WO2020177234A1 - 一种幽门螺旋杆菌o：6血清型o-抗原糖链的合成方法 - Google Patents

一种幽门螺旋杆菌o：6血清型o-抗原糖链的合成方法 Download PDF

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WO2020177234A1
WO2020177234A1 PCT/CN2019/091198 CN2019091198W WO2020177234A1 WO 2020177234 A1 WO2020177234 A1 WO 2020177234A1 CN 2019091198 W CN2019091198 W CN 2019091198W WO 2020177234 A1 WO2020177234 A1 WO 2020177234A1
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compound
reaction
helicobacter pylori
serotype
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尹健
胡静
田光宗
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins

Definitions

  • the invention specifically relates to a method for synthesizing the sugar chain of Helicobacter pylori O:6 serotype O-antigen, and belongs to the field of glycochemistry.
  • Hp Helicobacter pylori
  • MALT is closely related to the onset of gastric cancer.
  • the International Cancer Research Center classified it as a Class I carcinogen.
  • Hp is a group of prokaryotic microorganisms that are slender, flexible, curved in a spiral shape, and can move freely. Its characteristics are between bacteria and protozoa, and are mainly located in the deep layer of human gastric mucosa.
  • the epithelial cells of the gastric mucosa are mostly in gastric pits, epithelial folds and glandular cavities. Approximately 50% of the world’s population is infected with Helicobacter pylori. Among them, as high as 70% in developing countries, the infection rate in developed countries is relatively small, only 20%-30%. Children are susceptible to Helicobacter pylori. Children's infections in developing countries are mainly caused by low socioeconomic status and backward medical and health facilities. Improving personal hygiene habits has an important impact on the transmission of Hp.
  • the anti-Hp infection treatment plan is triple or quadruple therapy with bismuth or proton pump inhibitor combined with antibiotics.
  • these antibiotic-based treatments have many disadvantages, including long-term use can make Hp resistant to antibiotics, the risk of repeated infections and the high cost of antibiotic treatment. Therefore, there is an urgent need for new methods to prevent and cure Helicobacter pylori infection. .
  • research on Hp vaccine formulations mainly focus on protein components, while research on other components such as polysaccharides is relatively rare.
  • LPS surface lipopolysaccharide
  • Hp Hp lipopolysaccharide-based sugar conjugated in the body of mice caused immunity to produce antibodies that can recognize other serotypes of Hp.
  • O:6 serotype One of the Hp serotypes is composed of the non-reducing Lewis O-chain connected to heptose. The specific structure is as follows:
  • the research on the lipopolysaccharide of Helicobacter pylori is carried out by extracting from inactivated bacteria.
  • the disadvantage of this method is that the product obtained from one extraction is very small.
  • the extracted lipopolysaccharide is also The structure is not specific enough, and the structure is easy to be accompanied by similar impurities. The experiment repeatability is poor, and there is a certain interference to the research.
  • O:6 serotype O-antigen oligosaccharides have not been synthesized so far, in order to study its immune effect, we decided to synthesize Helicobacter pylori serotype O:6 antigen by chemical method and conduct immunological research on it.
  • the present invention provides a simple, rapid and effective chemical synthesis method for preparing H. pylori O:6 serotype O-antigen sugar chain fragments.
  • glucosamine, galactose, mannose and fucose are the starting materials, which are easily and conveniently obtained. Seven sugar building blocks are obtained through a series of chemical reactions, and then these sugar building blocks are used under the action of corresponding activating reagents. Based on the design of neighboring group participation effect, solvent effect, additive effect, etc., after a series of glycosylation reaction coupling, the Helicobacter pylori O:6 serotype O-antigen sugar chain is obtained, and the reducing end has an amino linking arm. Later, connexin is made into glycoconjugate vaccine for preparation.
