WO2025232105A1 - 一种绿脓杆菌o5血清型o-抗原寡糖的化学合成方法 - Google Patents

一种绿脓杆菌o5血清型o-抗原寡糖的化学合成方法

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WO2025232105A1
WO2025232105A1 PCT/CN2024/127397 CN2024127397W WO2025232105A1 WO 2025232105 A1 WO2025232105 A1 WO 2025232105A1 CN 2024127397 W CN2024127397 W CN 2024127397W WO 2025232105 A1 WO2025232105 A1 WO 2025232105A1
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mmol
arch
compound
reaction
pseudomonas aeruginosa
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French (fr)
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尹健
胡静
田光宗
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Jiangnan University
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Jiangnan University
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    • 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
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • 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
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • 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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to a chemical synthesis method for oligosaccharides of O5 serotype O-antigen from Pseudomonas aeruginosa, belonging to the field of chemical synthesis.
  • Pseudomonas aeruginosa also known as Pseudomonas aeruginosa
  • Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen and one of the main causes of hospital-acquired infections.
  • Pseudomonas aeruginosa infection can cause a series of complications such as sepsis and septicemia, posing a potentially fatal risk to immunocompromised individuals, including those with cystic fibrosis, HIV/AIDS, and those undergoing chemotherapy (K. Grimwood et al., Hum Vaccin Immunother 2015, 11, 14-20).
  • Pseudomonas aeruginosa According to the US Centers for Disease Control and Prevention (CDC), approximately 51,000 patients are infected with Pseudomonas aeruginosa annually, with a mortality rate of about 5.3% (M.E. Chirgwin et al., Materials 2019, 12, 4093). Pseudomonas aeruginosa exhibits strong resistance to multiple antibiotics (C.K. Elogne et al., Afr J Microbiol Res, 2018, 12, 62-67). In 2017, the World Health Organization (WHO) listed Pseudomonas aeruginosa as a "highest priority" pathogen in its first list of antibiotic-resistant pathogens.
  • WHO World Health Organization
  • Pseudomonas aeruginosa resistance has become one of the greatest threats to human health today (E. Tacconelli et al., World Health Organization, 2017). Since immunization strategies are not affected by antibiotic resistance mechanisms, the development of highly effective and safe vaccines for the treatment of drug-resistant pathogens is urgently needed.
  • researchers have conducted extensive studies on Pseudomonas aeruginosa vaccines, mainly including whole-cell vaccines and various subunit vaccines, but no vaccines currently available for human use have been approved for marketing (G.P. Priebe et al., Expert Rev Vaccines, 2014, 13, 507-519).
  • subunit vaccines containing Pseudomonas aeruginosa cell surface polysaccharides and membrane proteins can provide more effective immune protection.
  • LPS bacterial surface lipopolysaccharide
  • the O-antigen polysaccharide structure of serotype O5 has attracted attention due to its high structural specificity.
  • the polysaccharide structure consists of a complex modified polyaminotrisaccharide repeating unit: [ ⁇ 4)- ⁇ -D-ManpNAc3AmA-(1 ⁇ 4)- ⁇ -D-ManpNAc3AcA-(1 ⁇ 3)- ⁇ -D-FucpNAc-(1 ⁇ ] (I.Sadovskaya et al., Eur. J. Biochem. 1997, 255, 673-684).
  • polysaccharides with two consecutive ⁇ -D-ManpN3NA segments are uncommon in nature and have not been synthesized before.
  • the trisaccharide repeating segment are all linked by 1,2-cis-glycosidic bonds.
  • the O-antigen contains a unique functional group, the acetamidine (Am), which specifically attaches to the amino group at the C3 position of the 2,3-diamino-D-mannitol acid in the O-antigen trisaccharide.
  • This invention relates to the synthesis of a trisaccharide fragment of the O-antigen of Pseudomonas aeruginosa O5 serotype using three glycoblock chemical methods.
  • the fragment primarily comprises a 3-amino-D-glucose block, a D-glucuronic acid block, a D-fucoglycosamine block, and orthogonal modifications of 1,2- ⁇ -cis-glycosidic bonds, 1,2- ⁇ -cis-glycosidic bonds, and amino functional groups.
  • the 1,2-cis-glycosidic bond structure... Orthogonal modification of the amino functional groups is a key step in constructing the target trisaccharide.
  • This invention successfully solves the stereoselectivity problem in constructing 1,2- ⁇ -cis-glycosidic bonds through the synergistic effect of acyl long-range participation and additive effects.
  • Two stereochemical constructions of 1,2- ⁇ -cis-glycosidic bonds are achieved through SN2 nucleophilic substitution of the C2-position azido group.
  • the target trisaccharide (as shown in Formula VI) is obtained through C6-position hydroxyl oxidation and orthogonal modification with different amino functional groups, followed by deprotection.
  • This invention immobilizes the synthesized oligosaccharide on the chip surface via amino linkers and uses Pseudomonas aeruginosa patient serum to screen for oligosaccharide antigens for antigenicity studies of the synthesized oligosaccharide.
  • the first objective of this invention is to provide a method for synthesizing the O-antigen trisaccharide of O5 serotype Pseudomonas aeruginosa.
  • the method includes: constructing the O-antigen trisaccharide using 3-amino-D-glucose building blocks, D-glucuronic acid building blocks, and D-fucosamine building blocks; constructing a 1,2- ⁇ -cis-D-fucosamine glycosidic bond using the synergistic effect of acyl long-range participation and additive effects; constructing a 1,2- ⁇ -trans-glycosidic bond of two 2,3-di-amino-D-mannuronic acids using an SN2 substitution reaction of the C2-position azide group; and completing the orthogonal assembly of the five amino functional modifying groups in the O-antigen trisaccharide.
  • This invention relates to an oligosaccharide fragment of Pseudomonas aeruginosa O5 serotype O-antigen trisaccharide assembled with a linker arm, the chemical structure of which can be represented by general formula (VI):
  • Linker is -L-NH 2 , where L represents the connecting arm
  • the connecting arm L can be a chain structure containing 2-40 carbon atoms (including the carbon atoms of the side chain) with 0-6 heteroatoms.
  • the chain when the main chain length of the connecting arm is 4-8 atoms, the chain may contain 1, 2 or 3 heteroatoms (O, N and S). When the main chain length of the connecting arm is 9-14 atoms, the chain may contain 1, 2, 3, 4, 5 or 6 heteroatoms (O, N and S).
  • the linker arm -L- can be a cyclic structure that is wholly or partially fluorinated.
  • the linker arm -L- can contain a three-, four-, five-, or six-membered saturated carbon ring; it can also contain a five-membered unsaturated carbon ring (non-aromatic ring); it can also contain a four-, five-, or six-membered saturated oxygen heterocycle; it can also contain a four-, five-, or six-membered saturated nitrogen heterocycle; or it can contain a six-membered aromatic carbon ring.
  • the connecting arm -L- may also contain an amide bond and/or a urea group.
  • the connecting arms -L- may also contain one or more substituents, which may include: -F, -Cl, -CH3 , -C2H5, -C3H7 , -C5H9 , -C6H13 , -OCH3 , -OC2H5 , -CH2F , -CHF2 , -CF3 , -C( O ) -NH2 , -SCH3 , -SC2H5 , -NHC (O ) CH3 , -N ( CH3 ) 2 and -N ( C2H5 ) 2 .
  • substituents may include: -F, -Cl, -CH3 , -C2H5, -C3H7 , -C5H9 , -C6H13 , -OCH3 , -OC2H5 , -CH2F , -CHF2 , -CF3 , -C
  • the sugar chain structure synthesized in this invention contains basic (ethyl) and acidic (carboxyl) groups, which can form corresponding salts with organic or inorganic acids or bases.
  • the sugar chain structure synthesized in this invention contains both basic (ethyl) and acidic (carboxyl) groups, and can also be transferred through intramolecular proton migration, i.e., the proton of the acidic group is transferred to the basic group.
  • the general formula can be an amphoteric molecule containing -O- and -NH3 + .
  • This invention provides a chemical synthesis method for modifying the linker arm of a polysaccharide linker arm of Pseudomonas aeruginosa O5 serotype O-antigen (as shown in general formula VI), using three monosaccharide building blocks A, B, and C as raw materials, as shown in formulas (I) to (III), respectively:
  • PG 1 is H
  • PG 2 , PG 3 , PG 4 and PG 6 are temporary hydroxyl protecting groups, each independently selected from: acetyl (Ac), or benzoyl (Bz), or neopentyl (Piv), or chloroacetyl (ClAc), or acetylpropionyl (Lev), or 9-fluorenylmethoxycarbonyl (Fmoc), or allyloxycarbonyl (Alloc), or 2-naphthylmethyl (Nap), or p-methoxybenzyl (pMB), or allyl (All), or tert-butyldimethylsilyl (TBS), or triethylsilyl (TES);
  • PG 5 is a carboxyl protecting group, which can be benzyl (Bn), methyl (Me), ethyl (Et), tert-butyl (tBu), or allyl (All);
  • PG 7 is a temporary protecting group for the amino group, which can be trichloroethoxycarbonyl (Troc), phthaloyl (Phth), 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), etc.
  • PG 8 and PG 9 are hydroxyl protecting groups, which can be benzene methylene (PhCH), naphthyl methylene (NapCH), isopropyl ketal ((CH 3 ) 2 CH), etc.;
  • PG 10 and PG 11 are amino protecting groups, which can be benzyl (Bn) or benzyloxycarbonyl (Cbz);
  • the process includes the following:
  • the synthetic route of the method is as follows:
  • This invention utilizes D-glucuronic acid building blocks and D-fucosamine building blocks to construct O-antigen trisaccharides.
  • the stereoselective synthesis of the D-fucosamine 1,2- ⁇ -cis-glycosidic bond depends on the participation of a long-range acyl group and reagent regulation.
  • the synthesis of the two 2,3-diaminomannuronic acid 1,2- ⁇ -trans-glycosidic bonds is achieved through SN2 nucleophilic substitution of the C2-position azide group.
  • the method specifically includes: using 3,4-O-diacetylfucose as a raw material, reacting it with trimethyl azidosilane (TMS- N3 ) and diphenyldiselene ( Ph2Se2 ) under the action of diacetic acid iodobenzene (PhI(OAc ) 2 ) to generate 1-selenobenzene-2-azidofucose, then removing the acetyl group under alkaline conditions, selectively protecting the C3 hydroxyl group to 3-ONap, and protecting the C4 hydroxyl group with benzoyl group, and finally obtaining a highly stereoselective 1,2- ⁇ -cis-D-fucosamine glycosidic bond under the synergistic effect of additive effect ( Ph3PO ) and acyl group long-range participation effect (Bz).
  • TMS- N3 trimethyl azidosilane
  • Ph2Se2 diphenyldiselene
  • the method further includes: using glucose as a raw material, obtaining a C3-amino precursor by nucleophilic substitution of the C3-position azido group mediated by trifluorosulfonyl group, and similarly using SN2 nucleophilic substitution of the C2-position azido group for the C2-position amino precursor, thereby completing the efficient construction of two 2,3-diaminomannuronic acid 1,2- ⁇ -trans-glycosidic bonds.
  • the Lewis acid includes boron trifluoride ether ( BF3 ⁇ Et2O ), trifluoromethanesulfonic acid (TfOH), trimethylsilyl trifluoromethanesulfonate (TMSOTf), or silver salt silver trifluoromethanesulfonate (AgOTf), etc., and the thioglycoside is reacted by adding NIS together with the Lewis acid as a promoter.
  • the glycosylation reaction in step (1) is carried out in an environment of activating reagent Lewis acid, solvent, and molecular sieve.
  • the Lewis acid used as the activating agent for the glycosylation reaction in step (1) is boron trifluoride diethyl ether ( BF3 ⁇ Et2O ), trifluoromethanesulfonic acid (TfOH), trimethylsilyl trifluoromethanesulfonate (TMSOTf), or silver trifluoromethanesulfonate silver (AgOTf), etc., any one or more of these.
  • the glycosylation reaction in step (2) is carried out in an environment containing Lewis acid as an activating reagent, NIS (iodosuccinimide), solvent, and molecular sieve.
  • the amount of Lewis acid used as the activating agent for the glycosylation reaction in step (2) is 0.1 to 1 eq, and the amount of NIS is 1.2 to 1.5 eq.
  • the Lewis acid used as the activating agent for the glycosylation reaction in step (2) is any one or more of boron trifluoride diethyl ether ( BF3 ⁇ Et2O ), trifluoromethanesulfonic acid (TfOH), trimethylsilyl trifluoromethanesulfonate (TMSOTf), or silver trifluoromethanesulfonate silver (AgOTf).
  • the purpose of this invention is to provide a method for synthesizing a Pseudomonas aeruginosa O5 serotype O-antigen trisaccharide assembled with an amino linker arm.
  • the O5 serotype O-antigen oligosaccharide fragment prepared by the above method may be used in the development of Pseudomonas aeruginosa vaccines or Pseudomonas aeruginosa infectious drugs, etc.
  • This invention presents for the first time a chemical synthesis method for the O-antigen trisaccharide of Pseudomonas aeruginosa O5 serotype.
  • the method utilizes D-glucuronic acid and D-fucosamine building blocks to construct the O-antigen trisaccharide.
  • the stereoselective synthesis of the D-fucosamine 1,2- ⁇ -cis-glycosidic bond depends on the participation of a long-range acyl group and reagent regulation.
  • the synthesis of the two 2,3-diaminomannuronic acid 1,2- ⁇ -trans-glycosidic bonds is achieved through SN2 nucleophilic substitution of the C2-position azido group.
  • Figure 1 shows the trisaccharide repeat unit of the O-antigen of Pseudomonas aeruginosa O5 serotype.
  • Figure 2 shows compounds A, B, and C as represented by general formulas I, II, and III.
  • FIG. 3 shows compound F as shown in general formula VI.
  • Figure 4 shows the synthesis of sugar block 9.
  • Figure 5 shows the synthesis of sugar block 16.
  • Figure 6 shows the synthesis of sugar block 19.
  • Figure 7 shows the synthesis of trisaccharide 20.
  • Figure 8 shows the synthesis of the target trisaccharide 30.
  • Figure 9 shows an attempt to synthesize trisaccharide 35.
  • Figure 10 shows an attempt to synthesize trisaccharide 38.
  • Figure 11 shows an attempt to synthesize trisaccharides 43 and 44.
  • the yield calculation method of this invention is "product (mol) / reaction substrate (mol) * 100%".
  • the methods for identifying the compound structures in this invention include nuclear magnetic resonance (NMR) spectroscopy (400MHz, 600MHz), high-resolution mass spectrometry, optical rotation determination, and infrared spectroscopy. The results are listed in the specific synthesis of each compound.
  • the 2-O-Nap group in compound 13 was removed using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and its acetylpropionyl group was protected to obtain compound 5.
  • DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
  • the 4,6-O-benzyl group was hydrolyzed using 80% acetic acid solution to obtain diol 6.
  • the C6 hydroxyl group was oxidized using TEMPO/BAIB, followed by benzyl esterification of the carboxylic acid to obtain compound 7.
  • the temporary protecting group tert-butyldimethylsilyl (TBS) was assembled at the C4 hydroxyl group to obtain compound 8.
  • TBS tert-butyldimethylsilyl
  • Compound 2 Compound 1 (6.6 g, 16.01 mmol) was dissolved in methanol (40 mL) solution by stirring, and NaOMe (0.43 g) was added. The reaction was stirred at room temperature for 5 h, then the solution was neutralized with Amberlite IR 120 (H + ) ion exchange resin and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (PE/EA v/v 2:1) to obtain the corresponding diol compound. The above compound was azeotropically reacted with anhydrous toluene and dried under high vacuum for 30 min.
  • Compound 5 Compound 4 (2.77 g, 8.32 mmol) was dissolved in a dry DCM (40 mL) solution, and levulinic acid (LevOH) (1.45 g, 12.48 mmol), N,N-dicyclohexylcarbodiimide (DCC) (2.57 g, 12.48 mmol) and DMAP (1.52 g, 12.48 mmol) were added. The mixture was stirred at room temperature for 4 h until the starting material was completely consumed, and the reaction solution was diluted with DCM. The organic phase was washed with saturated NaHCO3 solution and saturated NaCl solution, and then back-extracted with DCM. The organic phase was then dried over anhydrous Na2SO4 and concentrated under vacuum.
  • LevOH levulinic acid
  • DCC N,N-dicyclohexylcarbodiimide
  • DMAP DMAP
  • Compound 7 Compound 6 (2.64 g, 7.70 mmol) was dissolved in DCM (77 mL) and H2O (15 mL) solution under argon protection. The system was cooled to 0 °C using an ice bath, and 2,2,6,6-tetramethylpiperidine oxide (TEMPO) (240 mg, 1.54 mmol) and diacetoxyiodobenzene (BAIB) (4.96 g, 15.4 mmol) were added. The mixture was stirred at room temperature for 4 h . The reaction progress was monitored by TLC until the starting material was completely consumed.
  • TEMPO 2,2,6,6-tetramethylpiperidine oxide
  • BAIB diacetoxyiodobenzene
  • the reaction solution was diluted with ethyl acetate (50 ml), and the organic phase was washed with saturated NaHCO3 solution and saturated NaCl solution, followed by DCM back-extraction. The organic phase was then dried over anhydrous Na2SO4 and concentrated under vacuum.
  • the crude product was purified by silica gel column chromatography (PE/EA v/v 10:1) to obtain colorless syrup 8 (1.73 g, 3.09 mmol, 89%).
  • dilute sugar 10 was obtained through acetylation, bromination, and elimination reactions. Subsequently, an azidization reaction was carried out under the action of diphenyldiselenylation ( Ph2Se2 ) and trimethylsilyl azide (TMSN3 ) to give compound 11 with an azide group at the C2 position . Then, deacetylation was performed under the action of sodium methoxide, followed by selective 2-naphthylmethyleneization on O3 to give compound 13.
  • Ph2Se2 diphenyldiselenylation
  • TMSN3 trimethylsilyl azide
  • Compound 11 Compound 10 (3.7 g, 17.3 mmol) was dissolved in dry DCM (86 mL), followed by the addition of diphenyldiselenoether (5.4 g, 17.3 mmol). The solution was cooled to -30 °C under argon atmosphere, and then BAIB (5.6 g, 17.3 mmol) and azidotrimethylsilane (3.98 g, 34.6 mmol) were added. The reaction progress was monitored by TLC until the starting material was exhausted. The mixture was washed with saturated NaHCO3 (3 ⁇ 100 mL) and back-extracted from DCM (3 ⁇ 50 mL). The organic phase was then dried over Na2SO4 and concentrated under vacuum.
  • Compound 12 Compound 11 (4.6 g, 11.1 mmol) was dissolved in methanol (55 mL), and NaOMe (0.30 g, 5.55 mmol) was added. The solution was stirred at room temperature for 5 h. The solution was neutralized with Amberlite IR 120 (H + ) ion exchange resin and then filtered through a cotton plug. The filtrate was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol v/v 100:1) to give product 12 (3.32 g, 10.1 mmol, 91%).
  • Compound 14 Compound 13 (127 mg, 0.27 mmol) was dissolved in anhydrous DMF (3 mL) and NaH (22 mg, 0.54 mmol, 60% dispersible in mineral oil) was added. The mixture was stirred at 0 °C for 10 min. Then, BnBr (50 ⁇ L, 0.41 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was added dropwise. The reaction mixture was then washed with water and brine, and back-extracted using DCM.
  • the benzoyl group was chosen as the temporary protecting group at the O4 position (Bz) because the ⁇ -configuration isomer of compound 15c was easier to isolate and had a higher yield (91%). Finally, the benzoyl group of compound 15c was removed using sodium methoxide, followed by benzylation to obtain compound 15.
  • This hemiacetal was dissolved in DCM (3 mL).
  • the reaction system was cooled to 0 °C, and 2,2,2-trifluoro-N-phenylacetylimine chloride (230 ⁇ L, 1.55 mmol) and DBU (140 ⁇ L, 0.93 mmol) were added.
