δ7.60–7.13(m,20H),7.12(d,J＝7.3Hz,1H,NHAc-H),6.69(d,J＝9.8Hz,1H,NHAc-H),5.23(d,J＝12.6Hz,1H,Cbz-H),5.15(d,J＝12.4Hz,1H,Bn-H),5.05(d,J＝12.3Hz,1H,Cbz-H),4.96–4.83(m,2H,1’-H,Bn-H),4.79(d,J＝15.7Hz,1H,Bn-H),4.72(d,J＝12.2Hz,1H,Bn-H),4.66–4.49(m,2H,Bn-H,2’-H),4.36(s,1H,OH-H),4.28(d,J＝9.5Hz,1H,2A-H),4.22(d,J＝16.0Hz,1H,Bn-H),4.03(d,J＝7.3Hz,2H,1”-H,NCH 2-H),4.00–3.84(m,4H,3’-H,1-H,6”-H,OCH 2-H),3.72(dd,J＝12.5,6.1Hz,1H,6”-H),3.64(q,J＝6.7Hz,1H,5-H),3.42(ddd,J＝26.0,12.9,5.7Hz,4H,5-H,2”-H,3”-H,4”-C),3.33(d,J＝4.6Hz,1H,3-H),3.31–3.19(m,2H,4’-H,5”-C),3.15(td,J＝9.7,4.0Hz,1H,OCH 2-H),2.88–2.74(m,2H,NH 2-H),2.09(s,3H,NHAc-Me),1.96(s,3H,NHAc-Me),1.72–1.57(m,2H,linker-CH 2)，1.32(s,J＝6.4,3H,6-Me),1.03(s,J＝6.3,3H,6-Me)。 13C NMR(101MHz,CDCl3)δ172.97,172.65,156.77,138.85,138.64,137.11,136.82,128.75,128.60,128.38,128.14,128.05,127.64,127.50,127.34,127.26,127.07,101.41,99.87,82.13,79.54,77.36,77.04,76.72,76.31,75.31,74.62,74.34,74.14,72.82,71.70,70.92,67.67,67.56,65.55,62.61,52.00,49.50,48.11,42.01,29.72,26.65,23.53,17.50,16.75。HRMS(ESI)m/z calcd for C 62H 77N 3NaO 20[M+Na] +1038.4570,found 1038.4538。
化合物24：将化合物23(12mg)溶于甲醇(3ml)、二氯甲烷(1ml)和水(1ml)的混合溶剂，并加入两滴冰乙酸，随后加入钯碳(10mg)。反应在BLT-2000中压氢化仪中进行，氢气压力为0.4MPa，反应时间12h。待反应结束后反应液用硅藻土过滤，浓缩后经CHROMAFIX C 18ec纯的得到化合物24(7mg)。 1H NMR(400MHz,Deuterium Oxide)δ5.03(s,1H,1’-H),4.51(d,J＝7.9Hz,1H,1”-H),4.37(d,J＝8.4Hz,1H,1-H),4.17(s,1H,2’-H),4.07(q,J＝6.6Hz,1H,5’-H),4.02(s,1H,3’-H),3.99–3.86(m,4H,2-H,4’-H,6”-H,OCH2-H),3.80–3.75(m,1H,5-H),3.67(ddt,J＝16.6,10.9,6.0Hz,3H,3-H,6”-H,OCH2-H),3.46(m,2H,3”-H,5”-H),3.34(t,J＝9.3Hz,1H,4”-H),3.26(t,J＝8.6Hz,1H,2”-H),3.05(t,J＝6.9Hz,2H,NCH 2-H),2.00(s,3H,NHAc-Me),1.98(s,3H,NHAc-Me),1.91(t,J＝6.4Hz,2H,CH 2-H),1.23(d,J＝6.9Hz,3H,6-Me),1.21(d,J＝6.8Hz,3H,6’-Me)。 13C NMR(101MHz,Deuterium Oxide)δ174.35,101.49,100.22,98.89,76.54,76.00,75.46,74.78,72.81,71.31,70.63,70.33,69.63,69.60,68.39,67.88,66.88,60.84,51.31,47.88,37.60,26.64,,22.20,22.17,15.39,15.29。HRMS(ESI)m/z calcd for C 25H 45N 3NaO 14[M+Na] +634.2794,found 634.2789。