  • the first object of the present invention is to provide a method for synthesizing H. pylori O:6 serotype O-antigen sugar chain fragments, which uses seven sugar building blocks to construct Helicobacter pylori O:6 serotype O- Antigen sugar chain fragments, the seven sugar building blocks are compounds represented by formulas 1-7:
  • PG 1 , PG 2 , PG 3 , PG 4 , PG 5 , PG 6 , PG 7 , PG 8 , PG 9 , PG 10 , PG 11 , PG 12 , PG 13 , PG 14 , PG 15 , PG 17 , PG 18 , PG 19 , PG 21 , PG 22 , PG 23 , PG 25 , PG 26 , PG 27 , PG 28 , PG 29 and PG 30 are independently selected from hydrogen, acyl, 2-naphthylmethyl and their derivatives , Benzyl and its derivatives, allyl and silyl groups;
  • PG 16 and PG 24 are independently selected from any one of hydrogen, acyl, alkoxycarbonyl and alkoxycarbonyl;
  • PG 20 is selected from any one of alkanoyl, diformyl, benzyloxycarbonyl and derivatives thereof;
  • the leaving groups LG in the structures of Formulas 1-7 are independently selected from any one of halogen, iminoester group, thio group and phosphonic acid group.
  • the PG 1 , PG 9 , PG 12 , PG 17 , PG 21 , PG 22 and PG 24 are temporary protecting groups for hydroxyl, selected from hydrogen (H) and acetyl (Ac) , Benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), levulinyl (Lev), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-naphthalene Any of methyl (Nap), p-methoxybenzyl (pMBn), or allyl (All).
  • the PG 2 , PG 3 , PG 4 , PG 6 , PG 7 , PG 8 , PG 11 , PG 13 , PG 14 , PG 18 , PG 25 , PG 26 , PG 29 And PG 30 is selected from hydrogen (H), acetyl (Ac), benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), allyloxycarbonyl (Alloc), benzyl (Bn) , 2-naphthylmethyl (Nap), p-methoxybenzyl (pMBn), or allyl (All).
  • the PG 16 and PG 24 are selected from hydrogen (H), acetyl (Ac), benzoyl (Bz), pivaloyl (Piv), chloroacetyl (ClAc), Any one of levulinyl (Lev), 9-fluorenylmethoxycarbonyl (Fmoc), and allyloxycarbonyl (Alloc).
  • the PG 5 , PG 10 , PG 15 , PG 19 , PG 23 , PG 27 and PG 28 are selected from hydrogen (H), acetyl (Ac), benzoyl (Bz ), pivaloyl (Piv), chloroacetyl (ClAc), allyloxycarbonyl (Alloc), benzyl (Bn), 2-naphthylmethyl (Nap), p-methoxybenzyl (pMBn), alkene Any one of propyl (All), tert-butyldimethylsilyl, tert-butyldiphenylsilyl and triethylsilyl.
  • the PG 20 is an amino protecting group selected from the group consisting of trichloroacetyl (TCA), trichloroacetyloxycarbonyl (Troc), phthaloyl (Phth), benzyloxy Any one of carbonyl (Cbz).
  • the method includes pre-synthesizing disaccharides, and then synthesizing H. pylori serotype O-antigen sugar chain fragments through the construction of glycosidic bonds.
  • the glycosidic bond is constructed by coupling a glycosyl donor and an acceptor with an active agent to realize D- ⁇ -D-Hep-(1-2) linkage.
  • the active agent includes one or more of NIS, NBS, DBU, and TMSOTf.
  • the active agent is preferably a mixture of NIS and TMSOTf.
  • the method includes synthesizing a disaccharide compound, and the synthetic route of the disaccharide compound is as follows:
  • the disaccharide utilizes sugar building block 1 as a glycosyl donor and sugar building block 8 as a glycosyl acceptor, which is coupled in an organic solvent to obtain D- ⁇ -D-Hep -(1-2) Linked disaccharide compound (Compound 9).
  • the molar ratio of sugar building block 1 to sugar building block 8 is (1-2):1
  • the organic solvent is one or more of dichloromethane, tetrahydrofuran, chloroform, and acetonitrile.
  • the disaccharide compound 9 is used to deprotect the glycosyl acceptor disaccharide compound 10, and then compound 2 is used as the glycosyl donor to obtain D- ⁇ -D-Hep-(1 -2) Linked trisaccharide compound 11.
  • the molar ratio of the disaccharide compound 10 to the compound 2 is 1: (1-2).