  • the reaction system was then heated to room temperature and stirred for 3 hours.
  • the reaction progress was monitored and analyzed by TLC until the raw materials were completely consumed.
  • the reaction solution was concentrated under reduced pressure at room temperature to obtain a crude product, which was then purified by silica gel column chromatography (PE/EA v/v 20:1 ⁇ 10:1) to obtain a pale yellow syrup 14a (153 mg, 0.26 mmol, 83%).
  • Donor 14a (20 mg, 0.034 mmol) and the linker N-benzyl-N-benzyloxycarbonyl-3-aminopropanol (15 mg, 0.051 mmol) were azeotropically dissolved in anhydrous toluene, and then dissolved in dry DCM (1 mL) under argon protection. Add [the following to the reaction flask]... Molecular sieves (flame-dried) and Ph3OP (67 mg, 0.24 mmol) were used. Then, TMSI (5 ⁇ L, 0.034 mmol, 1.0 eq) was slowly added dropwise to the reaction flask. The reaction was stirred at room temperature until TLC monitoring showed completion.
  • Compound 15 A solution of compound 15c (1.82 g, 2.55 mmol) dissolved in methanol (25 mL) was added to a solution containing NaOMe (70 mg, 1.28 mmol), and the solution was stirred at 40 °C for 6 h. The solution was neutralized with Amberlite IR 120 (H + ) ion exchange resin and filtered through a cotton plug. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography (PE/EA v/v 5:1 ⁇ 2:1) to obtain the corresponding product. The product was dissolved in DMF (13 mL), and NaH (60% dispersion in mineral oil) (205 mg, 5.10 mmol) was added.
  • the C3-position azide group was reduced with lithium aluminum hydride to obtain the corresponding amino sugar. Then, the C3-position amino group was selectively protected with a trichloroethoxycarbonyl (Troc) group to obtain compound 18. Subsequently, the C2-position hydroxyl group in compound 18 was protected with an acetylpropionyl group to obtain compound 19.
  • Troc trichloroethoxycarbonyl
  • Compound 18 Compound 17 (1.6 g, 4.01 mmol) was placed in a 20 mL THF solution at 0 °C, and LiAlH4 (460 mg, 12.03 mmol) was added. The reaction mixture was stirred at room temperature for 1.5 h until TLC showed complete conversion of the starting material. Then, H2O was added dropwise at 0 °C to quench the reaction, and the mixture was diluted with DCM. The reaction mixture was washed with H2O , saturated NaHCO3 solution, and brine. The organic phase was dried over anhydrous Na2SO4 , filtered, and concentrated under vacuum.
  • the crude product was purified by silica gel column chromatography (DCM/MeOH v/v 50:1 ⁇ 20:1) to obtain amino sugars.
  • the amino sugars were dissolved in THF (35 mL) and pyridine (0.97 mL, 12.03 mmol, 3 eq).
  • a mixture of THF (5 mL) containing TrocCl (0.55 mL, 4.01 mmol) was added dropwise at 0 °C.
  • the reaction was stirred at 0°C for 2 h until TLC showed complete conversion of the starting material.
  • the reaction was then quenched with methanol at 0°C.
  • reaction solution was concentrated under vacuum to obtain a crude product, which was purified by silica gel column chromatography (PE/EA v/v 5:1 ⁇ 2:1) to give a colorless syrupy product 18 (1.86 g, 3.41 mmol, 85%).
  • glycosylation was performed using glycosyl donor 9 and acceptor 16 as raw materials under the action of the activating reagent TMSOTf to obtain...
  • Disaccharide compound 20 was obtained.
  • the TBS at the C4 position of the disaccharide was removed using tetrabutylamine fluoride (TBAF) to obtain disaccharide acceptor 21.
  • monosaccharide donor 19 and disaccharide acceptor 21 were glycosylated under the catalysis of TMSOTf and NIS to obtain trisaccharide intermediate compound 22.
  • trisaccharide 28 containing an acetamidine group at the C3” position. Due to the increased polarity of trisaccharide 26, it was difficult to dissolve in dichloromethane but readily soluble in methanol. It was found that during dissolution, transfer, and storage in methanol, due to the effect of the basic acetamidine group, part of product 28 underwent an transesterification reaction from benzyl carboxylate to methyl carboxylate, yielding trisaccharide 29. Finally, the methyl ester on trisaccharide 29 was hydrolyzed at 0°C using lithium hydroxide, followed by complete deprotection via Pd/C hydrogenation to obtain the target trisaccharide 30.
  • Compound 25 Compound 24 (240 mg, 0.181 mmol) was placed in DCM (4.0 mL) solution and stirred. Trifluoroacetic acid (0.5 mL, 6.72 mmol) and H2O (33 ⁇ L, 1.81 mmol, 10 eq) were added. The reaction was stirred at room temperature for 4 h until TLC showed complete conversion of the starting material. The reaction solution was diluted with DCM, washed with water, saturated NaHCO3 solution, and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum.
  • Compound 26 Compound 25 (190 mg, 0.154 mmol) was dissolved in DCM (4 mL) and H2O (1 mL) solution under argon protection. The system temperature was lowered to 0 °C using an ice bath, and TEMPO (5 mg, 0.031 mmol) and BAIB (99 gm, 0.308 mmol) were added. The mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC until the starting material was completely consumed. The reaction was quenched dropwise with 10% (w/v ) Na2S2O3 solution, and the organic phase was washed with saturated NaHCO3 and saturated NaCl solutions, followed by DCM back-extraction.
  • Compound 28 Compound 27 (30 mg, 21.4 ⁇ L) was placed in acetic acid (2.0 mL) solution and stirred, and activated zinc powder (50 mg, 0.78 mmol) was added. After stirring at room temperature for 12 h, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography (DCM/MeOH v/v 20:1 ⁇ 10:1) to obtain the corresponding amino sugar. At 0 °C, the above amino sugar was placed in anhydrous pyridine (2 mL) solution and stirred, and benzylthioacetylimine hydrochloride (8.6 mg, 42.8 ⁇ mol) was added.
  • the reaction was stirred at 0 °C for 5 h. Subsequently, the reaction solution was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (DCM/MeOH v/v 10:1). The two-step reaction yielded colorless syrup product 28 (19 mg, 15.6 ⁇ mol, 73%).
  • the partially protected product was placed in a THF/MeOH/ H2O /AcOH solution (v/v/v/v 10:5:4:1, 2 mL), and 10% Pd/C (50 mg) was added.
  • the reaction was stirred in H2 environment for 24 h.
  • the mixture was then filtered and concentrated under vacuum, and eluted using a Sephadex LH-20 column ( H2O ).
  • the two-step reaction yielded a white solid 30 (4.5 mg, 5.88 ⁇ mol, 64%).
  • 1H NMR 600 MHz, Deuterium
  • intermediate compound 7 was used as a starting material.
  • the C4 hydroxyl group of compound 7 was benzyl-protected with benzyl bromide and silver oxide to obtain compound 31.
  • the allyl group at the reducing end of compound 31 was removed using palladium chloride ( PdCl2 ) to obtain the corresponding hemiacetal derivative, which was then converted into N-phenyltrifluoroimine ester donor 32.
  • PdCl2 palladium chloride
  • compound 32 Under the catalysis of TMSOTf, compound 32 reacted with p-toluenethiophenol to obtain thioglycoside donor 33.
  • disaccharide 21 underwent reduction of the azide groups at the C2 and C3' positions to aminoacetyl groups in the presence of zinc powder and acetic anhydride to obtain compound 34. Subsequently, using TMSOTf as an activator, disaccharide acceptor 34 was glycosylated with trifluoroacetylimine ester donor 32 and thioglycoside donor 33, respectively, in an attempt to assemble and synthesize trisaccharide 35.
  • N-phenyltrifluoroacetylimine donor 32 (0.45 g, 0.67 mmol) was placed in anhydrous DCM (7 mL), and TolSH (83 mg, 0.67 mmol) was added. Molecular sieve (flame dried). TMSOTf (23 ⁇ L, 0.134 mmol) was added dropwise under argon atmosphere. When TLC showed complete conversion of the starting material, the reactants were quenched with Et3N , then filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate v/v 5:1) to give a white solid 33 (343 mg, 0.57 mmol, 85%).
  • Compound 34 Compound 21 (1.70 g, 1.60 mmol) was placed in 16 mL of THF solution and stirred at room temperature. AcOH (91 ⁇ L, 1.60 mmol) and TBAF (2.4 mL, 2.4 mmol) were added, and the mixture was stirred for 5 h. The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography (PE/EA v/v 4:1 ⁇ 3:2) to give colorless syrup 34 (1.40 g, 1.47 mmol, 92%).
  • Compound 36 Condition 1: At -10°C, donor 32 (21 mg, 0.0316 mmol), acceptor 21 (20 mg, 0.0211 mmol) and Molecular sieves (flame-dried) were placed in a dry DCM solution (1.6 mL) and stirred. Under argon protection, TMSOTf (1.1 ⁇ L, 6.32 ⁇ mol) was added dropwise. When TLC showed complete conversion of the glycosyl donor, Et3N was added dropwise to quench the reaction, followed by filtration and vacuum concentration. The crude product was purified by silica gel column chromatography (PE/EA v/v 5:1 ⁇ 2:1) to obtain colorless syrup 36 (4.5 mg, 4.74 ⁇ mol, 15%).
  • reaction mixture was stirred at 0 °C for 2 hours until TLC showed complete conversion of the starting material, and then quenched by the addition of MeOH at 0 °C.
  • the mixture was concentrated under vacuum to the residue and purified by silica gel column chromatography (petroleum ether/ethyl acetate 5:1 ⁇ 2:1 v/v) to give product 37 (19 mg, 0.026 mmol, 31%) as a colorless syrup.
  • acceptor 16 and thioglycoside donor 39 under the action of TMSOTf and N-iodosuccinimide (NIS), acceptor 16 and thioglycoside donor 39 underwent glycosylation at temperatures ranging from -10°C to 0°C to obtain disaccharide 39 with complete ⁇ -selectivity in a yield of 79%.
  • Hydrolysis of the 4,6-O-benzyl group of compound 40 with trifluoroacetic acid (TFA) yielded diol 41 in a yield of 82%.
  • selective protection of the C6' hydroxyl group with tert-butyldimethylsilyl (TBDPS) failed to yield the desired product despite the reaction being carried out at 80°C.

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Abstract

本发明公开了一种绿脓杆菌O5血清型O-抗原寡糖的化学合成方法，属于化学合成领域。本发明利用D-葡萄糖醛酸砌块、D-岩藻糖胺砌块构建O-抗原三糖；其中D-岩藻糖胺1,2-α-顺式-糖苷键的立体选择性合成依赖于远程酰基参与和试剂调控，两种2,3-二氨基甘露糖醛酸1,2-β-反式-糖苷键的合成是通过C2位叠氮基的SN2亲核取代来实现；通过保护基团的选择组装，修饰基团的正交修饰，以及糖基供、受体反应活性的调控，成功完成了多功能化修饰的O-抗原目标三糖的制备。本发明方法原料廉价易得、制备方法简单易重复，在开发抗绿脓杆菌的疫苗中具有很好的应用前景。

Description

一种绿脓杆菌O5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杆菌O5血清型O-抗原寡糖的化学合成方法，属于化学合成领域。
背景技术
绿脓杆菌(Pseudomonas aeruginosa)，又称铜绿假单胞菌，是一类环境适应能力极强的条件致病菌，是引起医院感染的主要病原体之一。绿脓杆菌感染可引起脓毒症、败血症等一系列并发症，对囊性纤维化患者、艾滋病患者、化疗后的病人等免疫力低下人群造成致命伤害(K.Grimwood et al.,Hum Vaccin Immunother 2015,11,14-20)。根据美国疾控中心报告，每年约有51000名患者感染绿脓杆菌，死亡率约为5.3％(M.E.Chirgwin et al.,Materials 2019,12,4093)。绿脓杆菌对多种抗生素具有极强的抗药性(C.K.Elogne et al.,Afr J Microbiol Res,2018,12,62-67)，2017年世界卫生组织(WHO)首次发布的抗生素抗药性“优先病原体”清单中，绿脓杆菌被列为“最高等级”类别，绿脓杆菌耐药性问题已成为当今人类健康的最大威胁之一(E.Tacconelli et al.,WorldHealth Organization,2017)。由于免疫预防策略不受抗生素耐药性机制的影响，因此开发高效、安全的疫苗用于耐药性病原体的治疗已迫在眉睫。研究人员针对绿脓杆菌疫苗开展了大量的研究，主要包括全菌疫苗和多种亚单位疫苗，但目前尚未有人类可用的疫苗批准上市(G.P.Priebe et al.,Expert Rev Vaccines,2014,13,507-519)。相对于包含诸多无效抗原的全菌疫苗，绿脓杆菌细胞表面多糖和膜蛋白等亚单位疫苗可提供更有效的免疫保护。研究表明，以绿脓杆菌脂多糖作为免疫原开展疫苗研究表现出较好的保护作用，而免疫力最有效的抗原靶点是细菌表面脂多糖(LPS)的O-抗原多糖部分(S.J.Cryz et al.,Infect Immun,1984,44,508-513)，因此脂多糖O-抗原已广泛用于绿脓杆菌疫苗的开发(N.F.Abu-Baker et al.,Advances in Microbiology,2016,6,332-342)。
根据绿脓杆菌脂多糖O-抗原的不同，已鉴定出20种具有不同免疫学特异性的绿脓杆菌血清型(P.V.Liu et al.,J Clin Microbiol,1990,28,922-925)，在已经明确结构的绿脓杆菌20种血清型O-抗原中，O5血清型O-抗原与O2、O16、O18和O20血清型O-抗原在其三糖重复片段结构具有高度相似性。O5血清型的O-抗原多糖结构因其高度的结构特异性而受到关注。该多糖结构由一个复杂修饰多氨基三糖重复单元组成：[→4)-β-D-ManpNAc3AmA-(1→4)-β-D-ManpNAc3AcA-(1→3)-α-D-FucpNAc-(1→](I.Sadovskaya et al.,Eur.J.Biochem.1997,255,673-684)。值得注意的是，具有两个连续的β-D-ManpN3NA片段的多糖在自然界中并不常见，此前也并未合成过。此外，三糖重复片段的糖环上的大部分羟基都被氨基取代，仅有一个羟基暴露，从而形成高密度功能化的氨基糖苷。另一特别之处在于三糖重复片段都以1,2-顺式-糖苷键连接。在细菌O-抗原中主要存在着一种独特的功能基团乙脒基(Am)，它特异性地附着在O-抗原三糖2,3-二氨基-D-甘露醛酸C3位的氨基上。尽管近年来在立体选择性糖基化合成方法和组装复杂寡糖的策略方面取得了显著进展，但化学合成高密度功能化氨基糖苷仍充满挑战。其中难点包括：由于所有连接都是1,2-顺式-糖苷键，因此很难合成立体特异性三糖；稀有糖ManpNAc3NAcA在糖基化过程中反应活性差以及需要在氨基上正交组装乙脒基和乙酰基。因此，对于这一结构的全合成需要进行整体性的路线设计，包括保护基团的选择，修饰基团引入时机的选择，糖基化反应的效率与选择性等关键环节。