实施例7：考察溶剂对三糖24中顺式糖苷键构建的影响
利用糖环C-2位酰基辅助下的邻基参与效应，高效率的构建1,2-反式糖苷键已成为糖化学中较为成熟的方法，而2-位乙酰基作为糖化学保护策略中最易得、最有效的保护剂，一般被优先考虑，例如在本专利中化合物18的合成。然而对于1,2-顺式反应，目前还没有成熟的方法。参照实施例6，其他条件不变，改变溶剂组分，产物构型结果见表1。
表1 不同溶剂构建糖苷键的结果
Figure PCTCN2019119584-appb-000008
由表1可知，1,2-顺式糖苷键构建受溶剂影响很大，其中以无水二氯甲烷/乙醚/噻吩混合溶剂效果最佳，可以达到均一α构型产物，反应立体选择性可达100％。
实施例8：乙酰基组装时机对于糖链合成的影响
分别在单糖阶段对乙酰氨基进行组装，得到乙酰氨基二糖供体S21和乙酰氨基受体S22。以此开展糖苷化反应未能顺利完成目标三糖S23的合成，分析原因为供体二糖还原端2号位乙酰氨基被活化后形成稳定的噁唑啉环中间体，限制了糖苷化反应的进行。上述结果表明以非参与性的叠氮基作为乙酰氨基前体，将有利于提高目标寡糖合成的反应性和立体选择性。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以 权利要求书所界定的为准。

Claims (10)

  1. 一种绿脓假单胞菌O11血清型O抗原三糖的合成方法，其特征在于，所述方法包括：利用D-葡萄糖砌块、L-岩藻糖胺砌块、D-岩藻糖胺砌块构建O抗原三糖；其中，D-葡萄糖砌块或L-岩藻糖胺砌块与D-岩藻糖胺砌块通过1,2-α-顺式-糖苷键连接，D-葡萄糖砌块与L-岩藻糖胺砌块通过1,2-β-反式糖苷键连接，所述1,2-α-顺式-糖苷键的构建是在混合溶剂中进行的；所述混合溶剂包括二氯甲烷、乙醚、噻吩中的两种或两种以上。
  2. 根据权利要求1所述的方法，其特征在于，所述D-葡萄糖砌块的化学结构如式II所示，L-岩藻糖胺砌块的化学结构式如式III所示，D-岩藻糖胺砌块的化学结构式如式IV所示，
    Figure PCTCN2019119584-appb-100001
    其中，linker包括-(CH 2) n-N-Y 1Y 2，或者O-(CH 2) n-SY 1(Y 2)，或者O-(CH 2) n-N 3，n＝1～10；Y 1为氢(H)或者苄基(Bn)；Y 2为氢(H)或者苄甲氧羰基(Cbz)；
    R 1包括但不限定于氢(H)、酯基、乙酰基(Ac)、苯甲酰基(Bz)、新戊酰基(Piv)、氯乙酰基(ClAc)、乙酰丙酰基(Lev)、烯丙羰酰基(Alloc)；
    R 2，R 3，R 4为氢(H)或者酯基、醚基包括但不限于乙酰基(Ac)、苯甲酰基(Bz)、新戊酰基(Piv)、氯乙酰基(ClAc)、乙酰丙酰基(Lev)、烯丙羰酰基(Alloc)以及苄基(Bn)、对甲氧基苄基(pMBn)、烯丙基(All)、三苯甲基(Tr)、单甲氧基三苯甲基(Mmt)和硅醚类基团基团；
    R 5，R 6包括但不限于氢(H)、醚类基团、苄基(Bn)、对甲氧基苄基(pMBn)、烯丙基(All)、三苯甲基(Tr)、单甲氧基三苯甲基(Mmt)和硅醚类基团基团；
    R 7，R 8，R 9，R 10包括氢(H)、氮(N)或者乙酰基(Ac)。
  3. 根据权利要求1或2所述的方法，其特征在于，所述方法包括利用D-葡萄糖砌块和L-岩藻糖胺砌块合成二糖片段，其化学结构如式V所示，
    Figure PCTCN2019119584-appb-100002
  4. 根据权利要求3所述的方法，其特征在于，所述方法包括利用二糖片段与D-岩藻糖胺砌块合成三糖片段，其化学结构式如式I所示，
    Figure PCTCN2019119584-appb-100003