  • the preparation method of the trisaccharide compound 11 specifically includes: the disaccharide compound 9 selectively removes the 2-position protecting group of the disaccharide 3 to obtain the glycosyl acceptor 10, and then according to the mole
  • the glycosyl donor 2 and the glycosyl acceptor 10 are catalyzed by Lewis acid and coupled under stirring at -10°C to obtain the target trisaccharide compound 11 linked by D- ⁇ -D-Hep-(1-2).
  • the method further includes synthesizing a tetrasaccharide compound, and the synthetic route of the tetrasaccharide compound is as follows:
  • the molar ratio of sugar building block 3 to trisaccharide compound 12 is (1-2):1.
  • the method for synthesizing the tetrasaccharide compound 13 specifically includes: dissolving the sugar building block 3 and the trisaccharide compound 12 in dry dichloromethane according to the molar ratio, and adding acid-washed molecular sieves , Then catalyzed by Lewis acid, coupled with stirring at -10°C for 2-4 hours to prepare the target tetrasaccharide compound 13
  • the method further includes synthesizing a pentasaccharide compound, and the synthetic route of the pentasaccharide compound is as follows:
  • the sugar building block 3 is used as the glycosyl donor, the trisaccharide compound 14 is the glycosyl acceptor, and the tetrasaccharide compound 15 is obtained by coupling in an organic solvent; then, the tetrasaccharide compound 15 is used as The glycosyl donor, the trisaccharide compound 12 is the glycosyl acceptor, and the pentasaccharide compound 16 is obtained by coupling in an organic solvent.
  • the preparation method of the pentasaccharide compound 16 specifically includes:
  • molar ratio (1-2):1 dissolve sugar building block 3 and sugar building block 14 in an organic solvent, add molecular sieve and Lewis acid, and react at -10°C for 2-4 hours to obtain disaccharide donor 15;
  • the pentasaccharide compound 16 is obtained by coupling with 1-2 times the molar equivalent of the trisaccharide compound 12.
  • the method further includes synthesizing an octasaccharide compound, and the synthetic route of the octasaccharide compound is as follows:
  • the sugar building block 3 is used as the glycosyl donor, the disaccharide compound 17 is the glycosyl acceptor, and the trisaccharide compound 18 is obtained by coupling in an organic solvent; then, the trisaccharide compound 18 is used as As the glycosyl donor, the pentasaccharide compound 19 is the glycosyl acceptor, and the octasaccharide compound 20 is obtained by coupling in an organic solvent.
  • the preparation method of the octasaccharide compound 20 specifically includes:
  • molar ratio (1-2):1 dissolve sugar building block 3 and disaccharide compound 17 in an organic solvent, add molecular sieve and Lewis acid, and react at -10°C for 2-4 hours to obtain trisaccharide donor 18;
  • the pentasaccharide compound 19 is coupled with 1-2 times the molar equivalent to obtain the octasaccharide compound 20.
  • the method further includes synthesizing a tridecasaccharide compound, and the synthetic route of the tridecagar compound is as follows:
  • the method for synthesizing the trisaccharide compound includes:
  • Trisaccharide fragment 2 is used as the glycosyl acceptor 25 under Lewis acid catalysis and coupled with stirring at -10°C to obtain the undecose fragment 26.
  • the protective agents PG 21 and PG 24 are selectively removed to obtain the glycosyl Acceptor 27;
  • sugar building block 7 is used as the glycosyl donor, and the tridecane compound 28 is prepared by coupling reaction.
  • the preparation method of the tridecane compound 28 specifically includes:
  • the glycosyl donor 24 and the glycosyl acceptor 25 of equal molar ratio are catalyzed by Lewis acid and coupled under stirring at -10°C to obtain the undecose fragment 26, and the two temporary protective agents are selectively removed PG 21 and PG 24 to obtain the glycosyl acceptor 27; taking 4 moles of sugar building block 7 as the glycosyl donor and 1 mole of undecose 27 as the glycosyl acceptor, the glycosyl donor and the glycosyl acceptor Dissolve in dry dichloromethane/ether (1:1), add pickled The molecular sieve is then catalyzed by Lewis acid, coupled under stirring at -40°C, and reacted for 2-4 hours to prepare the target tridecane 28.