发明内容
本发明涉及的内容是通过三种糖砌块化学方法合成绿脓杆菌O5血清型O-抗原三糖片段，主要包括3-氨基-D-葡萄糖砌块、D-葡萄糖醛酸砌块、D-岩藻糖胺砌块以及1,2-α-顺式-糖苷键、1,2-β-顺式-糖苷键和氨基功能基团的正交修饰。其中1,2-顺式-糖苷键的构 建和氨基功能基团的正交修饰是该目标三糖的关键步骤。本发明通过酰基远程参与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。通过C2位叠氮基的SN2亲核取代实现两种1,2-β-顺式-糖苷键的立体构建。利用合成的三糖前体，通过C6位羟基氧化以及不同氨基功能基团的正交修饰，最后脱去保护得到的目标三糖如式Ⅵ。本发明通过氨基连接臂将合成寡糖固定于芯片表面，并利用绿脓杆菌病人血清筛选寡糖抗原应用于合成寡糖的抗原性研究。
本发明的第一个目的是提供一种绿脓杆菌的O5血清型O-抗原三糖的合成方法，所述方法包括：利用3-氨基-D-葡萄糖砌块、D-葡萄糖醛酸砌块、D-岩藻糖胺砌块构建O-抗原三糖；利用酰基远程参与效应和添加剂效应的协同作用构建D-岩藻糖胺的1,2-α-顺式-D-岩藻糖胺糖苷键，利用C2位叠氮基的SN2取代反应构建两种2,3-二-氨基-D-甘露糖醛酸的1,2-β-反式糖苷键的构建；完成O-抗原三糖中五个氨基功能修饰基团的正交组装。
本发明涉及一种绿脓杆菌O5血清型O-抗原三糖组装有连接臂的寡糖片段，此糖链化学结构式可表示为通式(VI)：
其中，Linker为-L-NH2，其中L表示连接臂；
本发明中连接臂L可以是含有0-6个杂原子的2-40碳原子数(包括侧链的碳原子数)的链式结构。
本发明中连接臂的主链长为4-8原子数时,链中可以包含1、2或3个杂原子(0，N和S当连接臂的主链长为9-14原子数时，链中可以包含1、2、3、4、5或6个杂原子(O，N和S)。
本发明中连接臂-L-可以全部或部分氟取代的环式结构。连接臂-L-可以包含一个三、四、五或六元饱和碳环；也可以包含一个五元不饱和碳环(非芳香环)；也可以包含四、五或六元饱和氧杂环；也可以包含一个四、五或六元饱和氮杂环；也可以包含一个六元芳香碳环。
本发明中连接臂-L-也可以包含酰胺键和/或脲基。
本发明中上述这些连接臂-L-中还可以含有一个或多个取代基团，这些取代基可以包括：-F，-Cl，-CH3，-C2H5，-C3H7，-C5H9，-C6H13，-OCH3，-OC2H5，-CH2F，-CHF2，-CF3，-C(O)-NH2，-SCH3，-SC2H5，-NHC(O)CH3，-N(CH3)2和-N(C2H5)2。
本发明中所合成糖链结构中含有碱性(乙基)和酸性(羧基)基团，它们可以与有机或无机的酸或碱形成相应的盐。
本发明中所合成糖链结构中因同时含有碱性(乙基)和酸性(羧基)基团，也可以通过分子内质子迁移，即酸性基团的质子转移至碱性基团，而通式我可以是含有-O-和-NH3 +的两性分子。
本发明提供了绿脓杆菌O5血清型O-抗原多糖的连接臂修饰糖链(如通式Ⅵ所示)的化学合成方法，以三个单糖砌块A，B，C为原料，分别如式(Ⅰ)～(Ⅲ)所示：
其中：
PG1为H；
PG2，PG3，PG4和PG6为羟基临时保护基，分别独立地选自：乙酰基(Ac)，或者苯甲酰(Bz)，或者新戊酰基(Piv)，或者氯乙酰(ClAc)，或者乙酰丙酰基(Lev)，或者9-芴甲氧羰基(Fmoc)，或者烯丙氧羰酰基(Alloc)，或者2-萘甲基(Nap)，或者对甲氧基苄基(pMB)，或者烯丙基(All)，或者叔丁基二甲基硅烷基(TBS)，或者三乙基硅烷基(TES)；
PG5为羧基保护基，可以是苄基(Bn)，甲基(Me)，乙基(Et)，叔丁基(tBu)，烯丙基(All)；
PG7为氨基临时保护基团，可以是三氯乙氧羰基(Troc)，邻苯二甲酰基(Phth)，9-芴甲氧羰基(Fmoc)，叔丁基氧羰基(Boc)等；
PG8，PG9为羟基保护基，可以是苯亚甲基(PhCH)，萘亚甲基(NapCH)，异亚丙基缩酮((CH3)2CH)等；
PG10，PG11为氨基保护基，可以是苄基(Bn)，苄氧羰基(Cbz)；
LG为用于糖基化反应的离去基团，如氟(F)或者氯(Cl)或者溴(Br)或者碘(I)或者三氯乙酰亚胺酯(CCl3C(＝NH)O-)或者N-苯基三氟乙酰亚胺酯糖苷(CF3C(＝NPh)O-)或者乙硫基(SEt)或者苯硫基(SPh)或者对甲苯硫基(STol)或者乙硫基(SEt)或者二丁基膦酸基(-P(＝O)-(OBu)2)等；
包括如下过程：
(1)利用单糖砌块B(D-葡萄糖醛酸砌块)与单糖砌块A(D-岩藻糖胺砌块)进行糖基化反应，合成得到式Ⅳ所示的二糖片段，
(2)将所得二糖片段先进行脱保护、脱去保护基团PG4，然后与单糖砌块C(3-氨基-D-葡萄糖砌块)进行糖基化，合成得到如式Ⅴ所示的三糖中间体片段，
(3)还原所得三糖中间体片段、脱保护PG3、PG6，并对单糖砌块B中脱保护后的2号位、以及单糖砌块C中脱保护后的2号位进行叠氮化；随后还原三糖中间体片段中的单糖砌块C的4、6号位，脱去PG8、PG9，并对6号位进行氧化，得到羧酸类基团；再对单糖砌块B中脱保护后的2号位、以及单糖砌块C中2号位的叠氮基团进行还原，得到乙酰氨基；再使用亚胺类试剂与单糖砌块C中的3号位构建乙脒结构，最后还原Linker中的氨基，得到式ⅤI所示的目标产物。
在本发明的一种实施方式中，所述方法的合成路线如下所示：
本发明利用D-葡萄糖醛酸砌块、D-岩藻糖胺砌块构建O-抗原三糖；其中D-岩藻糖胺1,2-α-顺式-糖苷键的立体选择性合成依赖于远程酰基参与和试剂调控，两种2,3-二氨基甘露糖醛酸1,2-β-反式-糖苷键的合成是通过C2位叠氮基的SN2亲核取代来实现；通过保护基团的选择组装，修饰基团的正交修饰，以及糖基供体和受体反应活性的调控，成功完成了多功能化修饰的O-抗原目标三糖及其寡糖模拟物的制备；将合成的O-抗原寡糖片段固定化于玻片表面，制得糖芯片，利用病人血清筛选有效抗原，确定O-抗原寡糖抗原表位；原料廉价易得、制备方法简单易重复，在开发抗绿脓杆菌的疫苗中具有很好的应用前景。
在本发明的一种实施方式中，所述方法具体包括：以3,4-O-二乙酰基岩藻糖烯为原料，在二乙酸碘苯(PhI(OAc)2)作用下，与叠氮基三甲基硅烷(TMS-N3)和二苯基二硒(Ph2Se2)生成1-硒苯-2-叠氮岩藻糖，然后碱性条件下脱除乙酰基，选择性保护C3位羟基为3-ONap，C4位羟基进行苯甲酰基保护，最后在添加剂效应(Ph3PO)和酰基远程参与效应(Bz)的协同作用下得到高立体选择性的1,2-α-顺式-D-岩藻糖胺糖苷键。
在本发明的一种实施方式中，所述方法还包括：以葡萄糖为原料，利用三氟加磺酰基介导的C3位叠氮基亲核取代得到C3位氨基前体，同样利用C2位叠氮基的SN2亲核取代C2位氨基前体，并完成两种2,3-二氨基甘露糖醛酸1,2-β-反式-糖苷键的高效构建。
在本发明的一种实施方式中，所述路易斯酸包括三氟化硼乙醚(BF3·Et2O)，或者三氟甲磺酸(TfOH)，或者三氟甲磺酸三甲基硅酯(TMSOTf)，或者为银盐三氟甲磺酸银(AgOTf)等，硫糖苷进行反应则是另外加入NIS与路易斯酸一并作为促进剂。
在本发明的一种实施方式中，所述步骤(1)中的糖基化反应是在活化试剂路易斯酸、溶剂、分子筛的环境下进行的。
在本发明的一种实施方式中，所述步骤(1)中的糖基化反应的活化试剂路易斯酸为三氟化硼乙醚(BF3·Et2O)，或者三氟甲磺酸(TfOH)，或者三氟甲磺酸三甲基硅酯(TMSOTf)，或者为银盐三氟甲磺酸银(AgOTf)等中任意一种或多种。。
在本发明的一种实施方式中，所述步骤(2)中的糖基化反应是在活化试剂路易斯酸、NIS(碘代丁二酰亚胺)、溶剂、分子筛的环境下进行的。
在本发明的一种实施方式中，所述步骤(2)中的糖基化反应的活化试剂路易斯酸用量为0.1～1eq，NIS用量为1.2～1.5eq。
在本发明的一种实施方式中，所述步骤(2)中的糖基化反应的活化试剂路易斯酸为三氟化硼乙醚(BF3·Et2O)，或者三氟甲磺酸(TfOH)，或者三氟甲磺酸三甲基硅酯(TMSOTf)，或者为银盐三氟甲磺酸银(AgOTf)等中任意一种或多种。。
本发明的目的是提供一种组装有氨基连接臂的绿脓杆菌O5血清型O-抗原三糖的合成方法，利用上述方法制备得到的O5血清型O-抗原寡糖片段可能用于绿脓杆菌疫苗或绿脓杆菌感染性药物的开发等。
有益效果：
本发明首次提出一种绿脓杆菌O5血清型O-抗原三糖的化学合成方法。本发明方法利用D-葡萄糖醛酸砌块、D-岩藻糖胺砌块构建O-抗原三糖；其中D-岩藻糖胺1,2-α-顺式-糖苷键的立体选择性合成依赖于远程酰基参与和试剂调控，两种2,3-二氨基甘露糖醛酸1,2-β-反式-糖苷键的合成是通过C2位叠氮基的SN2亲核取代来实现；通过保护基团的选择组装，修饰基团的正交修饰，以及糖基供体和受体反应活性的调控，成功完成了多功能化修饰的O-抗原目标三糖及其寡糖模拟物的制备；将合成的O-抗原寡糖片段固定化于玻片表面，制得糖芯片，利用病人血清筛选有效抗原，确定O-抗原寡糖抗原表位；原料廉价易得、制备方法简单易重复，在开发抗绿脓杆菌的疫苗中具有很好的应用前景。
附图说明
图1为绿脓杆菌O5血清型O-抗原三糖重复单元。
图2为通式Ⅰ、Ⅱ和Ⅲ中所示化合物A，B，C。
图3为通式Ⅵ中所示化合物F。
图4为糖砌块9的合成。
图5为糖砌块16的合成。
图6为糖砌块19的合成。
图7为三糖20的合成。
图8为目标三糖30的合成
图9为三糖35的合成尝试。
图10为三糖38的合成尝试。
图11为三糖43和44的合成尝试。
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将会理解，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，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购得的常规产品。
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(400MHz，600MHz)，高分辨质谱测定，旋光值测定，红外光谱测定，结果在每个化合物的具体合成中列出。
实施例1
糖砌块9的合成：
如图4所示，以烯丙基α-D-阿洛糖1为原料，利用甲醇钠脱除苯甲酰基(Bz)得到相应的二元醇，然后通过锡介导的区域选择性2-萘亚甲基化反应在2-OH组装萘亚甲基(Nap)，可得化合物2。随后对化合物2中的O3进行三氟乙酰化，接下来叠氮取代得到C3位为叠氮基的化合物3。利用2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)脱除化合物13的2-O-Nap，并对其进行乙酰丙酰基保护，可得到化合物5。随后，利用80％的乙酸溶液水解4,6-O-苄叉基，可得到二元醇6。随后，利用TEMPO/BAIB氧化C6位羟基，并进行羧酸的苄酯化，得到化合物7。对C4位羟基组装临时保护基叔丁基二甲基硅酰基(TBS)得到化合物8。最后，水解端基位烯丙基，并将其转化为三氟乙酰亚胺酯供体9。
具体试验操作和步骤：
化合物2：将化合物1(6.6g，16.01mmol)置于甲醇(40mL)溶液中搅拌溶解，加入NaOMe(0.43 g，8.01mmol)。反应在室温下搅拌5h后，用Amberlite IR 120(H+)离子交换树脂中和溶液并过滤。滤液减压浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 2:1)分离纯化，得到相应的二元醇化合物。将上述化合物与无水甲苯共沸，并在高真空下干燥30min。然后，在氮气的保护下加入无水甲苯(80mL)，再加入Bu2SnO(5.98g，24.02mmol)和分子筛(火焰干燥)。反应在110℃条件下回流，充分搅拌3h。将反应冷却至室温，加入2-溴甲基萘(5.32g，24.02mmol)和TBAB(7.74g，24.02mmol)，在60℃下搅拌5h。通过TLC监测显示反应进展。反应结束后，将反应混合物过滤并减压浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 6:1)分离纯化，得到糖浆状化合物2(5.88g，13.13mmol，82％)。[α]25 D＝+105.1°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ7.90–7.30(m,12H,Ar-H),5.95(dddd,J＝17.0,10.4,6.3,5.2Hz,1H,All-CH),5.50(s,1H,ArCH),5.37(dq,J＝17.2,1.5Hz,1H,All-CH2),5.25(dq,J＝10.4,1.3Hz,1H,All-CH2),4.97–4.88(m,2H,1-H,ArCH),4.77(d,J＝12.5Hz,1H,ArCH),4.47(dt,J＝6.3,2.8Hz,1H,3-H),4.32(dd,J＝10.2,5.1Hz,1H,6’-H),4.29–4.16(m,2H,All-CH2,5-H),4.07(ddt,J＝12.9,6.3,1.3Hz,1H,All-CH2),3.67(t,J＝10.2Hz,1H,6-H),3.53(t,J＝3.4Hz,1H,2-H),3.39(dd,J＝9.7,2.6Hz,1H,4-H),3.31(d,J＝7.0Hz,1H,3-OH)；13C NMR(101MHz,Chloroform-d)δ137.13,134.75,133.26,133.15,129.05,128.50,128.20,127.86,127.74,126.90,126.32,126.17,125.71,118.39,102.00,97.44,78.93,73.68,70.47,69.14,69.11,67.14,58.09；IR νmax(film)3510,2929,1601,1451,1316,1270,1177,1106,1070,1028,862,756,713cm-1；HR-ESI-MS(m/z):calcdfor C27H28O6Na+(M+Na+):471.1778,found:471.1783.
化合物3：在氮气环境下，将化合物2(5.80g，12.94mmol)置于无水DCM(65mL)中搅拌溶解。于-20℃加入吡啶(10.6mL，129.4mmol，10eq)。滴加Tf2O(4.35mL，25.88mmol)，搅拌溶剂，使其在4h内从-20℃升温至0℃。有机相经Na2SO4干燥后，在30℃真空条件下浓缩，得到黄色糖浆状的三氯酸盐产物。在氮气环境下，向上述三氯酸盐溶解于无水DMF(65mL)溶液中，于0℃加入TBAN3(11.04g，38.82mmol)，混合物在室温下充分搅拌过夜。通过TLC分析监测反应进展。反应结束后，反应液用EA(150mL)稀释，用水和盐水洗涤。有机相用过无水Na2SO4干燥后，减压浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 10:1)分离纯化，得到产物3(4.65g，9.83mmol，两步产率76％)。[α]25 D＝+173.0°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ7.89–7.34(m,12H,Ar-H),5.92(dddd,J＝17.1,10.3,6.6,5.2Hz,1H,All-CH),5.52(s,1H,ArCH),5.33(dd,J＝17.2,1.6Hz,1H,All-CH2),5.23(dd,J＝10.3,1.4Hz,1H,All-CH2),4.96(d,J＝12.2Hz,1H,ArCH),4.82(d,J＝12.2Hz,1H,ArCH),4.77(d,J＝3.6Hz,1H,1-H),4.24(dd,J＝10.3,4.9Hz,1H,6’-H),4.18(ddt,J＝12.9,5.2,1.5Hz,1H,All-CH2),4.09(t,J＝9.9Hz,1H,3-H),3.98(ddt,J＝12.8,6.6,1.3Hz,1H,All-CH2),3.89(td,J＝10.0,4.9Hz,1H,5-H),3.65(t,J＝10.3Hz,1H,6-H),3.47(dd,J＝9.9,3.6Hz,1H,2-H),3.39(t,J＝9.8Hz,1H,4-H)；13C NMR(101MHz,Chloroform-d)δ136.81,134.89,133.28,133.18,129.13,128.44,128.32,127.93,127.74,127.11,126.29,126.19,126.07,125.92,118.59,101.61,95.80,79.89,78.16,73.34,68.89,68.60,62.68,61.74；IR νmax(film)2868,2109,1451,1374,1268,1094,1071,1044,1028,995,929,751,713,699cm-1；HR-ESI-MS(m/z):calcd for C27H27N3O5Na+(M+Na+):496.1843,found:496.1848.
化合物4：将化合物3(4.62g，9.76mmol)溶解于DCM(90mL)和H2O(10mL)溶液中，加入DDQ(3.33g，14.64mmol)。室温下搅拌6.5h，通过TLC分析监测反应进展。滴加5％(w/v)Na2S2O3溶液淬灭，有机相通过饱和NaHCO3溶液和盐水洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。粗品用硅胶柱层析(PE/EA v/v 4:1)分离纯化得到化合物4(2.79g，8.39mmol，86％)。[α]25 D＝+162.1°(c 1.00,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.55–7.32(m,5H,Ar-H),5.93(dddd,J＝16.9,10.3,6.4,5.4Hz,1H,All-CH),5.56(s,1H,ArCH),5.34(dq,J＝17.2,1.5Hz,1H,All-CH2),5.27(dq,J＝10.3,1.2Hz,1H,All-CH2),4.94(d,J＝3.9Hz,1H,1-H),4.30(dd,J＝10.3,4.9Hz,1H,6’-H),4.29–4.23(m,1H,All-CH2),4.08(ddt,J＝12.6,6.3,1.3Hz,1H,All-CH2),3.89(td,J＝9.9,4.9Hz,1H,5-H),3.84(t,J＝9.8Hz,1H,3-H),3.73(t,J＝10.3Hz,1H,6-H),3.61(td,J＝10.0,3.9Hz,1H,2-H),3.48(t,J＝9.7Hz,1H,4-H),2.26(d,J＝10.4Hz,1H,2-OH)；13C NMR(101MHz,Chloroform-d)δ136.78,132.98,129.12,128.32,126.01,118.68,101.53,97.37,79.63,77.22,71.78,68.97,68.85,63.74,63.01；IR νmax(film)3345,2916,1870,2105,1368,1260,1152,1077,1058,1013,994,931,746,698,653cm-1；HR-ESI-MS(m/z):calcd for C16H19N3O5Na+(M+Na+):356.1217,found:356.1218.
化合物5：将化合物4(2.77g，8.32mmol)溶解于干燥的DCM(40mL)溶液中，加入乙酰丙酸(LevOH)(1.45g，12.48mmol)、N,N-双环己基碳二亚胺(DCC)(2.57g，12.48mmol)和DMAP(1.52g，12.48mmol)。在室温下搅拌4h，直到原料完全消耗，加入DCM稀释反应液。有机相通过饱和NaHCO3溶液和饱和NaCl溶液洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。粗品用硅胶柱层析(PE/EA v/v 8:1)分离纯化，得到化合物5(3.48g，8.07mmol，97％)。[α]25 D＝+110.8°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.54–7.33(m,5H,Ar-H),5.91(dddd,J＝16.9,10.3,6.2,5.3Hz,1H,All-CH),5.58(s,1H,ArCH),5.32(dq,J＝17.1,1.6Hz,1H,All-CH2),5.24(dq,J＝10.4,1.4Hz,1H,All-CH2),5.05(d,J＝3.7Hz,1H,1-H),4.74(dd,J＝10.3,3.7Hz,1H,2-H),4.29(dd,J＝10.4,4.9Hz,1H,6’-H),4.21(ddt,J＝12.9,5.3,1.5Hz,1H,All-CH2),4.12(t,J＝10.1Hz,1H,3-H),4.04(ddt,J＝12.9,6.2,1.4Hz,1H,All-CH2),3.95(td,J＝9.9,4.9Hz,1H,5-H),3.75(t,J＝10.3Hz,1H,6-H),3.54(t,J＝9.7Hz,1H,4-H),2.92–2.56(m,4H,CH2-Lev),2.20(s,3H,CH3CO)；13C NMR(101MHz,Chloroform-d)δ206.02,171.88,136.72,133.23,129.15,128.32,126.03,118.19,101.63,95.20,79.83,72.15,68.97,68.82,62.79,60.04,37.79,29.79,27.88；IR νmax(film)2923,2868,2109,1743,1718,1454,1368,1260,1208,1151,1123,1096,1072,1045,996,933,753,700,653cm-1；HR-ESI-MS(m/z):calcd for C21H25N3O7Na+(M+Na+):454.1585,found:454.1581.
化合物6：将化合物5(3.46g，8.02mmol)溶解于80％AcOH(40mL)溶液中，使用油浴锅将体系温度加热至55℃并搅拌。通过TLC分析监测反应进展，直至原料消耗殆尽。经减压浓缩去除大部分溶剂后，用硅胶柱层析(DCM/MeOH v/v 30:1)分离纯化，得到产物6(2.64g，7.70mmol，96％)。[α]25 D＝+96.6°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ5.90(dddd,J＝17.2,10.4,6.1,5.3Hz,1H,All-CH),5.31(dq,J＝17.2,1.6Hz,1H,All-CH2),5.23(dq,J＝10.4,1.4Hz,1H,All-CH2),5.03(d,J＝3.6Hz,1H,1-H),4.69(dd,J＝10.5,3.6Hz,1H,2-H),4.19(ddt,J＝13.0,5.3,1.5Hz,1H,All-CH2),4.02(ddt,J＝13.0,6.1,1.4Hz,1H,All-CH2),3.94(dd,J＝10.6,9.5Hz,1H,3-H),3.88–3.80(m,2H,6-H),3.73(dt,J＝9.8,3.5Hz,1H,5-H),3.62–3.52(m,1H,4-H),3.34(s,1H,4-OH),2.84–2.62(m,4H,CH2-Lev),2.37(br,1H,6-OH),2.20(s,3H,CH3CO)；13C NMR(101MHz,Chloroform-d)δ206.30,172.03,133.35,118.03,94.57,72.29,70.98,69.06,68.76,63.65,61.78,37.80,29.79,27.90；IR νmax(film)3395,2925,2108,1743,1716,1417,1362,1258,1207,1155,1040,929,840,770,607cm-1；HR-ESI-MS(m/z):calcd for C14H21N3O7Na+(M+Na+):366.1272,found:366.1275.
化合物7：在氩气保护下，将化合物6(2.64g，7.70mmol)溶解于DCM(77mL)和H2O(15mL)溶液中。使用冰浴将体系温度降至0℃，加入2,2,6,6-四甲基哌啶氧化物(TEMPO)(240mg，1.54mmol)和二乙酰氧基碘苯(BAIB)(4.96g，15.4mmol)。在室温下搅拌4h。通过TLC监测分析反应进展，直至原料消耗殆尽。滴加5％(w/v)Na2S2O3溶液淬灭，有机相通过饱和NaHCO3溶液和饱和NaCl溶液洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。将粗品在氮气的保护下溶解于无水DMF(77mL)中。在室温下，加入NaHCO3(3.23g，38.5mmol)和BnBr(1.83mL，15.4mmol)。当TLC显示原料完全消耗时，真空浓缩除去溶剂。用硅胶柱层析(PE/EA v/v 5:1)纯化。两步反应得到葡萄糖醛酸糖浆7(2.75g，6.16mmol，80％)。[α]25 D＝+74.6°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.41–7.32(m,5H,Ar-H),5.90(dddd,J＝17.0,10.4,6.2,5.3Hz,1H,All-CH),5.31(dq,J＝17.2,1.6Hz,1H,All-CH2),5.29(d,J＝12.3Hz,1H,ArCH),5.24(d,J＝12.2Hz,1H,ArCH),5.22(dq,J＝10.4,1.3Hz,1H,All-CH2),5.10(d,J＝3.6Hz,1H,1-H),4.68(dd,J＝10.6,3.6Hz,1H,2-H),4.27(d,J＝9.8Hz,1H,5-H),4.25–4.19(m,1H,All-CH2),4.07(ddt,J＝12.9,6.2,1.4Hz,1H,All-CH2),3.95(dd,J＝10.6,9.5Hz,1H,3-H),3.77(td,J＝9.7,2.8Hz,1H,4-H),3.17(s,1H,4-OH),2.87–2.55(m,4H,CH2-Lev),2.19(s,3H,CH3CO).13C NMR(400MHz,Chloroform-d)δ206.00,171.80,169.73,134.85,133.03,128.74,128.67,128.24,118.41,94.86,71.26,70.78,70.17,69.35,67.61,62.45,37.79,29.76,27.84；IR νmax(film)3480,2935,2110,1745,1717,1361,1259,1181,1155,1051,939,754,698,609cm-1；HR-ESI-MS(m/z):calcd for C21H25N3O8Na+(M+Na+):470.1534,found:470.1537.