  5. 根据权利要求1所述的方法，其特征在于，所述方法包括预先合成O抗原三糖前体，还原即得O抗原三糖，所述O抗原三糖前体的化学结构式为式VI，
    Figure PCTCN2019119584-appb-100004
  6. 根据权利要求1-5任一所述的方法，其特征在于，所述方法是利用葡萄糖合成得到D-岩藻糖胺，包括：以3,4,6-O-三乙酰基甘露糖烯为原料，经过叠氮化物、硒试剂得到1-硒苯-2叠氮葡萄糖，脱除乙酰基，然后6-C进行甲基化，最终得到D-岩藻糖胺类化合物。
  7. 一种组装有氨基连接臂的绿脓假单胞菌的O11血清型O抗原三糖化合物，其特征在于，所述化合物是利用权利要求1-6任一所述方法制备得到的。
  8. 一种糖-蛋白缀合物的制备方法，其特征在于，所述方法是包含绿脓假单胞菌的O11血清型O抗原三糖，所述绿脓假单胞菌的O11血清型O抗原三糖的制备方法为权利要求1-6任一所述的方法。
  9. 权利要求1-6所述合成方法在开发或制备绿脓假单胞菌疫苗或者绿脓假单胞菌感染导致的疾病的药物中的应用。
  10. 权利要求7所述化合物或者权利要求8所述的糖-蛋白缀合物的制备方法在开发或制备绿脓假单胞菌疫苗或者绿脓假单胞菌感染导致的疾病的药物中的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107709343A (zh) * 2015-06-08 2018-02-16 马克斯·普朗克科学促进学会 针对肺炎链球菌血清型5的疫苗
CN108558961A (zh) * 2018-01-29 2018-09-21 江南大学 类志贺邻单胞菌o51血清型o抗原寡糖化学合成方法
CN109627270A (zh) * 2018-12-18 2019-04-16 江南大学 一种绿脓假单胞菌o11血清型o抗原寡糖的化学合成方法

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Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107709343A (zh) * 2015-06-08 2018-02-16 马克斯·普朗克科学促进学会 针对肺炎链球菌血清型5的疫苗
CN108558961A (zh) * 2018-01-29 2018-09-21 江南大学 类志贺邻单胞菌o51血清型o抗原寡糖化学合成方法
CN109627270A (zh) * 2018-12-18 2019-04-16 江南大学 一种绿脓假单胞菌o11血清型o抗原寡糖的化学合成方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D'AMBRA, A. J. ET AL.: "Analysis by the Reductive-cleavage Method of a Polysaccharide Containing 2-acetamido-2，6-dideoxy-D and -L-galactopyranosyl Residues", CARBOHYDRATE RESEARCH, vol. 251, 3 January 1994 (1994-01-03), XP026661031, ISSN: 0008-6215, DOI: 20200108151350Y *
QIN, CHUNJUN ET AL.: "Total Synthesis of a Densely Functionalized Plesiomonas Shigelloides Serotype 51 Aminoglycoside Trisaccharide Antigen", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 140, no. 8, 29 January 2018 (2018-01-29), XP055627596, ISSN: 1520-5126, DOI: 20200108152211Y *

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