  • the second object of the present invention is to use the above method to synthesize a Helicobacter pylori O:6 serotype O-antigen oligosaccharide compound assembled with link arms, and the structure of the compound is shown in formula I:
  • the connecting arm Linker may be fully or partially substituted with fluorine.
  • the linker Linker can include a three-, four-, five-, or six-membered saturated carbocyclic ring; it can also include a five-membered unsaturated carbocyclic ring (non-aromatic ring); it can also include four, five, or Six-membered saturated oxygen heterocyclic ring; can also contain a four-, five- or six-membered saturated nitrogen heterocyclic ring; can also contain a six-membered aromatic carbocyclic ring.
  • the linker Linker may also include an amide bond and/or a urea group.
  • the linker Linker may contain one or more substituent groups, and these substituents may include: -F, -Cl, -CH 3 , -C 2 H 5 , -C 3 H 7 , -C 5 H 9 , -C 6 H 13 , -OCH 3 , -OC 2 H 5 , -CH 2 F, -CHF 2 , -CF 3 , -C(O)-NH 2 , -SCH 3 , -SC 2 H 5 , -NHC(O)CH 3 , -N(CH 3 ) 2 and -N(C 2 H 5 ) 2 .
  • the third object of the present invention is to provide a method for preparing a sugar-protein conjugate, which uses the above-mentioned Helicobacter pylori O:6 serotype O-antigen oligosaccharide compound assembled with link arms.
  • the fourth object of the present invention is to apply the above-mentioned Helicobacter pylori O:6 serotype O-antigen oligosaccharide compound assembled with link arms in the development or preparation of Helicobacter pylori vaccine or the treatment of diseases caused by Helicobacter pylori infection In the drug.
  • the method of the invention has simple steps, time-saving, labor-saving and low cost.
  • the present invention obtains Helicobacter pylori O:6 serotype O-antigen disaccharides, trisaccharides, pentasaccharides, batang and trisaccharides through chemical synthesis.
  • the present invention finds a method for selectively constructing a synthetic route for connecting various sugar building blocks through protective agent strategy, temperature effect, solvent effect and additive effect, and applies this method to Helicobacter pylori O:6 serotype O-antigen II Sugar, trisaccharide, pentasaccharide, Batang and Thirteen Sugar are in the synthesis.
  • the reducing ends of the synthetic H are described by the synthetic H.
  • pylori O:6 serotype O-antigen sugar chain fragments are assembled with amino linking arms, which can be formed into glycoconjugates with carrier proteins for immunological research, and are useful for the development of prevention and treatment of pylori.
  • Helicobacter has an important role.
  • Figure 1 Compounds represented by U 1 , U 2 , U 3 , U 4 , U 5 , U 6 , U 7 in general formula I;
  • Figure 2 Compounds shown in monosaccharide building blocks 1, 2, 3, 4, 5, 6 and 7;
  • Figure 8 Synthesis of Helicobacter pylori O:6 serotype O-antigen trisaccharide
  • Figure 12 The hydrogen NMR spectrum of Helicobacter pylori O:6 serotype O-antigen trisaccharide compound 36*;
  • Figure 13 The NMR spectrum of Helicobacter pylori O:6 serotype O-antigen trisaccharide compound 36*;
  • Figure 14 The NMR spectrum of Helicobacter pylori O:6 serotype O-antigen trisaccharide compound 36*;
  • Figure 15 Nuclear magnetic hydrocarbon correlation spectrum of Helicobacter pylori O:6 serotype O-antigen tridecose compound 36*.
  • N-iodosuccinimide N-iodosuccinimide
  • TfOH trifluoromethanesulfonic acid
  • sugar building block 13* For the synthesis of sugar building block 13*, first use the previously prepared intermediate compound 3 and 4 starting materials, under the action of dibutyl tin oxide (Bu 2 SnO), the 3-OH of compound 5* is selectively processed Bn protection gave compound 12*, and finally 2-OH was protected with acetyl group to give heptose building block 13*.