化合物8：向葡萄糖醛酸7(1.55g，3.47mmol)加入无水DMF(3.5mL)，在氩气保护下充分搅拌反应液。随后向反应瓶中加入叔丁基二甲基硅烷基氯(1.05g，6.94mmol)、咪唑(472mg，6.94mmol)和4-二甲氨基吡啶(43mg，0.35mmol)。在80℃的条件下搅拌过夜。通过TLC监测分析反应进展，直 至原料消耗殆尽。然后，加入甲醇(3ml)淬灭反应。用乙酸乙酯(50ml)稀释反应液，有机相通过饱和NaHCO3溶液和饱和NaCl溶液洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 10:1)分离纯化，得到无色糖浆8(1.73g，3.09mmol，89％)。[α]25 D＝+74.5°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.40–7.32(m,5H,Ar-H),5.90(dddd,J＝16.8,10.2,6.3,5.3Hz,1H,All-CH),5.30(dd,J＝17.2,1.6Hz,1H,All-CH2),5.23(d,J＝12.3Hz,1H,ArCH),5.25–5.20(m,1H,All-CH2),5.13(d,J＝12.4Hz,1H,ArCH),5.07(d,J＝3.6Hz,1H,1-H),4.75(dd,J＝10.3,3.6Hz,1H,2-H),4.23–4.20(m,1H,All-CH2),4.20(d,J＝9.4Hz,1H,5-H),4.03(ddt,J＝13.0,6.3,1.3Hz,1H,All-CH2),3.82(t,J＝9.8Hz,1H,3-H),3.76(t,J＝9.3Hz,1H,4-H),2.84–2.73(m,2H,CH2-Lev),2.71–2.60(m,2H,CH2-Lev),2.19(s,3H,CH3CO),0.85(s,9H,SiC(CH3)3),0.15(s,3H,SiCH3),0.01(s,3H,SiCH3)；13C NMR(151MHz,CDCl3)δ205.90,171.79,168.70,134.98,133.17,128.63,128.49,128.26,118.43,94.88,72.21,72.18,70.94,69.27,67.38,64.49,37.80,29.74,27.88,25.72,18.05,-4.43,-5.02；IR νmax(film)＝2930,2857,2109,1748,1721,1456,1404,1361,1252,1182,1136,1052,939,838,781,749,697,669cm-1；HR-ESI-MS(m/z):calcd for C27H39N3O8SiNa+(M+Na+):584.2399,found:584.2401.
化合物9：向化合物8(1.54g，2.74mmol)的MeOH/DCM(v/v，2/1，30mL)溶液中加入PdCl2(485mg，2.74mmol)，在40℃下搅拌反应6h。将混合物过滤后真空浓缩，硅胶柱层析(PE/EA v/v 2:1)分离纯化，得到相应的半缩醛，为淡黄色糖浆。将此淡黄色糖浆溶解于DCM(30mL)溶液中。在0℃条件下，向反应液加入2,2,2-三氟-N-苯基乙酰亚胺氯(2.05mL，13.7mmol)和DBU(1.23mL，8.22mmol)。反应体系温度升至室温，搅拌3h。通过TLC监测分析反应进展，直至原料消耗殆尽。将反应液真空浓缩，粗品经硅胶柱层析(PE/EA v/v 20:1→10:1)分离化纯，得到淡黄色糖浆9(1.43g，2.06mmol，75％)。[α]25 D＝+29.9°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.39–6.77(m,10H,Ar-H),5.28(d,J＝12.3Hz,1H,ArCH),5.20–5.15(m,1H,2-H),5.13(d,J＝12.3Hz,1H,ArCH),4.10–3.78(m,2H,3-H,5-H),3.62–3.44(m,1H,4-H),2.85–2.79(m,2H,CH2-Lev),2.70–2.59(m,2H,CH2-Lev),2.18(s,3H,CH3CO),0.84(s,9H,SiC(CH3)3),0.16(s,3H,SiCH3),0.01(s,3H,SiCH3)；13C NMR(151MHz,Chloroform-d)δ205.73,171.02,166.98,142.94,134.69,128.78,128.67,128.63,128.34,124.56,119.26,94.44,77.40,70.86,70.45,67.63,67.48,37.86,29.66,27.72,25.64,17.97,-4.53,-5.09；IR νmax(film)＝2930,2857,2109,1753,1720,1597,1489,1405,1363,1327,1257,1212,1162,1137,1090,910,839,779,754,696,584cm-1；HR-ESI-MS(m/z):calcd for C32H39F3N4O8Na+(M+Na+):715.2381,found:715.2377.
实施例2
糖砌块16的合成：
如图5所示，以D-岩藻糖作为原料，经乙酰化、溴代以及消除反应得到稀糖10。随后在二苯二硒化(Ph2Se2)和三甲基硅基叠氮(TMSN3)作用下进行叠氮化反应，得到C2位为叠氮基的化合物11。随后，在甲醇钠的作用下脱乙酰并在O3上进行选择性2-萘亚甲基化反应，得到化合物13。利用溴化苄和氢化钠(NaH)在N,N-二甲基甲酰胺(DMF)溶剂中进行O4的苄基化反应得到化合物14。随后，供体14与N-苄基-N-苄氧羰基-3-氨基丙醇在乙醚/二氯甲烷中进行糖基化反应，得到化合物15。最后，在DDQ的作用下脱除2-萘亚甲基(Nap)得到糖基受体16。
具体试验操作和步骤：
化合物10：在氮气环境下，将D-岩藻糖(5.0g，30.5mmol)溶解于Ac2O(25.0mL，0.266mol)中。将反应体系温度降至0℃，加入HClO4(50μL，0.83mmol)。在室温下搅拌30min后，向反应液中加入100mL冰水，随后有机相通过饱和NaHCO3溶液(3×100mL)和饱和NaCl溶液(100mL)洗涤，DCM(3×50mL)反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。有机层浓缩至15mL，直接用于下一步。
在氮气环境下，将过乙酰葡萄糖溶解于干燥的DCM(15mL)中。将反应体系温度降至0℃，持续30min滴加HBr-AcOH(33％w/w，10mL，57.8mmol)。体系温度升至室温，搅拌7h后，用DCM(50mL)稀释反应液，用冰水(3×100mL)、饱和NaHCO3(3×100mL)、盐水(100mL)洗涤，并用Na2SO4干燥。在高真空下浓缩后，粗品直接进行下一步，无需进一步纯化。
在氮气环境下，将粗品溶解于乙酸乙酯(100mL)中，加入饱和NaH2PO4(50mL)。通过TLC监测反应完全后，通过硅藻土过滤溶液，滤液用饱和NaHCO3(3×100mL)洗涤，乙酸乙酯(3×50mL)反萃。 随后有机相用Na2SO4干燥并浓缩。浓缩得到的粗品经硅胶柱层析(PE/EA v/v 20:1)分离纯化，得到无色糖浆10(4.11g，19.2mmol，63％)。[α]25 D＝+22.1°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ6.46(dd,J＝6.3,1.9Hz,1H,1-H),5.58(ddd,J＝4.1,2.1,1.0Hz,1H,4-H),5.29(dt,J＝4.7,1.6Hz,1H,2-H),4.64(dt,J＝6.4,2.0Hz,1H,3-H),4.30–4.12(m,1H,5-H),2.16(s,3H,CH3CO),2.02(s,3H,CH3CO),1.28(d,J＝6.6Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ170.72,170.42,146.12,98.27,71.54,66.30,65.07,20.87,20.71,16.53；IR νmax(film)1747,1650,1372,1243,1163,1091,1073,1028,989,924,892,851,761cm-1；HR-ESI-MS(m/z):calcdfor C10H14O5Na+(M+Na+):237.0733,found:237.0734.
化合物11：将化合物10(3.7g，17.3mmol)溶于干燥的DCM(86mL)中，然后加入二苯基二硒醚(5.4g，17.3mmol)。在氩气环境下将溶液冷却至-30℃，然后加入BAIB(5.6g，17.3mmol)和叠氮基三甲基硅烷(3.98g，34.6mmol)。通过TLC监测分析反应进展，直至原料消耗殆尽。用饱和NaHCO3(3×100mL)洗涤，DCM(3×50mL)反萃。随后有机相用Na2SO4干燥并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1)分离纯化，得到微黄色糖浆状产物11(5.0g，12.1mmol，70％)。[α]25 D＝+204.3°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.68–7.49(m,2H,Ar-H),7.41–7.13(m,3H,Ar-H),5.95(d,J＝5.4Hz,1H,1-H),5.32(dd,J＝3.3,1.2Hz,1H,4-H),5.13(dd,J＝10.8,3.2Hz,1H,3-H),4.57–4.44(m,1H,5-H),4.24(dd,J＝10.8,5.4Hz,1H,2-H),2.17(s,3H,CH3CO),2.07(s,3H,CH3CO),1.09(d,J＝6.5Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ170.44,169.83,134.77,129.29,128.15,84.54,71.74,70.27,67.54,58.88,20.78,15.93；IR(film):ν＝2110,1747,1368,1233,1084,1020,954,908,741,692cm-1；HR-ESI-MS(m/z):calcd for C16H19N3O5SeNa[M+Na]+436.0382,found436.0385.
化合物12：将化合物11(4.6g，11.1mmol)溶解于甲醇(55mL)中，加入NaOMe(0.30g，5.55mmol)，溶液在室温下搅拌5h。溶液用Amberlite IR 120(H+)离子交换树脂中和，随后通过棉塞过滤。真空浓缩滤液，粗品经硅胶柱层析(二氯甲烷甲醇v/v 100:1)分离纯化，得到产物12(3.32g，10.1mmol，91％)。[α]D 25＝+247.6°(c＝1.00,CHCl3)；1HNMR(400MHz,Methanol-d4)δ＝7.66–7.21(m,5H,Ar-H),5.94(d,J＝5.3Hz,1H,1-H),4.32(q,J＝6.5Hz,1H,5-H),4.04(dd,J＝10.0,5.2Hz,1H,2-H),3.79–3.71(m,2H,3-H,4-H),1.18(d,J＝6.5Hz,3H,6-H)；13C NMR(100MHz,Methanol-d4)δ135.88,130.02,128.70,86.91,72.91,72.66,70.62,62.92,16.41；IR νmax(film)2106,1093,1058,988,827,761,689,670,633cm-1；HR-ESI-MS(m/z):calcd for C12H15N3O3SeNa+(M+Na+):352.0171,found:352.0176.
化合物13：将二元醇12(1.76g，5.35mmol)与无水甲苯共沸，并在高真空下干燥30min。然后，在氮气保护下加入无水甲苯(50mL)，再加入Bu2SnO(2.0g，8.03mmol)和分子筛(火焰干燥)。反应在回流条件下搅拌2h。将反应冷却至室温，加入2-溴甲基萘(1.78g，8.03mmol)和TBAB(2.59g，8.03mmol)。在60℃下搅拌4h。过滤反应液，减压浓缩除去溶剂。粗品经硅胶柱层析(PE/EA v/v 6:1)分离纯化，得到白色固体化合物13(1.95g，4.16mmol，78％)。[α]D 25＝+125.9°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ7.91–7.26(m,12H,Ar-H),5.90(d,J＝5.3Hz,1H,1-H),4.89(t,J＝11.9Hz,2H,ArCH),4.29(q,J＝6.6Hz,1H,5-H),4.20(dd,J＝10.1,5.3Hz,1H,2-H),3.90(dd,J＝3.3,1.4Hz,1H,4-H),3.75(dd,J＝10.2,3.1Hz,1H,3-H),1.25(d,J＝6.5Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ134.46,133.27,133.24,129.11,128.65,128.55,128.01,127.80,127.79,127.03,126.40,126.32,125.69,85.22,79.24,72.32,68.64,60.34,29.71,16.05；IR νmax(film)3500,3054,2975,2925,2111,1578,1509,1476,1438,1346,1271,1215,1163,1091,1065,1021,999,936,857,817,759,670,692,630,573cm-1；HR-ESI-MS(m/z):calcd for C23H23N3O3SeNa+(M+Na+):492.0797,found:492.0799.
化合物14：将化合物13(127mg，0.27mmol)在无水DMF(3mL)中的溶液中加入NaH(22mg，0.54mmol，在矿物油中分散度为60％)，在0℃下搅拌10min。然后在反应液中加入BnBr(50μL，0.41mmol)，在室温下搅拌4h。反应结束后，将混合物冷却至0℃，逐滴加入甲醇。随后反应液通过水和盐水洗涤，DCM反萃。有机相经无水Na2SO4干燥，真空浓缩，得到粗品，经硅胶柱层析(PE/EA v/v 20:1)分离纯化，得到化合物14(135mg，0.24mmol，89％)。[α]D 25＝+202.8°(c＝1.00,CHCl3)；1H NMR(400MHz,Chloroform-d)δ7.91–7.24(m,17H,Ar-H),5.94(d,J＝5.3Hz,1H,1-H),4.93(m,3H,ArCH),4.64(d,J＝11.5Hz,1H,ArCH),4.39(dd,J＝10.3,5.3Hz,1H,2-H),4.22(q,J＝6.5Hz,1H,5-H),3.78(dd,J＝10.4,2.7Hz,1H,3-H),3.73(d,J＝2.8Hz,1H,4-H),1.13(d,J＝6.5Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ138.14, 134.98,134.38,133.31,133.11,129.01,128.72,128.38,128.30,128.12,127.98,127.75,127.64,126.62,126.25,126.10,125.67,85.56,80.65,75.97,75.04,72.67,69.45,61.09,29.70,16.54；IR νmax(film)3056,2976,2882,2108,1718,1602,1578,1509,1496,1476,1454,1438,1346,1297,1269,1213,1158,1102,1080,1065,1022,999,970,895,856,816,739,691,671,631,596,566cm-1；HR-ESI-MS(m/z):calcd for C30H29N3O3SeNa+(M+Na+):582.1266,found:582.1268.
化合物15：在-20℃下，向无水DCM/Et2O(v/v 3:1，1mL)中加入化合物14(20mg，0.034mmol)在氩气的保护下搅拌。随后加入N-苄基-N-苄氧羰基-3-氨基丙醇(15mg，0.051mmol)和分子筛(火焰干燥)。将反应体系温度降至0℃，加入NIS(9.2mg，0.041mmol)和TMSOTf(1.27μL，0.007mmoL)。反应在0℃下搅拌3h，直到TLC检测显示原料完全转化。反应液用DCM稀释并过滤。有机相用10％(w/v)Na2S2O3、饱和NaHCO3溶液和饱和NaCl溶液洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 10:1→5:1)分离纯化，得到产物15(20mg，0.029mmol，84％，α:β＝1:1.1)。
化合物16：将化合物26(1.45g，2.07mmol)溶于DCM/H2O(v/v 9:1，40mL)中，加入DDQ(0.71g，3.11mmol)。在室温下搅拌6h后，用5％(w/v)Na2S2O3溶液淬灭，然后用饱和NaHCO3和盐水洗涤，用DCM反萃。有机相用无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→4:1)分离纯化，得到化合物11(0.92g，1.64mmol，79％)。[α]25 D＝+108.1°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.62–7.04(m,15H,Ar-H),5.17(d,J＝5.8Hz,2H,ArCH),4.79(d,J＝11.7Hz,1H,ArCH),4.82–4.72(m,1H,1-H),4.66(d,J＝11.5Hz,1H,ArCH),4.56(d,J＝15.5Hz,1H,ArCH),4.51–4.40(m,1H,ArCH),4.08–3.73(m,2H,3-H,5-H),3.71–3.52(m,2H,CH2-Linker,4-H),3.51–3.21(m,4H,2-H,CH2-Linker),1.94–1.68(m,2H,CH2-Linker),1.22(d,J＝6.1Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ156.72,156.25,137.82,136.73,128.71,128.58,128.24,128.15,128.01,127.90,127.36,98.24(C-1),80.17,76.20,68.59,67.30,66.48,65.81,60.91,50.87,44.68,43.83,28.37,27.87,16.79；IR νmax(film)3444,2925,2106,1695,1496,1496,1454,1421,1359,1221,1165,1127,1103,1042,968,914,735,698cm-1；HR-ESI-MS(m/z):calcd for C31H36N4O6Na+(M+Na+):583.2527,found:583.2524.
实施例3
1,2-顺式-α-D-岩藻糖胺糖苷的合成优化：
如表1所示，为提高D-岩藻糖苷的α-糖苷键选择性，在不同条件下对一系列糖基供体进行了尝试。以TMSOTf作为活化剂在乙醚/二氯甲烷中探究三氟乙酰亚氨酯供体14a反应效率，得到的产物15在糖苷键选择性上略有提高，达到α:β＝1.5:1。随后，利用TMSI-Ph3PO试剂将供体14a与N-苄基-N-苄氧羰基-3-氨基丙醇进行糖基化，得到了产物15，α-构型选择性显著提高(α/β＝4.2:1)。鉴于酰基的远程参与效应和试剂效应相结合的协同策略，可增强1,2-顺式-α-氨基糖苷的立体选择性。因此，对化合物13的O4位上分别组装Ac、Bz和Lev，分别得到供体14b、14c和14d，以利用其酰基远程参与效应生成α-D-岩藻糖苷。最后，在远程参与和溶剂效应的协同作用下，得到了目标糖苷15a、15b和15c，并都具有较高的产率以及优异的立体选择性，α/β选择性分别为7.5:1、13.5:1和12.1:1。最终，选择苯甲酰基作为O4位Bz作为临时保护基，原因是化合物15c的α-构型异构体更易分离得到，且具有较高的产率(91％)。最后，利用甲醇钠脱除将化合物15c的苯甲酰基，并进行苄基化反应得到化合物15。
表1 1,2-顺式-α-D-岩藻糖胺糖苷的合成优化

具体试验操作和步骤：
化合物14a：室温下，向在THF/H2O(v/v 4:1，3mL)中搅拌的化合物14(130mg，0.31mmol)混合液中加入NBS(166mg，0.93mmol)，搅拌2.5h。当TLC显示起始原料完全转化时，用DCM稀释混合液，用10％(w/v)Na2S2O3和盐水洗涤。有机相经无水Na2SO4干燥过滤，减压浓缩。粗品经硅胶柱层析(PE/EA v/v 4:1→2:1)分离纯化，得到相应的半缩醛，为无色糖浆。将此半缩醛溶解于DCM(3mL)中。反应体系温度降至0℃，加入2,2,2-三氟-N-苯基乙酰亚胺氯(230μL，1.55mmol)和DBU(140μL，0.93mmol)。随后反应体系温度升至室温并搅拌3h。通过TLC监测分析反应进展，直至原料消耗殆尽。反应液在室温下减压浓缩得到粗品，粗品经硅胶柱层析(PE/EA v/v 20:1→10:1)分离纯化，得到淡黄色糖浆14a(153mg，0.26mmol，83％)。[α]25 D＝+33.7°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ8.13–6.41(m,17H,Ar-H),4.98(d,J＝11.6Hz,1H,ArCH),4.89(s,2H,ArCH),4.70(d,J＝11.5Hz,1H,ArCH),4.17–3.99(m,1H,5-H),3.58(s,1H,4-H),3.56–3.28(m,2H,2-H,3-H),1.20(d,J＝6.1Hz,3H,6-H)；13CNMR(100MHz,Chloroform-d)δ143.43,137.97,135.09,133.25,133.16,129.43,128.70,128.47,128.42,128.36,127.96,127.91,127.79,126.78,126.41,126.36,126.21,125.71,124.29,120.47,119.33,95.97,80.88,74.94,74.68,72.90,71.90,62.17,29.72,16.70；IR νmax(film)2931,2114,1720,1598,1549,1489,1452,1318,1285,1211,1163,1107,1082,1028,910,856,818,777,753,732,695cm-1；HR-ESI-MS(m/z):calcd for C32H29F3N4O4Na+(M+Na+):613.2033,found:613.2037.