  • dibutyl tin oxide Bu 2 SnO
  • Compound 12* Compound 5* (600 mg, 1.14 mmol) and Bu 2 SnO (426 mg, 1.71 mmol) were dissolved in dry toluene (5.7 mL), and the reaction was refluxed for 4 hours. During this process, the Dean-Stark device was used to remove Toluene-water azeotropic mixture ( ⁇ 3mL), then the reaction system was cooled to room temperature, concentrated and dried using vacuum. The above residue was dissolved in CH 3 CN (3 mL), and then CsF (260 mg, 1.71 mmol) and BnBr (200 ⁇ L, 1.71 mmol) were added. The reaction was stirred at 70° C. for 10 hours.
  • the glycosylation reaction conditions in the reducing-end trisaccharide shown in Figure 5 are optimized (Table 1), and the optimal glycosylation reaction conditions are determined as follows: the glycosyl donor and acceptor are steamed three times in toluene; add Anhydrous DCM, reaction concentration of 0.1M, activated or Molecular sieve: After the mixture is cooled to -10°C and stirred for 15 minutes, the activation reagents TMSOTf (0.12eq) and NIS (1.2eq) are added, and the reaction time is 3h. After the reaction, the reaction was terminated with triethylamine (Et 3 N). The reaction solution was filtered, diluted with DCM, washed with saturated NaHCO 3 , dried with anhydrous Na 2 SO 4 , concentrated, and separated and purified by silica gel column chromatography.
  • the conditions for removing the acetyl group are as follows: the starting material is dissolved in MeOH/THF (v/v, 1:1), the reaction concentration is 0.05M, and 0.5 equivalent of MeONa (5M in MeOH) is added. The reaction temperature was at room temperature, and after the reaction was detected by TLC, the pH of the reaction solution was neutralized with Amerlite IR 120 (H + ) resin to reach 7. It is filtered, concentrated and purified by silica gel column chromatography.
  • the conditions for removing the Lev group are as follows: the starting material is dissolved in CH 2 Cl 2 / MeOH (20/1, 0.1M), hydrazine acetate (2eq) is added, and the reaction is stirred at room temperature for 3 hours . After the completion of the reaction was detected by TLC, an appropriate amount of DCM was added to dilute, and then the reaction mixture was washed with saturated NaHCO 3 and brine, dried over anhydrous Na 2 SO 4 , concentrated, and separated and purified by silica gel column chromatography to obtain de-Lev receptor.
  • Compound 35* The glycosyl donor 34* (7 mg, 0.0107 mmol) and the glycosyl acceptor 33* (15 mg, 0.00268 mmol, 1 eq) were mixed, dissolved in toluene and steamed twice. Add dry DCM/Et 2 O (v/v, 1:1) (0.2mL), add activated or Molecular sieve. Add 10eq of thiophenol, reduce the reaction temperature to -40°C, stir for 15min, then add the activation reagent TMSOTf (0.4eq), and stir the reaction at -40°C for 3h. After TLC detects that the reaction is complete, add an appropriate amount of Et 3 N to terminate the reaction.
  • This method uses monosaccharide building block 37* as the acceptor, reacts with glycosylation donor 38* under the action of activating reagent TMSOTf to obtain disaccharide 39*, and then uses CH 3 ONa to remove acyl Bz and Lev to obtain disaccharide Acceptor 40*, then react with 4 equivalents of glycosyl donor 34* to obtain fully protected Le y tetrasaccharide.
  • compound 28* was used as a glycosylation acceptor to react with the donor Le y tetrasaccharide to obtain pentasaccharide 42*.
  • Compound 39* Dissolve compound 37* (270mg, 0.50mmol) and 38* (435mg, 0.60mmol) in anhydrous CH 2 Cl 2 (10mL), add freshly activated Molecular sieve, lower the temperature to -10°C, and then add TMSOTf (11 ⁇ L, 0.06mmol) dropwise under argon protection. After reacting at -10°C for 3 hours, TLC detects that the reaction is complete, adding an appropriate amount of Et 3 N to quench the reaction, the reaction solution is filtered and concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate: 20/1 ⁇ 8/1) Disaccharide compound 39* (330 mg, 63%) was obtained.
  • Compound 40* Compound 39* (214 mg, 0.20 mmol) was dissolved in THF/MeOH (1/1, v/v, 2 mL), and sodium methoxide (20 mg, 0.40 mmol) was added at room temperature. After reacting at room temperature overnight, TLC detects the completion of the reaction of the raw materials, adding methanol to dilute, using resin to neutralize excess sodium methoxide, adjusting the pH to 0, the solution is filtered and concentrated, and dried under vacuum. The above crude compound was dissolved in pyridine (20 mL), 10% AcOH (2 mL) was added, and the mixture was heated to reflux for 16 h.
  • Compound 42* Dissolve donor tetrasaccharide 41* (35mg, 0.019mmol) and acceptor 28* (21mg, 0.038mmol) in anhydrous CH 2 Cl 2 (1mL) and add Molecular sieve and NIS (5mg, 0.023mmol), the reaction temperature was reduced to -20°C, and then TfOH (0.34 ⁇ L, 0.0038mmol) was added dropwise under the protection of argon. React at -20°C for 3h.
  • Compound 45* Dissolve the donor tetrasaccharide 44* (20mg, 0.0114mmol) and acceptor 28* (12mg, 0.0228mmol) in anhydrous CH 2 Cl 2 (1.1 mL) and add Molecular sieve and NIS (3mg, 0.0137mmol), the reaction temperature was reduced to -15°C, and then TfOH (0.11 ⁇ L, 0.0011mmol) was added dropwise under the protection of argon. React at -20°C for 3h.
  • the hemiacetal obtained above was dissolved in CH 2 Cl 2 (2 mL), CCl 3 CN (16 ⁇ L, 0.11 mmol) and DBU (10 ⁇ L, 0.066 mmol) were added at 0° C., and the reaction was stirred at room temperature for 2 h. After the completion of the reaction detected by TLC, the reaction solution was concentrated at 30°C, and then separated and purified by silica gel column chromatography (n-hexane/ethyl acetate: 5/1 ⁇ 3/1) to obtain compound 46* (21 mg, 42%).
  • the inventors have drawn two important conclusions: one is that the branched chain structure of the glycosylation donor increases the steric hindrance of the reaction, which leads to the failure of the reaction; the other is the glycosylation