化合物15：将供体14a(20mg，0.034mmol)和连接臂N-苄基-N-苄氧羰基-3-氨基丙醇(15mg，0.051mmol)与甲苯共沸并真空干燥过夜。然后将这些物质溶解于干燥的DCM/Et2O(v/v 3:1，1mL)中，并加入分子筛(火焰干燥)。将反应体系温度冷却至0℃。在氩气保护下，向反应液中加入TMSOTf(1.27μL，0.007mmol)。反应液在0℃下搅拌，直到TLC监测到反应结束。然后滴加三乙胺淬灭，过滤分子筛并用DCM稀释。有机相用饱和NaHCO3洗涤，随后通过无水Na2SO4干燥有机相并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 10:1→5:1)分离纯化，得到化合物15(21mg，0.03mmol，87％，α:β＝1.5:1)。
将供体14a(20mg，0.034mmol)和连接物N-苄基-N-苄氧羰基-3-氨基丙醇(15mg，0.051mmol)与无水甲苯共沸，然后在氩气保护下溶解于干燥的DCM(1mL)中。向反应瓶中加入分子筛(火焰干燥)和Ph3OP(67mg，0.24mmol)。然后向反应瓶中缓慢滴加TMSI(5μL，0.034mmol，1.0eq)。反应在室温下搅拌，直到TLC监测显示反应结束。用饱和Na2S2O3淬灭反应，过滤掉固体分子筛并用DCM洗涤。有机相用水和盐水洗涤，通过无水Na2SO4干燥。随后真空浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 10:1→5:1)分离纯化，得到化合物15(22mg，0.031mmol，91％，α:β＝4.2:1)。
15α:[α]25 D＝+74.6°(c 1.00,CH3Cl).1H NMR(600MHz,Chloroform-d)δ8.05–7.05(m,22H,Ar-H),5.17(d,J＝9.7Hz,2H,ArCH),4.93(d,J＝11.5Hz,1H,ArCH),4.89–4.75(m,3H,ArCH,1-H),4.63(d,J＝11.5Hz,1H,ArCH),4.58–4.39(m,2H,ArCH),4.00–3.88(m,1H,4-H),3.88–3.73(m,2H,2-H,5-H),3.72–3.54(m,2H,3-H,CH2-Linker),3.51–3.26(m,3H,CH2-Linker),1.97–1.69(m,2H,CH2-Linker),1.13(d,J＝7.5Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ156.70,156.22,138.24,137.87,136.81,135.19,133.32,133.09,128.55,128.49,128.34,128.29,128.26,127.98,127.96,127.85,127.75,127.73,127.31,126.53,126.21,126.03,125.69,98.26(C-1),77.70,76.25,74.94,72.39,67.25,66.69,65.74,59.69,50.85,50.55,44.63,43.81, 28.34,27.85,16.75；IR νmax(film)2920,2107,1697,1496,1454,1421,1359,1218,1169,1124,1044,965,914,857,818,749,699cm-1；HR-ESI-MS(m/z):calcd for C42H44N4O6Na+(M+Na+):723.3153,found:723.3157.
15β:[α]25 D＝-30.8°(c 1.00,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.89–7.15(m,22H,Ar-H),5.21–5.10(m,2H,ArCH),4.96(d,J＝11.7Hz,1H,ArCH),4.86(s,2H,ArCH),4.70(d,J＝11.6Hz,1H,ArCH),4.59–4.45(m,2H,ArCH),4.17–4.02(d,J＝8.0Hz,1H,1-H),3.98–3.84(m,1H,CH2-Linker),3.83–3.75(m,1H,2-H),3.54(dd,J＝2.9,1.0Hz,1H,4-H),3.52–3.41(m,1H,CH2-Linker),3.41–3.27(m,4H,3-H,5-H,CH2-Linker),1.96–1.77(m,2H,CH2-Linker),1.17(d,J＝6.3Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ156.79,138.21,137.96,135.17,133.22,133.08,128.50,128.44,128.35,128.24,127.92,127.88,127.83,127.75,127.72,127.37,127.23,126.64,126.26,126.08,125.75,102.08(C-1),80.68,75.00,74.75,72.69,70.55,67.15,63.20,50.88,44.65,43.60,28.49,27.99,16.84.IR νmax(film)2935,2110,1697,1603,1496,1474,1454,1420,1362,1271,1218,1172,1111,1067,1028,988,893,857,818,754,698,690,664,604cm-1；HR-ESI-MS(m/z):calcd for C42H44N4O6Na+(M+Na+):723.3153,found:723.3156.
化合物15a：将供体14b(49mg，0.09mmol)和连接臂HO(CH2)3NBnCbz(40mg，0.136mmol)与甲苯共沸，然后在氩气环境下溶解于干燥的DCM(2mL)中。向反应瓶中加入分子筛(火焰干燥)和Ph3OP(200mg，0.72mmol)。然后向反应瓶中缓慢滴加TMSI(13μL，0.09mmol，1.0eq)。反应在室温下搅拌，直到TLC监测显示反应结束。用饱和Na2S2O3淬灭反应，过滤掉固体分子筛并用DCM洗涤。有机相用水和盐水洗涤，通过无水Na2SO4干燥。随后真空浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 10:1→5:1)分离纯化，得到化合物15a(52mg，0.079mmol，88％，α:β＝7.5:1)。
化合物15b：将供体14c(65mg，0.10mmol)和连接臂HO(CH2)3NBnCbz(45mg，0.15mmol)与甲苯共沸，然后在氩气下溶解在干燥的DCM(2mL)中。向反应瓶中加入分子筛(火焰干燥)和Ph3OP(222mg，0.8mmol)。然后向反应瓶中缓慢滴加TMSI(15μL，0.10mmol，1.0eq)。反应在室温下搅拌，直到TLC监测显示反应结束。用饱和Na2S2O3淬灭反应，过滤掉固体分子筛并用DCM洗涤。有机相用水和盐水洗涤，通过无水Na2SO4干燥。随后真空浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v10:1→5:1)分离纯化，得到化合物15b(60mg，0.085mmol，85％，α:β＝13.5:1)。
化合物15c：将供体14d(1.70g，2.81mmol)和连接臂HO(CH2)3NBnCbz(1.26g，4.22mmol)与甲苯共沸，然后在氩气下溶解在干燥的DCM(28mL)中。向反应瓶中加入分子筛(火焰干燥)和Ph3OP(6.26g，22.5mmol)。然后向反应瓶中缓慢滴加TMSI(0.42mL，2.81mmol，1.0eq)。反应在室温下搅拌，直到TLC监测显示反应结束。用饱和Na2S2O3淬灭反应，过滤掉固体分子筛并用DCM洗涤。有机相用水和盐水洗涤，通过无水Na2SO4干燥。随后真空浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v10:1→5:1)分离纯化，得到化合物15c(1.83g，2.56mmol，91％，α:β＝12.1:1)。[α]25 D＝+159.9°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ8.21–7.08(m,22H,Ar-H),5.73–5.59(m,1H,4-H),5.18(s,2H,ArCH),5.00–4.81(m,2H,ArCH,1-H),4.73–4.61(m,1H,ArCH),4.59–4.37(m,2H,ArCH),4.16–3.94(m,2H,3-H,5-H),3.78–3.58(m,2H,2-H,CH2-Linker),3.53–3.27(m,3H,CH2-Linker),1.85(s,2H,CH2-Linker),1.18(d,J＝6.1Hz,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ166.14,156.65,156.23,137.77,136.61,134.65,133.29,133.19,133.02,131.46,129.86,129.61,128.55,128.47,128.16,128.00,127.92,127.87,127.60,127.33,127.25,127.01,125.95,125.86,98.19(C-1),74.18,71.40,69.93,67.27,65.97,65.21,59.33,50.84,50.51,44.52,43.71,28.23,27.78,16.33；IR νmax(film)2936,2108,1719,1698,1601,1496,1471,1452,1421,1358,1314,1269,1219,1169,1112,1052,1026,968,857,819,752,712,670cm-1；HR-ESI-MS(m/z):calcd for C42H42N4O7Na+(M+Na+):737.2946,found:737.2951.
化合物15：将化合物15c(1.82g，2.55mmol)溶解于甲醇(25mL)中的溶液中加入NaOMe(70mg，1.28mmol)，溶液在40℃条件下搅拌6h。溶液用Amberlite IR 120(H+)离子交换树脂中和并通过棉塞过滤。减压浓缩得到粗品，随后经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到相应的产物。将上述产物溶于DMF(13mL)中，加入NaH(在矿物油中的60％分散液)(205mg，5.10mmol)。然后在0℃下加入BnBr(0.45mL，3.82mmol)。将体系温度升至室温，在室温下充分搅拌3h。TLC监测反应结束后，用甲醇淬灭，并用水和盐水洗涤，DCM反萃。有机相用无水Na2SO4干燥并真空浓缩得到粗品。粗品经硅胶柱层析(PE/EA v/v 10:1→5:1)分离纯化，经两步反应得到化合物15(1.47g，2.1mmol，82％)。 [α]25 D＝+74.6°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ8.05–7.05(m,22H,Ar-H),5.17(d,J＝9.7Hz,2H,ArCH),4.93(d,J＝11.5Hz,1H,ArCH),4.89–4.75(m,3H,ArCH,1-H),4.63(d,J＝11.5Hz,1H,ArCH),4.58–4.39(m,2H,ArCH),4.00–3.88(m,1H,4-H),3.88–3.73(m,2H,2-H,5-H),3.72–3.54(m,2H,3-H,CH2-Linker),3.51–3.26(m,3H,CH2-Linker),1.97–1.69(m,2H,CH2-Linker),1.13(d,J＝7.5Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ156.70,156.22,138.24,137.87,136.81,135.19,133.32,133.09,128.55,128.49,128.34,128.29,128.26,127.98,127.96,127.85,127.75,127.73,127.31,126.53,126.21,126.03,125.69,98.26(C-1),77.70,76.25,74.94,72.39,67.25,66.69,65.74,59.69,50.85,50.55,44.63,43.81,28.34,27.85,16.75；IR νmax(film)2920,2107,1697,1496,1454,1421,1359,1218,1169,1124,1044,965,914,857,818,749,699cm-1；HR-ESI-MS(m/z):calcd for C42H44N4O6Na+(M+Na+):723.3153,found:723.3157.
实施例4
糖砌块19的合成：
如图6所示，以对甲苯硫基β-D-葡萄糖17为原料，利用四氢铝锂还原C3位叠氮基得到相应的氨基糖，然后对C3位氨基进行三氯乙氧羰基(Troc)的选择性保护，得到化合物18。随后对化合物18中C2位羟基进行乙酰丙酰基保护，可得到化合物19。
具体试验操作和步骤：
化合物18：在0℃下，将化合物17(1.6g，4.01mmol)置于THF(20mL)溶液中，加入LiAlH4(460mg，12.03mmol)。反应液在室温下搅拌1.5h，直到TLC显示原料完全转化，然后在0℃下，滴加H2O淬灭，并用DCM稀释。反应液用H2O、饱和NaHCO3溶液和盐水洗涤。有机相通过无水Na2SO4干燥，过滤并真空浓缩。粗品经硅胶柱层析(DCM/MeOHv/v 50:1→20:1)分离纯化，得到氨基糖。将氨基糖溶解于THF(35mL)和吡啶(0.97mL，12.03mmol，3eq)中。然后在0℃下，逐滴加含有TrocCl(0.55mL，4.01mmol)的THF(5mL)混合液。反应在0℃下搅拌2h，直到TLC显示起始原料完全转化，随后在0℃下加入甲醇淬灭反应。将反应液真空浓缩得到粗品，用硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆状的产物18(1.86g，3.41mmol，85％)。[α]25 D＝+57.3°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.51–7.10(m,9H,Ar-H),5.48(s,1H,ArCH),5.35–5.14(m,1H,NH),4.72(s,2H,CH2-Troc),4.61(d,J＝9.4Hz,1H,1-H),4.37(dd,J＝10.6,4.5Hz,1H,6’-H),3.85–3.79(m,1H,3-H),3.75(t,J＝10.2Hz,1H,6-H),3.63–3.49(m,3H,2-H,4-H,5-H),3.38(br,1H,2-OH),2.36(s,3H,ArCH3)；13C NMR(101MHz,Chloroform-d)δ155.26,138.84,136.69,133.71,129.89,129.28,128.32,127.42,126.15,101.75,95.27,89.82,74.69,71.81,71.53,68.58,58.35,21.20；IR νmax(film)3328,2881,1715,1542,1493,1451,1372,1318,1245,1168,1109,1071,1031,1006,810,749,733,698cm-1；HR-ESI-MS(m/z):calcd for C23H24Cl3NO6SNa+(M+Na+):570.0282,found:570.0279.
化合物19：将化合物18(1.84g，3.36mmol)置于干燥的DCM(33mL)中搅拌，加入乙酰丙酸(LevOH)(585mg，5.04mmol)、N,N-二环己基碳酰亚胺(DCC)(1.04g，5.04mmol)和DMAP(615mg，5.04mmol)。反应液在室温下搅拌4h。直到起始原料完全消耗，用DCM稀释反应液。有机相用饱和NaHCO3溶液和盐水洗涤，无水Na2SO4干燥并过滤，滤液真空浓缩得到粗品。用硅胶柱层析(PE/EA v/v 10:1→4:1)分离纯化，得到化合物19(2.06g，3.19mmol，95％)。[α]25 D＝+41.3°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.46–7.10(m,9H,Ar-H),5.47(s,1H,ArCH),5.12(d,J＝9.0Hz,1H,NH),4.90(t,J＝9.8Hz,1H,2-H),4.76(d,J＝12.1Hz,1H,CH2-Troc),4.73(d,J＝9.8Hz,1H,1-H),4.68(d,J＝12.0Hz,1H,CH2-Troc),4.37(dd,J＝10.7,4.4Hz,1H,6’-H),4.02(q,J＝9.4Hz,1H,3-H),3.75(t,J＝10.0Hz,1H,6-H),3.62–3.48(m,2H,4-H,5-H),2.83–2.73(m,2H,CH2-Lev),2.69–2.60(m,2H,CH2-Lev),2.35(s,3H,CH3CO),2.19(s,3H,ArCH3)；13C NMR(151MHz,Chloroform-d)δ205.95,171.82,154.35,138.83,136.74,133.87,129.77,129.14,128.23,127.59,126.13,101.56,95.56,87.07,77.92,74.49,72.00,70.54,68.48,56.75,37.89,29.79,28.01,21.20；IR νmax(film)3326,2880,1742,1721,1551,1375,1318,1262,1245,1156,1103,1069,1030,811,751,734,697cm-1；HR-ESI-MS(m/z):calcd for C28H30Cl3NO8SNa+(M+Na+):668.0650,found:668.0652.
实施例5
三糖中间体化合物22的合成：
如图7所示，以糖基供体9和受体16为原料，在活化试剂TMSOTf的作用下进行糖苷化，得 到二糖化合物20。随后，利用四丁基氟化胺(TBAF)脱除二糖C4位TBS，得到二糖受体21。最后，单糖供体19和二糖受体21在TMSOTf和NIS催化作用下进行糖基化，得到三糖中间体化合物22。
具体试验操作和步骤：
化合物20：在-10℃条件下，将供体9(1.43g，2.06mmol)、受体16(0.77g，1.37mmol)和分子筛溶于干燥的DCM(20mL)中。在氩气的保护下，滴加TMSOTf(75μL，0.412mmol)。当TLC检测显示起始原料完全转化时，用Et3N淬灭，然后过滤并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆20(1.71g，1.61mmol，78％)。[α]25 D＝+24.5°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.41–7.13(m,20H,Ar-H),5.21–5.12(m,4H,ArCH),5.03–4.97(m,1H,2’-H),4.83(d,J＝11.4Hz,1H,ArCH),4.81–4.73(m,1H,1-H),4.72–4.66(m,1H,1’-H),4.54(d,J＝15.5Hz,1H,ArCH),4.51–4.44(m,1H,ArCH),4.40(d,J＝11.4Hz,1H,ArCH),3.99–3.88(m,2H,5’-H,3-H),3.84(td,J＝9.2,1.3Hz,1H,4’-H),3.78–3.63(m,1H,5-H),3.65(dd,J＝10.6,3.7Hz,1H,2-H),3.57–3.52(m,1H,4-H),3.53–3.46(m,1H,3’-H),3.46–3.36(m,1H,CH2-Linker),3.38–3.26(m,1H,CH2-Linker),2.89–2.67(m,4H,CH2-Lev),2.16(s,3H,CH3CO),1.93–1.71(m,2H,CH2-Linker),1.06–0.96(m,3H,6-H),0.86(s,9H,SiC(CH3)3),0.18(s,3H,SiCH3),0.03(s,3H,SiCH3)；13C NMR(151MHz,Chloroform-d)δ205.95,171.33,167.26,156.65,156.26,138.49,137.87,136.77,134.83,102.48(C-1’),98.26(C-1),78.96,78.50,77.10,75.11,71.77,70.89,67.75,67.39,67.27,66.46,65.80,59.50,50.89,50.62,44.66,43.89,37.89,31.94,29.71,29.69,29.69,29.68,27.75,25.70,24.76,22.69,18.02,16.34,14.10,-4.45,-5.01；IR νmax(film)2917,2850,2107,1750,1697,1455,1418,1362,1255,1217,1134,1041,838,780,752,698cm-1；HR-ESI-MS(m/z):calcd for C55H69N7O13SiNa+(M+Na+):1086.4615,found:1086.4619.
化合物21：在室温下，将化合物20(1.70g，1.60mmol)溶于THF(16mL)中，加入AcOH(91μL，1.60mmol)和TBAF(2.4mL，2.4mmol)，搅拌5h。有机相用Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(PE/EA v/v 4:1→3:2)分离纯化，得到无色糖浆21(1.40g，1.47mmol，92％)。[α]25 D＝+21.6°(c 1.00,CH3Cl).1H NMR(600MHz,Chloroform-d)δ7.40–7.13(m,20H,Ar-H),5.26–5.11(m,4H,ArCH),4.87(dd,J＝10.3,7.9Hz,1H,2’-H),4.80(d,J＝11.4Hz,1H,ArCH),4.79–4.72(m,1H,1-H),4.72–4.65(m,1H,1’-H),4.57–4.42(m,2H,ArCH),4.38(d,J＝11.4Hz,1H,ArCH),4.01–3.88(m,2H,5’-H,3-H),3.85(t,J＝9.5Hz,1H,4’-H),3.77–3.64(m,1H,5-H),3.69–3.63(m,2H,2-H,4-H),3.65–3.54(m,1H,CH2-Linker),3.59(t,J＝9.9Hz,1H,3’-H),3.51–3.25(m,3H,CH2-Linker),2.87–2.64(m,4H,CH2-Lev),2.17(s,3H,CH3CO),1.91–1.71(m,2H,CH2-Linker),0.99(d,J＝6.3Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ206.05,171.31,156.23,138.53,137.83,134.45,128.97,128.82,128.58,128.50,128.17,128.02,127.82,127.57,127.38,127.22,102.61(C-1”),98.24(C-1),79.06,75.14,74.35,70.90,70.81,67.96,67.25,66.47,65.80,65.59,59.30,50.85,50.56,44.67,43.84,37.91,29.75,27.75,16.38；IR νmax(film)3445,2919,2108,1750,1697,1454,1422,1362,1314,1218,1161,1042,914,752,698cm-1；HR-ESI-MS(m/z):calcd for C49H55N7O13Na+(M+Na+):972.3750,found:972.3756.
化合物22：在-10℃下，硫苷供体19(0.56g，0.87mmol)、受体21(0.55g，0.58mmol)和分子筛置于干燥的DCM(6mL)中搅拌，加入NIS(235mg，1.04mmol)和TMSOTf(30μL，0.174mmoL)。反应在0℃下搅拌5h，直到TLC检测显示糖基供体完全转化。反应液用DCM稀释并过滤。滤液用10％(w/v)Na2S2O3、饱和NaHCO3溶液和盐水洗涤。有机相用无水Na2SO4干燥并过滤，滤液真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆22(648mg，0.44mmol，76％)。[α]25 D＝+10.2°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.46–7.16(m,25H,Ar-H),5.47(s,1H,ArCH),5.41(d,J＝11.8Hz,1H,ArCH),5.21–5.14(m,2H,ArCH),5.09(d,J＝11.8Hz,1H,ArCH),4.98(d,J＝9.2Hz,1H,NH),4.87–4.81(m,2H,2’-H,2”-H),4.82(d,J＝11.4Hz,1H,ArCH),4.80–4.74(m,1H,1-H),4.78(d,J＝12.3Hz,1H,CH2-Troc),4.74–4.67(m,2H,1’-H,CH2-Troc),4.54(d,J＝15.5Hz,1H,ArCH),4.52–4.47(m,1H,ArCH),4.46(d,J＝8.1Hz,1H,1”-H),4.43(d,J＝11.4Hz,1H,ArCH),4.34(dd,J＝10.7,5.0Hz,1H,6”-H),4.10–4.03(m,2H,5’-H,6’-H),4.00–3.91(m,1H,3-H),3.88(q,J＝10.3Hz,1H,3”-H),3.77-3.64(m,1H,5-H),3.72(t,J＝10.4Hz,1H,6”-H),3.65(dd,J＝10.8,3.5Hz,1H,4-H),3.63–3.51(m,4H,3’-H,CH2-Linker,2-H,4”-H),3.50–3.29(m,3H,CH2-Linker),3.18(td,J＝9.6,5.0Hz,1H,5”-H),2.86–2.49(m, 8H,CH2-Lev),2.18(s,3H,CH3CO),2.17(s,3H,CH3CO)1.92–1.71(m,2H,CH2-Linker),1.05–0.95(m,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ206.08,206.02,172.10,171.31,166.67,154.37,138.40,137.83,136.75,134.57,129.34,129.17,129.02,128.66,128.65,128.57,128.51,128.26,128.22,128.01,127.83,127.67,127.38,127.24,126.13,102.51(C-1’),101.60(ArCH),100.52(C-1”),98.23(C-1),95.54(CCl3-Troc),78.81,78.03,76.23,74.98,74.48,71.83,70.46,68.39,67.82,67.59,67.25,66.38,65.79,64.61,59.40,54.95,50.56,44.61,37.91,37.79,29.83,29.75,27.84,27.73,27.63,16.37；IR νmax(film)3323,2924,2111,1719,1543,1455,1420,1364,1314,1274,1239,1152,1081,1029,917,820,752,699cm-1；HR-ESI-MS(m/z):calcd for C70H77Cl3N8O21Na+(M+Na+):1493.4161,found:1493.4168.
实施例6
目标三糖30的合成：
如图8所示，以三糖中间体化合物22为原料，在乙酸肼的作用下同时脱除化合物22中的两个乙酰丙酰基，得到二元醇23。随后，使用三氟酸酐(Tf2O)和吡啶将化合物23中的两个羟基转化为三氟甲磺酰基，然后用四丁基叠氮化铵(TBAN3)在轴向位置进行置换，得到化合物24。然后，在三氟乙酸(TFA)介导下水解4,6-O-苄叉基后，将得到的二元醇25的C6”位羟基进行选择性氧化，生成相应的羧酸，然后使用溴化苄和碳酸氢钠(NaHCO3)进行选择性苄酯化反应，得到化合物26。在吡啶中利用硫代乙酸(AcSH)还原叠氮基，得到乙酰氨基糖27。随后，在55℃下使用过量的锌粉在乙酸中去除三糖27上的Troc基团，然后使用S-苄基硫代乙酰亚氨酸盐酸盐在吡啶中反应，得到C3”位含有乙脒基的三糖28。由于三糖26极性增大，使其难以溶解于二氯甲烷中，而易溶于甲醇中，发现其在甲醇中溶解、转移和保存的过程中，由于碱性乙脒基的作用，部分产物28发生了从羧酸苄酯转化为羧酸甲酯的酯交换反应，得到三糖29。最后，在0℃的利用氢氧化锂对三糖29上的甲酯进行水解，随后利用Pd/C氢化反应实现全脱保护，得到目标三糖30。
具体试验操作和步骤：
化合物23：将化合物22(620mg，0.422mmol)置于DCM/MeOH(v/v 20:1，4.2mL)溶液中搅拌，加入乙酸肼(117mg，1.27mmol)。反应液在室温下搅拌4.5h，直到TLC显示起始原料完全转化。用DCM稀释反应液，并用饱和NaHCO3溶液和盐水洗涤，有机相用无水Na2SO4干燥并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆产物23(457mg，0.359mmol，85％)。[α]25 D＝+35.8°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.49–7.14(m,25H,Ar-H),5.48(s,1H,ArCH),5.29(d,J＝11.9Hz,1H,ArCH),5.23(s,1H,NH),5.19–5.11(m,3H,ArCH),4.86–4.78(m,1H,1-H),4.77(d,J＝11.7Hz,1H,ArCH),4.77–4.72(m,2H,CH2-Troc),4.58–4.49(m,1H,1’-H),4.50(d,J＝7.4Hz,1H,1”-H),4.43(d,J＝11.6Hz,1H,ArCH),4.40(dd,J＝10.7,5.0Hz,1H,6”-H),4.08–4.02(m,1H,5’-H),4.03–3.94(m,1H,3-H),3.91(t,J＝9.5Hz,1H,4’-H),3.80–3.65(m,3H,6”-H,2-H,5-H),3.72(dd,J＝10.1,7.6Hz,1H,3”-H),3.65–3.56(m,1H,CH2-Linker),3.65–3.60(m,2H,4”-H,4-H),3.59–3.51(m,2H,2”-H,3’-H),3.43–3.39(m,1H,5”-H),3.48–3.28(m,3H,CH2-Linker),3.38(dd,J＝9.9,7.7Hz,1H,2’-H),2.90(s,1H,2’-OH),1.91–1.70(m,2H,CH2-Linker),1.04–0.94(m,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ167.89,155.05,138.35,137.79,136.82,134.56,129.24,129.08,128.85,128.59,128.54,128.51,128.40,128.31,128.24,128.03,127.85,127.74,127.41,127.25,126.17,105.07(C-1’),104.34(C-1”),101.78(ArCH),97.87(C-1),95.39(CCl3-Troc),80.27,75.02,74.67,74.46,73.11,72.47,68.50,68.16,67.86,67.28,66.88,66.36,65.89,59.35,56.90,27.82,27.23,16.35.IR νmax(film)3390,2925,2112,1743,1683,1537,1497,1454,1423,1363,1239,1220,1176,1082,1029,916,819,752,698cm-1；HR-ESI-MS(m/z):calcd for C60H65Cl3N8O17Na+(M+Na+):1297.3425,found:1297.3431.
化合物24：在氮气保护下，将化合物23(440mg，0.345mmol)置于干燥的DCM(6.0mL)和吡啶(0.56mL，6.9mmol，20eq)的混合溶液中搅拌。反应体系温度降至-20℃，加入，滴加含有Tf2O(0.35mL，2.07mmol)的干燥DCM(1mL)混合液。搅拌反应液，使其在4h内从-20℃升温至0℃。有机相经无水硫酸钠干燥后，在30℃条件下真空浓缩，得到黄色糖浆状的三氯酸盐产物。在氮气环境下，将上述三酸盐溶解于无水DMF(7mL)溶液中。于0℃加入TBAN3(0.98g，3.45mmol)。反应在室温下搅拌过夜。通过TLC监测分析反应进展。反应液用乙酸乙酯(20mL)稀释，用水和盐水洗涤，有机相经无 水Na2SO4干燥并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 2:1)分离纯化。通过两步反应得到产物24(334mg，0.252mmol，73％)。[α]25 D＝-8.4°(c＝1.00,CHCl3)；1H NMR(600MHz,Chloroform-d)δ7.60–7.05(m,25H,Ar-H),5.46(s,1H,ArCH),5.29(d,J＝11.6Hz,1H,ArCH),5.25(d,J＝8.5Hz,1H,NH),5.22–5.12(m,3H,ArCH),4.99(d,J＝11.4Hz,1H,ArCH),4.92–4.81(m,1H,1’-H),4.81–4.72(m,3H,CH2-Troc,1-H),4.66(s,1H,1”-H),4.59–4.43(m,3H,ArCH),4.35(dd,J＝11.1,4.9Hz,1H,6”-H),4.22(t,J＝9.6Hz,1H,4’-H),4.14–4.08(m,1H,5’-H),4.08–3.97(m,1H,3-H),3.96–3.85(m,3H,3”-H,2-H,2’-H),3.83(s,1H,2”-H),3.78(t,J＝10.4Hz,1H,6”-H),3.75–3.68(m,1H,5-H),3.68–3.53(m,2H,4-H,CH2-Linker),3.56(t,J＝9.7Hz,1H,4”-H),3.52–3.26(m,4H,CH2-Linker,3’-H),3.22(td,J＝9.9,4.9Hz,1H,5”-H),1.90–1.70(m,2H,CH2-Linker),1.01–0.92(m,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ167.18,154.03,138.34,137.84,136.85,134.67,129.40,129.28,129.18,129.04,128.75,128.70,128.59,128.52,128.27,128.24,128.13,128.02,127.89,127.81,127.75,127.39,127.20,126.14,102.01(C-1’),101.95(C-1”),101.40(ArCH),97.52(C-1),95.38(CCl3-Troc),80.80,78.25,75.77,75.02,74.66,74.57,74.25,68.36,68.17,68.01,67.23,66.41,65.75,63.57,62.68,62.23,60.40,59.43,52.08,50.77,50.52,44.57,43.72,28.20,27.83,16.39；IR νmax(film)2920,2108,1743,1693,1455,1275,1234,1178,1090,1045,1000,905,822,753,699cm-1；HR-ESI-MS(m/z):calcd for C60H63Cl3N14O15Na+(M+Na+):1347.3555,found:1347.3559.
化合物25：将化合物24(240mg，0.181mmol)置于DCM(4.0mL)溶液中搅拌，加入三氟乙酸(0.5mL，6.72mmol)和H2O(33μL，1.81mmol，10eq)。反应在室温下搅拌4h，直到TLC显示起始原料完全转化。用DCM稀释反应液，用水、饱和NaHCO3溶液和盐水洗涤，有机相经无水Na2SO4干燥并真空浓缩。粗品经硅胶柱层析(DCM/MeOH v/v 50:1→20:1)分离纯化，得到无色糖浆25(199mg，0.161mmol，89％)。[α]25 D＝-5.5°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.42–7.14(m,20H,Ar-H),5.53(d,J＝7.8Hz,1H,NH),5.28–5.11(m,4H,ArCH),4.99(d,J＝11.5Hz,1H,ArCH),4.93–4.79(m,1H,1’-H),4.82(d,J＝11.9Hz,1H,CH2-Troc),4.82–4.70(m,1H,1-H),4.77(d,J＝12.0Hz,1H,CH2-Troc),4.62(s,1H,1”-H),4.56(d,J＝11.5Hz,1H,ArCH),4.55–4.42(m,2H,ArCH),4.21(t,J＝9.6Hz,1H,4’-H),4.09(d,J＝8.8Hz,1H,5’-H),4.07–3.97(m,1H,3-H),3.97–3.86(m,1H,2-H),3.93–3.86(m,1H,2’-H),3.92(dd,J＝12.2,3.1Hz,1H,6”-H),3.83(dd,J＝12.3,3.9Hz,1H,6”-H),3.79–3.66(m,1H,5-H),3.66–3.55(m,5H,4”-H,4-H,CH2-Linker,2”-H,3”-H),3.53–3.26(m,4H,3’-H,CH2-Linker),3.15(dt,J＝8.1,3.5Hz,1H,5”-H),2.84(br,1H,4”-OH),2.28(br,1H,6”-OH),1.93–1.70(m,2H,CH2-Linker),1.02–0.91(m,3H,6-H)；13CNMR(151MHz,Chloroform-d)δ167.20,156.61,156.25,155.16,138.28,137.79,136.57,134.67,129.34,129.25,128.98,128.78,128.59,128.50,128.23,128.02,127.81,127.75,127.42,127.37,127.17,101.86(C-1’),101.25(C-1”),97.56(C-1),95.24(CCl3-Troc),80.71,78.19,75.02,74.92,74.55,74.47,67.97,67.24,66.46,66.38,65.73,63.32,62.42,62.06,61.88,59.40,54.55,50.74,50.49,44.54,43.74,28.16,27.75,16.37.IR νmax(film)3411,2926,2109,1738,1697,1516,1455,1423,1362,1273,1168,1076,1045,820,737,699cm-1；HR-ESI-MS(m/z):calcd for C53H59Cl3N14O15Na+(M+Na+):1259.3242,found:1259.3247.
化合物26：在氩气保护下，将化合物25(190mg，0.154mmol)溶解于DCM(4mL)和H2O(1mL)溶液中。使用冰浴将体系温度降至0℃，加入TEMPO(5mg，0.031mmol)和BAIB(99gm，0.308mmol)。在室温下搅拌4h。通过TLC监测分析反应进展，直至原料消耗殆尽。滴加10％(w/v)Na2S2O3溶液淬灭，有机相通过饱和NaHCO3溶液和饱和NaCl溶液洗涤，DCM反萃。随后通过无水Na2SO4干燥有机相并真空浓缩。将粗品在氮气的保护下溶解于无水DMF(3mL)中。在室温下，加入NaHCO3(65mg，0.77mmol)和BnBr(55μL，0.462mmol)。当TLC显示原料完全消耗时，真空浓缩除去溶剂。用硅胶柱层析(PE/EA v/v 2:1)纯化。两步反应得到糖浆26(2.75g，6.16mmol，80％)。[α]25 D＝-12.5°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.43–7.13(m,25H,Ar-H),5.36(d,J＝8.4Hz,1H,NH),5.27(d,J＝12.2Hz,1H,ArCH),5.26–5.22(m,1H,ArCH),5.21(d,J＝12.2Hz,1H,ArCH),5.19–5.12(m,3H,ArCH),4.98(d,J＝11.5Hz,1H,ArCH),4.90–4.77(m,1H,1’-H),4.84(d,J＝12.0Hz,1H,CH2-Troc),4.80–4.72(m,1H,1-H),4.72(d,J＝12.0Hz,1H,CH2-Troc),4.64(s,1H,1”-H),4.56(d,J＝11.5Hz,1H,ArCH),4.56–4.43(m,2H,ArCH),4.22(t,J＝9.6Hz,1H,4’-H),4.11–3.96(m,2H,5’-H,3-H),3.95–3.83(m,2H,2-H,2’-H),3.77–3.61(m,6H,4”-H,5-H,4-H,2”-H,3”-H,5”-H),3.63–3.53(m,1H,CH2-Linker),3.53–3.27(m,4H,3’-H, CH2-Linker),3.17(br,1H,4”-OH),1.89–1.70(m,2H,CH2-Linker),0.96(t,J＝8.5Hz,3H,6”-H)；13C NMR(151MHz,Chloroform-d)δ168.67,167.18,156.60,156.22,154.55,138.32,137.80,136.83,136.59,134.71,129.33,129.23,128.97,128.78,128.63,128.58,128.49,128.43,128.21,128.01,127.80,127.72,127.38,127.16,101.73(C-1’),101.39(C-1”),97.56(C-1),95.24(CCl3-Troc),80.65,78.12,75.34,74.96,74.89,74.87,74.48,67.95,67.79,67.22,66.37,65.71,63.35,62.62,61.66,59.39,53.75,50.77,50.49,44.54,43.72,28.17,27.74,16.38.IR νmax(film)3367,2925,2110,1743,1697,1517,1455,1362,1273,1176,1127,1075,1044,1001,823,753,698cm-1；HR-ESI-MS(m/z):calcd for C60H63Cl3N14O16Na+(M+Na+):1363.3504,found:1259.3501.
化合物27：在氮气保护下，将化合物26(56mg，0.042mmol)置于吡啶(2.1mL)溶液中搅拌。反应体系温度降至0℃，加入AcSH(2.1mL)。反应在室温下搅拌，直到TLC检测显示起始原料完全转化。用DCM稀释反应液，用水、饱和NaHCO3溶液和盐水洗涤，有机相经无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(DCM/MeOH v/v 50:1→20:1)分离纯化，得到无色糖浆产物27(48mg，0.034mmol，81％)。[α]25 D＝+16.2°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.91–7.74(m,1H,NH),7.45–7.16(m,25H,Ar-H),6.96–6.86(m,1H,NH),6.86–6.79(m,1H,NH),6.80–6.72(m,1H,NH),6.08–5.86(m,1H,NH),5.29(d,J＝11.9Hz,1H,ArCH),5.23–5.04(m,5H,ArCH),4.91–4.83(m,2H,1’-H,ArCH),4.80(d,J＝12.0Hz,1H,CH2-Troc),4.72(d,J＝12.1Hz,1H,CH2-Troc),4.71–4.66(m,1H,3’-H),4.65–4.59(m,2H,1-H,ArCH),4.54(d,J＝11.8Hz,1H,ArCH),4.38(d,J＝15.9Hz,1H,ArCH),4.31–4.19(m,3H,3”-H,-H,1”-H),4.09(d,J＝9.8Hz,1H,5”-H),3.98(q,J＝10.6,9.5Hz,1H,4”-H),3.90–3.82(m,2H,4’-H,5-H),3.79–3.71(m,3H,3-H,4-H,CH2-Linker),3.68(s,1H,2’-H),3.65–3.61(m,1H,CH2-Linker),3.55(d,J＝9.5Hz,1H,5’-H),3.33–3.23(m,2H,CH2-Linker,2-H),3.23–3.15(m,1H,CH2-Linker),2.13(s,3H,CH3CO),2.02(s,3H,CH3CO),1.89(s,3H,CH3CO),1.78(s,3H,CH3CO),1.08(d,J＝6.4Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ175.35,172.52,172.22,171.43,168.43,167.65,157.71,156.53,138.68,137.33,136.40,135.02,134.75,129.51,129.38,129.00,128.71,128.65,128.55,128.47,128.24,128.15,127.83,127.70,127.61,127.22,101.44(C-1’),98.02(C-1”),97.82(C-1),95.26(CCl3-Troc),80.59,79.40,74.96,74.21,69.58,67.79,67.48,67.33,66.76,63.43,57.96,52.78,51.84,51.22,49.65,48.50,42.66,27.20,23.50,23.22,23.15,22.78,16.64；IRνmax(film)3306,2926,1743,1668,1537,1455,1423,1373,1240,1180,1093,1050,819,736,698cm-1；HR-ESI-MS(m/z):calcd for C68H79Cl3N6O20Na+(M+Na+):1427.4307,found:1427.4311.