  • the activity of the donor and the acceptor are not matched, the activity of the donor is relatively high, and the activity of the acceptor is relatively low, which leads to the failure of the reaction. Therefore, the main solution strategy is to reduce the steric hindrance of the reaction and reduce the reactivity of the donor.

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Abstract

本发明公开了一种幽门螺旋杆菌O：6血清型O-抗原糖链的合成方法，属于糖化学领域。。本发明设计在幽门螺旋杆菌O:6血清型O-抗原糖链还原末端组装氨基连接臂，可以将合成的寡糖链偶联载体分子或固定化于相应基质。利用廉价易得的D-葡萄糖胺，D-半乳糖，D-甘露糖和L-岩藻糖为起始原料，经过一系列的化学反应得到七种糖基化砌块，然后利用这些糖砌块在相应的活化试剂的作用下，经过一系列的糖苷化反应偶联得到多个幽门螺旋杆菌O:6血清型O-抗原寡糖链。本发明制备的寡糖链原料廉价易得、制备方法简单易重复，将会在幽门螺旋杆菌的新型药物和疫苗开发等方面具有良好的应用前景。

Description

一种幽门螺旋杆菌O：6血清型O-抗原糖链的合成方法 技术领域
本发明具体涉及一种幽门螺旋杆菌O：6血清型O-抗原糖链的合成方法，属于糖化学领域。
背景技术
自1983年Warren.Marshall首先从胃炎病人胃内分离出幽门螺杆菌(Helicobacter pylori,Hp)后，各国学者经过广泛深入的研究发现Hp与慢性活动性胃炎、胃十二指肠溃疡、胃粘膜相关性淋巴瘤(MALT)及胃癌的发病有密切关系，1994年国际癌症研究中心将其归为I类致癌因子。作为一种革兰氏阴性细菌，Hp是一群细长、柔韧、弯曲呈螺旋状、能自由运动的原核细胞微生物，它的特性介于细菌与原虫之间，主要位于人的胃黏膜的深层、胃黏膜上皮细胞，以胃小凹、上皮褶皱及腺腔内为多。世界上大约50％的人口感染了幽门螺杆菌，其中发展中国家高达70％，而发达国家感染率相对较小只有20％-30％。儿童是幽门螺杆菌的易感人群，在发展中国家儿童的感染主要是社会经济地位低下和医疗卫生设施落后造成的，提高个人卫生习惯对Hp的传播具有重要影响。
目前，抗Hp感染治疗方案是以铋剂或质子泵抑制剂联合应用抗生素的三联或四联疗法。但是这些基于抗生素的治疗有许多缺点，包括长期服用可以使Hp对抗生素产生抗性，具有重复感染的危险及抗生素治疗费用的昂贵，因此，迫切需要新的方法来预防和根治幽门螺杆菌的感染。研究表明Hp疫苗可能成为控制这一全球性感染的最有效的方法，目前对Hp疫苗的配方研究主要是蛋白质成份，而其它成分如多糖的研究相对较少。然而，研究表明发展糖类疫苗抵抗Hp的感染是非常合理的，以多糖为基础的共轭疫苗已经成功地用于防止系统性感染和抑制宿主定植。目前对肠道病原体的研究是基于其表面脂多糖(LPS)的调查研究，可以作为候选疫苗为人类所使用。LPS是幽门螺旋杆菌细胞表面主要的抗原组成成分，结构鉴定研究表明LPS是由O-链多糖、核心结构和脂质A三部分组成，LPS结构如下所示：
O-Chain→Core→Lipid A～Cell。
在Hp早期的研究中，Penner和他的同事根据LPS分子抗原性的差异发展了一套血清型系统，根据O-链多糖的结构差异定义了六种不同的血清型(O:1-O:6)。Mario和他的同事研究证明以多价的Hp脂多糖为基础的糖缀合在物小鼠体内引起免疫产生的抗体可以识别其他血清型Hp。O:6血清型Hp血清型中的一种，是由非还原端的Lewis O-链与庚糖连接组成，具体结构如下所示：
Figure PCTCN2019091198-appb-000001
目前，针对幽门螺旋杆菌脂多糖的研究都是通过从灭活细菌中提取，这类方法的不足在于一次提取得到的产物极少，另外受细菌基因表达和修饰的特性，提取得到的脂多糖也存在结构不够专一，并且容易附带结构类似杂质的特点，实验重复性差，对研究存在一定的干扰。由于O:6血清型O-抗原寡糖至今无人合成，为了研究其免疫效果，我们决定通过化学方法合成幽门螺杆菌O:6血清型抗原，并对其进行免疫学研究。然而，在复杂的糖化学合成过程中，糖苷键的构建是糖合成中最基本但是也最为棘手和关键的问题，由于糖类化合物结构的多样性，立体化学的复杂性，因此与其他结构的有机化合物不同，糖的合成的方法学依然是不成熟和不完善的，被认为是有机化学领域中唯一存在众多方法(数十种之多)但又有没有一个 具有普适性的方法为大家公认的领域。因为糖模块的结构复杂、顺式糖苷键选择性低，难以实现该结构合成的构建，从而制约了O:6血清型O-抗原寡糖化学合成方法的研究。