化合物28：将化合物27(30mg，21.4μL)置于乙酸(2.0mL)溶液中搅拌，加入已活化的锌粉(50mg，0.78mmol)。在室温下搅拌12h后，用硅藻土过滤反应混合物，滤液真空浓缩。得到的粗品经硅胶柱层析(DCM/MeOH v/v 20:1→10:1)分离纯化，得到相应的氨基糖。在0℃下，将上述氨基糖置于无水吡啶(2mL)溶液中搅拌，加入苄基硫代乙酰亚胺盐酸盐(8.6mg，42.8μmol)。反应在0℃下搅拌5h。随后真空浓缩反应液，粗品经硅胶柱层析(DCM/MeOH v/v 10:1)分离纯化。经两步反应得到无色糖浆产物28(19mg，15.6μmol，73％)。[α]25 D＝-2.7°(c 1.00,CH3Cl)；1H NMR(600MHz,Methanol-d4)δ7.43–7.17(m,25H,Ar-H),5.37(d,J＝12.3Hz,1H,ArCH),5.26(s,2H,ArCH),5.19–5.10(m,3H,ArCH),5.08(s,1H,1’-H),4.99–4.90(m,1H,1-H),4.88(d,J＝11.4Hz,1H,ArCH),4.65–4.58(m,1H,1”-H),4.57–4.53(m,1H,2-H),4.53–4.50(m,2H,),4.47–4.45(m,1H,2’-H),4.45–4.39(m,1H,2”-H),4.35(d,J＝11.3Hz,1H,ArCH),4.26–4.15(m,2H,3-H,5-H),4.13–4.06(m,2H,5’-H,4-H),4.01–3.89(m,2H,3”-H,3’-H),3.83–3.76(m,2H,5-H,4’-H),3.66–3.59(m,1H,4”-H),3.59–3.48(m,1H,CH2-Linker),3.46–3.33(m,3H,CH2-Linker),2.20(s,3H,CH3CO),2.02(s,3H,CH3CO),1.95–1.90(m,3H,CH3CO),1.85(s,3H,CH3CO),1.81(s,3H,CH3CO),1.05–0.95(m,3H,6-H)；13C NMR(151MHz,Methanol-d4)δ175.03,174.27,173.88,173.71,170.65,169.77,167.49,140.34,139.29,138.10,136.71,136.56,129.77,129.66,129.21,129.17,128.93,128.53,128.33,127.99,103.49(C-1),100.69(C-1’),99.03(C”-1),80.53,78.19,76.46,76.14,68.81,68.47,67.73,67.58,66.05,58.19,53.03,52.26,51.35,50.21,45.66,45.49,29.43,28.80,23.04,22.87,22.78,19.24,16.97；IR νmax(film)3291,2931,1743,1657,1550,1454,1374,1293,1244,1177,1112,1051,739,699,599cm-1；HR-ESI-MS(m/z):calcd for C67H82N7O18 +(M+H+):1272.5711,found:1272.5714.
化合物30：在0℃下，将化合物29(11mg，9.20μmol)置于THF(1.0mL)溶液中，加入1M LiOH水溶液(20μL)。TLC显示起始原料完全转化后，用DCM稀释混合物，用水和盐水洗涤。经过滤后，滤 液减压浓缩得到粗品。粗品用硅胶柱层析(DCM/MeOH v/v 10:1)分离纯化，得到半脱保护产物。将半保护产物置于THF/MeOH/H2O/AcOH(v/v/v/v 10:5:4:1，2mL)溶液中，加入10％Pd/C(50mg)。反应在H2环境下搅拌24h。随后过滤并真空浓缩，通过Sephadex LH-20柱(H2O)洗脱。经两步反应得到白色固体30(4.5mg，5.88μmol，64％)。1H NMR(600MHz,Deuterium Oxide)δ4.98(s,1H,1’-H),4.84(s,1H,1-H),4.77(s,1H,1”-H),4.51(d,J＝3.3Hz,1H,2”-H),4.41–4.38(m,1H,2’-H),4.24(dd,J＝11.7,3.4Hz,1H,2-H),4.16–4.12(m,1H,3’-H),4.05(q,J＝6.4Hz,1H,5-H),4.03–3.98(m,3H,3-H,4-H,5”-H),3.97–3.92(m,2H,5’-H,3”-H),3.85–3.81(m,2H,4’-H,4”-H),3.78(dt,J＝11.3,6.4Hz,1H,CH2-Linker),3.54(dt,J＝11.3,6.1Hz,1H,CH2-Linker),3.11(t,J＝7.5Hz,2H,CH2-Linker),2.20(s,3H,CH3C(N)),2.05(s,3H,CH3CO),2.05(s,3H,CH3CO),2.02–1.97(m,2H,CH2-Linker),1.99(s,3H,CH3CO),1.97(s,3H,CH3CO),1.23(d,J＝6.5Hz,3H,6-H)；13C NMR(151MHz,Deuterium Oxide)δ174.82,174.26,174.20,173.93,166.06,163.13,162.89,99.90(C-1’),98.56(C-1”),97.23(C-1),78.23,77.39,76.93,75.13,70.00,66.54,65.70,64.94,56.20,51.95,51.35,49.49,47.75,37.14,26.74,21.92,21.91,21.80,21.75,18.53,15.35；HR-ESI-MS(m/z):calcd for C31H52N7O16 +(M+H+):778.3465,found:778.3466.
对比例1
三糖35的合成尝试：
如图9所示，中间体化合物7为原料，在溴化苄和氧化银的作用下对化合物7的C4位羟基进行苄基保护，得到化合物31。随后，利用氯化钯(PdCl2)脱除化合物31还原端的烯丙基，得到相应的半缩醛衍生物，然后将其转化为N-苯基三氟亚胺酯供体32。在TMSOTf的催化作用下，化合物32与对甲苯硫酚反应，得到硫苷供体33。中间体二糖21在锌粉和醋酸酐作用下，将C2和C3’位的叠氮基还原为氨基乙酰基，得到化合物34。随后，以TMSOTf为活化剂，将二糖受体34分别与三氟乙酰亚氨酯供体32和硫苷供体33进行糖基化反应，尝试组装合成三糖35。
具体试验操作和步骤：
化合物31：在氮气下保护下，将葡萄糖醛酸苄酯7(1.2g,2.68mmol)溶解于无水DCM(27mL)中，在0℃条件下加入BnBr(1.59mL,13.4mmol)和Ag2O(1.86g,8.04mmol)。将反应混合物升温至30℃并搅拌24小时。通过硅藻土过滤并浓缩后，通过硅胶柱色谱法(石油醚/乙酸乙酯10:1v/v)纯化残余物，得到白色固体产物31(1.14g，2.12mmol，79％)。[α]25 D＝+68.4°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.40–7.16(m,10H,Ar-H),5.94–5.81(m,1H,All-CH),5.28(dq,J＝17.2,1.6Hz,1H,All-CH2),5.20(s,2H,ArCH),5.07(d,J＝3.6Hz,1H,1-H),4.75–4.67(m,2H,2-H,ArCH),4.47(d,J＝10.5Hz,1H,ArCH),4.30(d,J＝9.8Hz,1H,5-H),4.20(ddt,J＝13.0,5.3,1.5Hz,1H,All-CH2),4.07–4.01(m,1H,All-CH2),4.00(t,J＝10.1Hz,1H,3-H),3.66(t,J＝9.7Hz,1H,4-H),2.88–2.57(m,4H,CH2-Lev),2.19(s,3H,CH3CO)；13C NMR(101MHz,Chloroform-d)δ206.06,171.85,168.82,140.90,137.13,134.96,133.01,128.66,128.59,128.51,128.44,128.42,128.17,128.05,127.68,127.00,118.48,77.99,75.07,71.51,70.33,69.14,67.51,65.40,63.04,37.80,29.82,27.86；IR νmax(film)＝2926,2108,1747,1716,1455,1362,1181,1154,1053,739,697cm-1；HR-ESI-MS(m/z):calcd for C28H31N3O8Na+(M+Na+):560.2003,found:560.2005.
化合物32：将化合物31(0.64g,1.19mmol)溶于在MeOH/DCM(2/1,v/v,24mL)中，加入PdCl2(210mg,1.19mmol)并将反应在35℃下搅拌4.5小时。将混合物过滤并真空浓缩，通过硅胶柱色谱法(石油醚/乙酸乙酯2:1v/v)纯化，得到相应的半缩醛。在0℃下，向上述半缩醛的DCM(12mL)溶液中添加2,2,2-三氟-N-苯基亚氨基乙酰氯(0.89mL,5.95mmol)和DBU(0.53mL,3.57mmol)。将反应升温至室温并搅拌3小时。将溶液真空浓缩至残余物，将其通过硅胶柱色谱法(石油醚/乙酸乙酯20:1→10:1v/v)纯化，得到淡黄色糖浆32(0.62g,0.93mmol,78％,两步反应)。[α]25 D＝+31.1°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.43–6.76(m,15H,Ar-H),5.23(d,J＝12.2Hz,1H,ArCH),5.19(d,J＝12.2Hz,1H,ArCH),5.13–5.04(m,1H,2-H),4.68(d,J＝10.5Hz,1H,ArCH),4.49(d,J＝10.5Hz,1H,ArCH),4.17–3.89(m,1H,5-H),3.83(t,J＝9.5Hz,1H,4-H),3.75–3.63(m,1H,3-H),2.84–2.78(m,2H,CH2-Lev),2.67–2.62(m,2H,CH2-Lev),2.18(s,3H,CH3CO)；13C NMR(151MHz,Chloroform-d)δ205.76,171.05,167.20,142.92,136.71,134.69,128.78,128.73,128.70,128.64,128.48,128.25,128.22, 124.57,119.25,94.49,77.29,75.54,75.14,70.60,67.81,65.67,37.88,29.67,27.74；IR νmax(film)＝2919,2109,1749,1719,1597,1489,1455,1405,1365,1327,1212,1161,1087,1028,910,778,755,696cm-1.
化合物33：在-20℃，N-苯基三氟乙酰亚氨酯供体32(0.45g，0.67mmol)置于无水DCM(7mL)中，加入TolSH(83mg，0.67mmol)和分子筛(火焰干燥)。在氩气环境下，滴加TMSOTf(23μL，0.134mmol)。当TLC显示起始物质完全转化时，用Et3N淬灭反应物，然后过滤并真空浓缩。粗品经硅胶柱层析(石油醚/乙酸乙酯v/v 5:1)分离纯化，得到白色固体33(343mg，0.57mmol，85％)。[α]25 D＝-14.32(c 1.0,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.36(d,J＝6.5Hz,5H,Ar-H),7.30–7.24(m,5H,Ar-H),7.18(d,J＝7.8Hz,2H,Ar-H),7.05(d,J＝7.7Hz,2H,Ar-H),5.23–5.16(m,2H,ArCH),4.81(dd,J＝10.8,8.9Hz,1H,2-H),4.67(d,J＝10.5Hz,1H,ArCH),4.56(d,J＝9.9Hz,1H,1-H),4.49(d,J＝10.5Hz,1H,ArCH),3.95(d,J＝9.5Hz,1H,5-H),3.70(t,J＝9.6Hz,1H,4-H),3.62(t,J＝9.7Hz,1H,3-H),2.83(td,J＝6.6,2.1Hz,2H,Lev-H),2.69(q,J＝6.9Hz,2H,Lev-H),2.32(s,3H,Lev-CH3),2.21(d,J＝1.8Hz,3H,Tol(CH3))；13C NMR(151MHz,Chloroform-d)δ205.94,171.15,167.25,138.74,136.97,134.92,133.76,129.72,128.66,128.62,128.53,128.43,128.21,128.16,128.10,127.64,87.00,78.55,77.55,75.06,70.04,67.87,67.56,37.83,29.84,27.99,21.18；IR νmax(film)＝2919,2107,1748,1718,1494,1455,1401,1363,1262,1201,1174,1150,1072,1020,910,810,752,698,580cm-1；HR-ESI-MS(m/z):calcd for C32H33N3O7SNa+(M+Na+):626.1932,found:626.1929.
化合物34：在室温下，将化合物21(1.70g，1.60mmol)置于THF(16mL)溶液中搅拌，加入AcOH(91μL，1.60mmol)和TBAF(2.4mL，2.4mmol)，搅拌5h。有机相用无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(PE/EA v/v 4:1→3:2)分离纯化，得到无色糖浆34(1.40g，1.47mmol，92％)。[α]25 D＝+37.8°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.40–7.14(m,20H,Ar-H),6.71–6.57(m,2H,NH),5.31–5.14(m,4H,ArCH),4.88(d,J＝11.6Hz,1H,ArCH),4.78(t,J＝7.1Hz,1H,2’-H),4.75(d,J＝6.4Hz,1H,1’-H),4.67–4.56(m,3H,2-H,1-H,ArCH),4.53(d,J＝11.6Hz,1H,ArCH),4.40(d,J＝15.9Hz,1H,ArCH),4.07(d,J＝9.2Hz,1H,5’-H),4.01–3.90(m,2H,3’-H,4’-H),3.83–3.71(m,2H,5-H,3-H),3.69–3.57(m,3H,4-H,CH2-Linker),3.39–3.22(m,2H,CH2-Linker),2.90(ddd,J＝18.4,9.8,4.4Hz,1H,CH2-Lev),2.81–2.71(m,1H,CH2-Lev),2.69–2.55(m,2H,CH2-Lev),2.15(s,3H,CH3CO),2.04(s,3H,CH3CO),2.01(s,3H,CH3CO),1.81–1.58(m,2H,CH2-Linker),1.08(d,J＝6.4Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ206.81,174.23,173.56,170.80,168.55,156.43,138.80,137.42,136.46,135.27,128.69,128.58,128.41,128.28,128.14,128.13,127.88,127.69,127.52,127.25,101.48(C-1’),97.88(C-1),79.69,78.94,75.78,74.92,73.02,70.37,67.43,67.20,66.74,63.58,57.57,49.72,48.34,42.92,38.04,29.68,27.69,27.25,23.38,22.72,16.58；IRνmax(film)3305,2924,1747,1661,1549,1454,1423,1372,1218,1177,1127,1072,1042,751,699,593cm-1；HR-ESI-MS(m/z):calcdfor C53H63N3O15Na+(M+Na+):1004.4151,found:1004.4157.
化合物35：
条件一：在-10℃下，供体32(21mg，0.0306mmol)、受体34(20mg，0.0204mmol)和分子筛(火焰干燥)置于干燥的DCM(1.5mL)溶液中搅拌。在氩气保护下，滴加TMSOTf(1.1μL，0.006mmol)。当TLC检测显示糖基供体完全转化时，滴加Et3N将反应淬灭，然后过滤并真空浓缩。粗品经硅胶柱层析分离纯化，并用MALDI-TOF和NMR进行检测。未反应的受体和供体降解产物表明糖基化反应失败。
条件二：在-20℃下，硫苷供体33(18mg，0.0306mmol)、受体34(20mg，0.0204mmol)和分子筛置于干燥的DCM(1.5mL)中搅拌，加入NIS(8.3mg，0.0367mmol)和TMSOTf(1.1μL，0.006mmoL)。反应在0℃下搅拌5h，直到TLC检测显示糖基供体完全转化。反应液用DCM稀释并过滤。滤液用10％(w/v)Na2S2O3、饱和NaHCO3溶液和盐水洗涤。有机相用无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析分离纯化，仅得到痕量产物。大量未反应的受体残留表明糖基化反应失败。
对比例2
三糖38的合成尝试：
如图10所示，为了提高二糖受体反应活性，尝试使用叠氮未还原的二糖8作为受体，进行三糖的组装尝试。首先，尝试以三氟乙酰亚氨酯32和硫代糖苷33作为糖基供体分别与二糖受体8进行糖基化反应， 得到仅具有β-构型的三糖27，产率分别为15％和21％。虽然产率并不理想，但二糖受体的反应性得到了改善，而且硫代糖苷供体的反应性高于三氟乙酰亚胺酯供体。为了实现C3”位氨基的正交修饰，我们尝试在-10℃至0℃下，以TMSOTf和NIS为活化剂，进行硫代糖苷供体37与二糖受体21的糖基化反应，尝试合成三糖38。然而目标三糖29产率并不理想，仅为3％，反应得到了供体分解的副产物以及糖基受体21。
具体试验操作和步骤：
化合物36：条件一：在-10℃下，供体32(21mg，0.0316mmol)、受体21(20mg，0.0211mmol)和分子筛(火焰干燥)置于干燥的DCM(1.6mL)溶液中搅拌。在氩气保护下，滴加TMSOTf(1.1μL，6.32μmol)。当TLC检测显示糖基供体完全转化时，滴加Et3N将反应淬灭，然后过滤并真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆36(4.5mg，4.74μmol，15％)。
条件二：在-20℃下，硫苷供体33(19mg，0.0316mmol)、受体21(20mg，0.0211mmol)和分子筛置于干燥的DCM(1.5mL)中搅拌，加入NIS(8.5mg，0.038mmol)和TMSOTf(1.1μL，6.62μmoL)。反应在0℃下搅拌5h，直到TLC检测显示糖基供体完全转化。反应液用DCM稀释并过滤。滤液用10％(w/v)Na2S2O3、饱和NaHCO3溶液和盐水洗涤。有机相用无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(PE/EA v/v 5:1→2:1)分离纯化，得到无色糖浆36(9.5mg，6.64μmol，21％)。[α]25 D＝+18.9°(c 0.50,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.38–7.14(m,30H,Ar-H),5.32(d,J＝11.8Hz,1H,ArCH),5.20–5.13(m,4H,ArCH),5.11(d,J＝11.9Hz,1H,ArCH),4.85(dd,J＝10.4,7.9Hz,1H,2’-H),4.80(d,J＝11.4Hz,1H,ArCH),4.82–4.74(m,1H,1-H),4.75(dd,J＝10.3,7.9Hz,1H,2”-H),4.73–4.66(m,1H,1’-H),4.62(d,J＝10.7Hz,1H,ArCH),4.55–4.46(m,2H,ArCH),4.44(J＝10.4Hz,1H,ArCH),4.44(d,J＝8.3Hz,1H,1”-H),4.40(d,J＝11.5Hz,1H,ArCH),4.14–4.07(m,1H,5’-H),3.98(t,J＝9.5Hz,1H,4’-H),3.96–3.88(m,1H,3-H),3.77(d,J＝9.6Hz,1H,5”-H),3.71–3.75(m,1H,5-H),3.69(t,J＝9.6Hz,1H,4”-H),3.67–3.64(m,2H,2-H),3.64–3.58(m,1H,CH2-Linker),3.59–3.54(m,2H,3’-H,4-H),3.48–3.35(m,2H,CH2-Linker),3.31(dd,J＝11.6,8.2Hz,2H,3”-H,CH2-Linker),2.87–2.50(m,8H,CH2-Lev),2.19(s,3H,CH3CO),2.16(s,3H,CH3CO),1.89–1.73(m,2H,CH2-Linker),1.04–0.94(m,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ206.17,205.93,171.23,167.06,166.88,138.43,137.83,137.04,134.82,134.71,130.04,129.74,129.26,129.19,128.90,128.71,128.65,128.64,128.58,128.40,128.19,128.10,128.04,127.84,127.62,127.42,127.25,102.54(C-1’),100.31(C-1”),98.15(C-1),78.85,77.65,76.66,75.21,74.93,74.91,74.69,70.96,70.63,67.72,67.26,66.39,65.83,65.80,64.61,59.38,50.59,37.95,37.70,29.87,29.86,27.77,27.60,27.23,27.17,24.74,22.70,16.38；IR νmax(film)2918,2109,1749,1715,1372,1218,1146,1042,751,699cm-1；HR-ESI-MS(m/z):calcd for C74H80N10O20Na+(M+Na+):1451.5443,found:1451.5451.
化合物37：在氮气下保护下，向溶有化合物33(50mg,0.083mmol)的AcOH/THF(1:2v/v,3mL)溶液中加入锌粉(106mg,1.66mmol)。将混合物在37℃搅拌12小时。将溶液过滤并真空浓缩以获得粗产物。将粗产物溶解于THF(7mL)、吡啶(20μL,0.249mmol,3当量)中，然后在0℃下滴加TrocCl(11.4μL,0.083mmol)的THF(1mL)溶液。将反应混合物在0℃下搅拌2小时直至TLC显示起始原料完全转化，然后通过在0℃下添加MeOH猝灭。将混合物真空浓缩成残余物并通过硅胶柱色谱法纯化(石油醚/乙酸乙酯5:1→2:1v/v)得到呈无色糖浆的产物37(19mg,0.026mmol,31％)。[α]25 D＝-25.3°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.47–6.94(m,14H,Ar-H),5.21(s,2H,CH2-Troc),4.88(d,J＝9.4Hz,1H,NH),4.76(d,J＝12.1Hz,1H,CH2-Troc),4.75(t,J＝9.7Hz,1H,2-H),4.70(d,J＝12.1Hz,1H,CH2-Troc),4.64(d,J＝9.8Hz,1H,1-H),4.48(d,J＝11.0Hz,1H,ArCH),4.44(d,J＝11.1Hz,1H,ArCH),4.03(d,J＝9.4Hz,1H,5-H),3.94(q,J＝9.8Hz,1H,3-H),3.74(t,J＝9.7Hz,1H,4-H),2.81–2.68(m,2H,CH2-Lev),2.64–2.57(m,2H,CH2-Lev),2.33(s,3H,CH3CO),2.18(s,3H,ArCH3)；13C NMR(151MHz,Chloroform-d)δ205.86,171.78,167.67,154.25,138.82,137.05,134.95,134.03,129.71,128.66,128.60,128.53,128.41,128.11,128.03,127.22,95.47,86.74,79.10,76.52,74.64,74.26,69.82,67.53,58.36,37.80,29.78,27.97,21.21；IR νmax(film)3334,2923,1742,1719,1544,1495,1455,1401,1364,1317,1288,1257,1205,1157,1074,1029,814,735,698cm-1；HR-ESI-MS(m/z):calcd for C35H36Cl3NO9SNa+(M+Na+):774.1069,found:774.1072.
化合物38：在-10℃下，硫苷供体37(24mg，0.0316mmol)、受体21(20mg，0.0211mmol)和分子筛置于干燥的DCM(1.6mL)中搅拌，加入NIS(9mg，0.038mmol)和TMSOTf(1.1μL，6.32μmoL)。反应在0℃下搅拌5h，直到TLC检测显示糖基供体完全转化。反应液用DCM稀释并过滤。滤液用10％(w/v)Na2S2O3、饱和NaHCO3溶液和盐水洗涤。有机相用无水Na2SO4干燥，真空浓缩。粗品经硅胶柱层析(PE/EA v/v 3:1→1:1)分离纯化，得到无色糖浆38(1.5mg，0.95μmol，3％)。1H NMR(600MHz,Chloroform-d)δ7.38–7.10(m,30H,Ar-H),5.35(d,J＝11.8Hz,1H,ArCH),5.21–5.12(m,4H,ArCH),5.07(d,J＝11.8Hz,1H,ArCH),4.86(dd,J＝10.3,7.9Hz,1H,2’-H),4.83–4.74(m,3H,1-H,CH2-Troc),4.76–4.70(m,2H,2”-H,ArCH),4.70–4.65(m,1H,1’-H),4.55–4.48(m,2H,ArCH),4.46–4.39(m,4H,1”-H,ArCH),4.06–3.97(m,2H),3.97–3.87(m,1H),3.81(q,J＝9.8Hz,1H),3.79–3.70(m,3H),3.66(dd,J＝10.9,3.7Hz,1H),3.66–3.61(m,1H,CH2-Linker),3.59–3.54(m,2H),3.48–3.29(m,3H,CH2-Linker),2.85–2.65(m,6H,CH2-Lev),2.58–2.46(m,2H,CH2-Lev),2.17(s,3H,CH3CO),2.16(s,3H,CH3CO),1.90–1.71(m,2H,CH2-Lev),1.03–0.95(m,3H,6-H)；HR-ESI-MS(m/z):calcd for C77H83Cl3N8O22Na+(M+Na+):1599.4580,found:1599.4586.
对比例3
三糖43和44的合成尝试：
如图11所示，在TMSOTf和N-碘代琥珀酰亚胺(NIS)的作用下，受体16和硫代糖苷供体39在-10℃至0℃温度下进行糖苷化反应，得到具有完全β选择性的二糖39，产率为79％。利用三氟乙酸(TFA)水解化合物40的4,6-O-苄叉基得到二元醇41，产率为82％。随后，对C6'位羟基进行选择性的叔丁基二甲基硅烷基(TBDPS)保护时，尽管反应在80℃的条件下进行，仍未能得到所需产物。考虑到可通过采用叔丁基二甲基硅烷(TBS)对C6’位羟基进行选择性保护，以降低空间立体位阻的影响。随后，在50℃的下进行了反应，成功得到了产物42，产率为69％。但遗憾的是，即使将催化剂TMSOTf的当量增至0.6当量，糖基供体37与二糖受体42的糖基化反应依旧未能成功进行。此外，反应活性更强的供体19与二糖受体42的糖苷化反应也不能得到所需的三糖。通过对分解的供体副产物和未反应的受体分析，上述糖基化反应的失败可能是由于O6位TBS的空间位阻导致的。
具体试验操作和步骤：
化合物40：在氩气保护和-10℃条件下，向溶有硫供体39(610mg，1.22mmol)、受体16(380mg，0.68mmol)和新活化的MS的无水DCM(6.8mL)的混合溶液中添加NIS(330mg，1.46mmol)和TMSOTf(44μL，0.244mmol)。将反应混合物在0℃搅拌4.5小时直至TLC显示完全转化，将混合物用DCM稀释并过滤，并将滤液依次用10％(w/v)的Na2S2O3溶液、饱和NaHCO3和食盐水洗涤。将有机层经无水Na2SO4干燥，过滤并真空浓缩。将残余物通过硅胶柱色谱法(石油醚/乙酸乙酯5:1→2:1v/v)纯化，得到产物40(501mg，0.54mmol，79％)。[α]25 D＝+28.0°(c 1.00,CH3Cl)；1H NMR(400MHz,Chloroform-d)δ7.56–7.09(m,20H,Ar-H),5.59(s,1H,ArCH),5.23–5.12(m,1H,ArCH),4.96(dd,J＝9.9,7.8Hz,1H,2’-H),4.88(d,J＝11.5Hz,1H,ArCH),4.86–4.80(m,1H,1-H),4.80–4.76(m,1H,1’-H),4.62–4.31(m,4H,ArCH,6’-H),4.06–3.93(m,1H,CH2-Linker),3.88–3.74(m,3H,3’-H,6’-H,5-H),3.57–3.26(m,5H,4’-H,5’-H,CH2-Linker,3-H,4-H),3.56–3.41(m,2H,2-H,CH2-Linker),3.37–3.30(m,1H,CH2-Linker),2.91–2.63(m,4H,CH2-Lev),2.16(s,3H,CH3CO),1.90–1.82(m,2H,CH2-Linker),1.14–1.05(m,3H,6-H)；13C NMR(101MHz,Chloroform-d)δ206.01,171.40,138.40,137.82,136.50,129.28,128.69,128.60,128.51,128.39,128.35,128.22,128.03,127.83,127.72,127.42,127.23,126.02,102.63(C-1’),101.60,98.34(C-1),79.41,77.35,77.35,77.03,76.72,74.95,71.90,68.52,67.27,66.52,65.89,63.11,59.54,50.49,37.92,29.77,27.75,16.44；IR νmax(film)2933,2108,1753,1698,1497,1454,1421,1360,1315,1218,1125,1092,1043,1002cm-1；HR-ESI-MS(m/z):calcd for C49H55N7O12Na+(M+Na+):956.3801,found:956.3805.
化合物41：向溶有化合物40(460mg,0.49mmol)的DCM(5.0mL)溶液中加入三氟乙酸(0.5mL,6.72mmol)和H2O(88μL,4.90mmol,10eq.)。将反应混合物在室温下搅拌4小时直至TLC显示起始材料完全转化。将混合物用DCM稀释并用水、饱和NaHCO3溶液和饱和食盐水洗涤，经Na2SO4 干燥并真空浓缩。通过硅胶柱色谱法(DCM/MeOH 50:1→20:1v/v)纯化残余物，得到呈无色糖浆状的化合物41(340mg，0.402mmol，82％)。[α]25 D＝+27.2°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.46–7.13(m,15H,Ar-H),5.22–5.10(m,2H,ArCH),4.91(dd,J＝9.7,7.8Hz,1H,2’-H),4.88(d,J＝11.6Hz,1H,ArCH),4.86–4.77(m,1H,1-H),4.77–4.69(m,1H,1’-H),4.59(d,J＝11.6Hz,1H,ArCH),4.57–4.41(m,2H,ArCH),4.12–3.94(m,1H,3-H),3.90–3.74(m,3H,5-H,6’-H),3.72(dd,J＝10.8,3.6Hz,1H,2-H),3.67–3.53(m,4H,4-H,CH2-Linker,3’-H,4’-H),3.49–3.38(m,2H,5’-H,CH2-Linker),3.38–3.27(m,1H,CH2-Linker),2.84–2.66(m,4H,CH2-Lev),2.15(s,3H,CH3CO),1.90–1.74(m,2H,CH2-Linker),1.10(d,J＝6.4Hz,3H,6-H)；13C NMR(151MHz,Chloroform-d)δ206.31,171.52,156.38,138.44,137.68,136.49,128.60,128.56,128.50,128.20,128.05,127.80,127.69,127.42,127.20,101.86(C-1),98.27(C-1’),78.86,77.46,76.10,74.80,71.83,69.75,67.35,66.83,66.60,65.38,62.29,59.77,50.31,44.34,37.87,29.74,28.22,16.47；IR νmax(film)3462,2935,2107,1750,1696,1497,1454,1423,1362,1314,1220,1161,1126,1044cm-1；HR-ESI-MS(m/z):calcd for C42H51N7O12Na+(M+Na+):868.3488,found:868.3493.
化合物42：向溶有41(140mg，0.166mmol)的无水DMF(0.3mL)溶液中加入叔丁基二甲基氯硅烷(30mg，0.20mmol)、咪唑(23mg，0.33mmol)和4-二甲基氨基吡啶(3mg，0.02mmol)。将反应混合物在50℃下搅拌过夜，然后通过添加甲醇(3mL)猝灭反应。将混合物用乙酸乙酯(30mL)稀释并用水、饱和NaHCO3溶液和饱和食盐水洗涤。将合并的有机相经Na2SO4干燥，过滤并浓缩。通过快速二氧化硅柱色谱法(石油醚/乙酸乙酯10:1v/v)纯化粗产物得到呈无色油状的42(110mg，0.115mmol，69％)。[α]25 D＝+19.7°(c 1.00,CH3Cl)；1H NMR(600MHz,Chloroform-d)δ7.40–7.15(m,15H,Ar-H),5.22–5.14(m,2H,ArCH),4.92–4.88(m,2H,ArCH,2’-H),4.84–4.76(m,1H,1-H),4.73–4.65(m,1H,1’-H),4.57–4.43(m,3H,ArCH),4.02–3.93(m,1H,3-H),3.93–3.86(m,1H,6’-H),3.86–3.73(m,1H,5-H),3.81(dd,J＝10.3,6.0Hz,1H,6’-H),3.69(dd,J＝10.8,3.6Hz,1H,2-H),3.68–3.65(m,1H,4-H),3.66–3.54(m,3H,CH2-Linker,3’-H,4’-H),3.51–3.30(m,4H,5’-H,CH2-Linker),2.86–2.68(m,4H,CH2-Lev),2.16(s,3H,CH3CO),1.91–1.74(m,2H,CH2-Linker),1.10(d,J＝6.6Hz,3H,6-H),0.89(s,9H,SiC(CH3)3),0.12–0.06(m,6H,Si(CH3)2)；13C NMR(151MHz,Chloroform-d)δ206.09,171.50,156.23,138.53,137.81,136.60,128.58,128.50,128.15,128.02,127.82,127.59,127.38,127.21,102.01(C-1’),98.33(C-1),79.05,77.84,75.03,74.71,72.15,71.59,67.27,66.60,66.52,65.82,64.41,59.61,50.55,44.67,37.92,29.75,27.80,25.78,18.17,16.42,-5.49；IR νmax(film)3419,2929,2108,1754,1704,1472,1455,1423,1361,1317,1253,1153,1130,1047,970cm-1；HR-ESI-MS(m/z):calcd for C48H65N7O12SiNa+(M+Na+):982.4353,found:982.4355.
化合物43：在-10℃和氩气保护下，向溶有供体37(32mg，0.042mmol)、二糖受体42(20mg，0.021mmol)和新活化MS的无水DCM(0.5mL)混合物中加入NIS(11mg,0.05mmol)和TMSOTf(1.5μL,8.4μmoL)。将反应混合物在0℃下搅拌10小时直至TLC显示糖基供体完全转化，用Et3N猝灭反应，然后过滤并真空浓缩。将残余物通过硅胶柱色谱纯化，并利用MALDI-TOF和NMR分析。未反应的受体和供体降解产物表明糖基化反应失败。
化合物44：在-10℃和氩气保护下，向溶有供体19(21mg，0.042mmol)、二糖受体42(20mg，0.021mmol)和新活化MS的无水DCM(0.5mL)混合物中加入NIS(11mg，0.05mmol)和TMSOTf(1.5μL,8.4μmoL)。将反应混合物在0℃下搅拌8小时直至TLC显示糖基供体完全转化，用Et3N猝灭反应，然后过滤并真空浓缩。将残余物通过硅胶柱色谱纯化，并利用MALDI-TOF和NMR分析。未反应的受体和供体降解产物表明糖基化反应失败。
上面结合具体实施例对本发明进行了示例性的描述，显然本发明的实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (10)

  1. 一种绿脓杆菌O5血清型O-抗原三糖组装有连接臂的寡糖片段的化学合成方法，其特征在于，所述绿脓杆菌O5血清型O-抗原三糖组装有连接臂的寡糖片段的结构如通式VI所示：
    其中，Linker为-L-NH2，其中L表示连接臂；连接臂L为含有0-6个杂原子的2-40碳原子数的链式结构、取代或未取代的三-六元环式结构、酰胺键、或脲基；
    是以三个单糖砌块A，B，C为原料，分别如式(Ⅰ)～(Ⅲ)所示：
    其中：
    PG1为H；
    PG2，PG3，PG4和PG6为羟基临时保护基，分别独立地选自：乙酰基，或者苯甲酰，或者新戊酰基，或者氯乙酰，或者乙酰丙酰基，或者9-芴甲氧羰基，或者烯丙氧羰酰基，或者2-萘甲基，或者对甲氧基苄基，或者烯丙基，或者叔丁基二甲基硅烷基，或者三乙基硅烷基；
    PG5为羧基保护基，选自苄基，甲基，乙基，叔丁基，烯丙基；
    PG7为氨基临时保护基团，选自三氯乙氧羰基，邻苯二甲酰基，9-芴甲氧羰基，叔丁基氧羰基；
    PG8，PG9为羟基保护基，选自苯亚甲基，萘亚甲基，异亚丙基缩酮；
    PG10，PG11为氨基保护基，选自苄基，苄氧羰基；
    LG为用于糖基化反应的离去基团，选自氟，或者氯，或者溴，或者碘，或者三氯乙酰亚胺酯，或者N-苯基三氟乙酰亚胺酯糖苷，或者乙硫基，或者苯硫基，或者对甲苯硫基，或者乙硫基，或者二丁基膦酸基；
    包括如下过程：
    (1)利用单糖砌块B与单糖砌块A进行糖基化反应，合成得到式Ⅳ所示的二糖片段，
    (2)将所得二糖片段先进行脱保护、脱去保护基团PG4，然后与单糖砌块C进行糖基化，合成得到如式Ⅴ所示的三糖中间体片段，
    (3)还原所得三糖中间体片段、脱保护PG3、PG6，并对单糖砌块B中脱保护后的2号位、以及单糖砌块C中脱保护后的2号位进行叠氮化；随后还原三糖中间体片段中的单糖砌块C的4、6号位，脱去PG8、PG9，并对6号位进行氧化，得到羧酸类基团；再对单糖砌块B中脱保护后的2号位、以及单糖砌块C中2号位的叠氮基团进行还原，得到乙酰 氨基；再使用亚胺类试剂与单糖砌块C中的3号位构建乙脒结构，最后还原Linker中的氨基，得到式ⅤI所示的目标产物。
  2. 根据权利要求1所述的化学合成方法，其特征在于，合成路线如下所示：
  3. 根据权利要求1所述的化学合成方法，其特征在于，所述步骤(1)中的糖基化反应是在活化试剂路易斯酸、溶剂、分子筛的环境下进行的。
  4. 根据权利要求3所述的化学合成方法，其特征在于，所述步骤(1)中的糖基化反应的活化试剂路易斯酸选自如下任意一种或多种：三氟化硼乙醚，三氟甲磺酸，三氟甲磺酸三甲基硅酯，银盐三氟甲磺酸银。
  5. 根据权利要求1所述的化学合成方法，其特征在于，所述步骤(2)中的糖基化反应是在活化试剂路易斯酸、NIS、溶剂、分子筛的环境下进行的。
  6. 根据权利要求5所述的化学合成方法，其特征在于，所述步骤(2)中的糖基化反应的活化试剂路易斯酸用量为0.1～1eq，NIS用量为1.2～1.5eq。
  7. 根据权利要求5所述的化学合成方法，其特征在于，所述步骤(2)中的糖基化反应的活化试剂路易斯酸选自如下任意一种或多种：三氟化硼乙醚，三氟甲磺酸，三氟甲磺酸三甲基硅酯，银盐三氟甲磺酸银。
  8. 一种组装有氨基连接臂的绿脓杆菌O5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1-7任一所述方法制备得到的，结构如下所示：
  9. 权利要求8所述的组装有氨基连接臂的绿脓杆菌O5血清型O-抗原三糖化合物在开发或制备绿脓杆菌疫苗中的应用。
  10. 权利要求8所述的组装有氨基连接臂的绿脓杆菌O5血清型O-抗原三糖化合物在开发或制备治疗性绿脓杆菌感染药物中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN118546188A (zh) * 2024-05-10 2024-08-27 江南大学 一种绿脓杆菌o5血清型o-抗原寡糖的化学合成方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN118546188A (zh) * 2024-05-10 2024-08-27 江南大学 一种绿脓杆菌o5血清型o-抗原寡糖的化学合成方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LARKIN ANGELYN, CHANG MICHELLE M, WHITWORTH GARRETT E, IMPERIALI BARBARA: "Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis", NATURE CHEMICAL BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 9, no. 6, 1 June 2013 (2013-06-01), New York, pages 367 - 373, XP093370065, ISSN: 1552-4450, DOI: 10.1038/nchembio.1249 *
NING, YUNZHAN: "Synthesis of High-Density Aminooligosaccharide Fragments of the O Antigen from Pseudomonas aeruginosa Serotype O5", CHINA MASTER’S THESES FULL-TEXT DATABASE, no. 2, 17 June 2021 (2021-06-17), XP093370076 *
SADOVSKAYA IRINA, BRISSON JEAN‐ROBERT, LAM JOSEPH S., RICHARDS JAMES C., ALTMAN ELEONORA: "Structural elucidation of the lipopolysaccharide core regions of the wild‐type strain PAO1 and O‐chain‐deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5", EUROPEAN JOURNAL OF BIOCHEMISTRY, PUBLISHED BY SPRINGER-VERLAG ON BEHALF OF THE FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES, HOBOKEN, USA, vol. 255, no. 3, 1 August 1998 (1998-08-01), Hoboken, USA, pages 673 - 684, XP093370069, ISSN: 0014-2956, DOI: 10.1046/j.1432-1327.1998.2550673.x *
TIAN GUANGZONG, HU JING, QIN CHUNJUN, LI LINGXIN, NING YUNZHAN, ZHU SHENGYONG, XIE SUQING, ZOU XIAOPENG, SEEBERGER PETER H., YIN J: "Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-Man p N3NA", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ACS PUBLICATIONS, vol. 146, no. 27, 10 July 2024 (2024-07-10), pages 18427 - 18439, XP093370063, ISSN: 0002-7863, DOI: 10.1021/jacs.4c03814 *

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