发明内容
为了解决上述问题，本发明提供了一种简单、快速有效的制备幽门螺旋杆菌O:6血清型O-抗原糖链片段的化学合成方法。
本发明简便易得的葡萄糖胺，半乳糖，甘露糖和岩藻糖为起始原料，经过一系列的化学反应得到七个糖砌块，然后利用这些糖砌块在相应的活化试剂的作用下，基于邻基参与效应、溶剂效应、添加剂效应等设计，经过一系列的糖苷化反应偶联得到幽门螺旋杆菌O:6血清型O-抗原糖链，同时还原端带有氨基的连接臂，为以后连接蛋白制成糖缀合物疫苗做准备。
本发明的第一个目的是提供一种合成幽门螺旋杆菌O:6血清型O-抗原糖链片段的方法，所述方法是利用七个糖砌块构建幽门螺旋杆菌O:6血清型O-抗原糖链片段，所述七个糖砌块分别为式1～7所示化合物：
Figure PCTCN2019091198-appb-000002
其中，PG 1，PG 2，PG 3，PG 4，PG 5，PG 6，PG 7，PG 8，PG 9，PG 10，PG 11，PG 12，PG 13，PG 14，PG 15，PG 17，PG 18，PG 19，PG 21，PG 22，PG 23，PG 25，PG 26，PG 27，PG 28，PG 29和PG 30分别独立的选自氢、酰基、2-萘甲基及其衍生物、苄基及其衍生物、烯丙基和硅烷基中任意一种；
PG 16和PG 24分别独立的选自氢、酰基、烷氧羰基和烷氧羰酰基中任意一种；
PG 20选自烷酰基、二甲酰基、苄氧羰基及其衍生物中任意一种；
式1～7结构中的离去基团LG分别独立的选自卤素、亚胺酯基、硫基和膦酸基中任意一种。
在本发明的一种实施方式中，所述PG 1，PG 9，PG 12，PG 17，PG 21，PG 22和PG 24为羟基临时保护基，选自氢(H)、乙酰基(Ac)、苯甲酰(Bz)、新戊酰基(Piv)、氯乙酰(ClAc)、乙酰丙酰基(Lev)、9-芴甲氧羰基(Fmoc)、烯丙氧羰酰基(Alloc)、2-萘甲基(Nap)、对甲氧基苄基(pMBn)或者烯丙基(All)中任意一种。
在本发明的一种实施方式中，所述PG 2，PG 3，PG 4，PG 6，PG 7，PG 8，PG 11，PG 13，PG 14，PG 18，PG 25，PG 26，PG 29和PG 30选自氢(H)、乙酰基(Ac)、苯甲酰(Bz)、新戊酰基(Piv)、氯乙酰(ClAc)、烯丙氧羰酰基(Alloc)、苄基(Bn)、2-萘甲基(Nap)、对甲氧基苄基(pMBn)或者烯丙基(All)中任意一种。
在本发明的一种实施方式中，所述PG 16、PG 24选自氢(H)、乙酰基(Ac)、苯甲酰(Bz)、新戊酰基(Piv)、氯乙酰(ClAc)、乙酰丙酰基(Lev)、9-芴甲氧羰基(Fmoc)、烯丙氧羰酰基(Alloc)中任意一种。
在本发明的一种实施方式中，所述PG 5，PG 10，PG 15，PG 19，PG 23，PG 27和PG 28选自氢(H)、乙酰基(Ac)、苯甲酰(Bz)、新戊酰基(Piv)、氯乙酰(ClAc)、烯丙氧羰 酰基(Alloc)、苄基(Bn)、2-萘甲基(Nap)、对甲氧基苄基(pMBn)、烯丙基(All)、叔丁基二甲基硅烷基、叔丁基二苯基硅烷基和三乙基硅烷基中任意一种。
在本发明的一种实施方式中，所述PG 20为氨基保护基团，选自三氯乙酰基(TCA)、三氯乙酰氧羰基(Troc)、邻苯二甲酰基(Phth)、苄氧羰基(Cbz)中任意一种。
在本发明的一种实施方式中，所述LG为用于糖基化反应的离去基团，选自氟(F)、氯(Cl)、溴(Br)、碘(I)、三氯乙酰亚胺酯(CCl 3C(＝NH)O-)、N-苯基三氟乙酰亚胺酯糖苷(CF 3C(＝NPh)O-)、乙硫基(SEt)、苯硫基(SPh)、对甲苯硫基(STol)、二丁基膦酸基(-P(＝O)-(OBu) 2)中任意一种。
在本发明的一种实施方式中，所述方法包括预先合成二糖，然后通过糖苷键的构建，合成幽门螺旋杆菌O:6血清型O-抗原糖链片段。
在本发明的一种实施方式中，所述糖苷键的构建是利用活性试剂偶联糖基供体和受体，实现D-α-D-Hep-(1-2)连接。
在本发明的一种实施方式中，所述活性试剂包括NIS、NBS、DBU、TMSOTf中的一种或多种。
在本发明的一种实施方式中，所述活性试剂优选NIS和TMSOTf两种混合。
在本发明的一种实施方式中，所述方法包括合成二糖化合物，所述二糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000003
在本发明的一种实施方式中，所述二糖是利用糖砌块1为糖基供体，糖砌块8为糖基受体，在有机溶剂中偶联得到D-α-D-Hep-(1-2)连接的二糖化合物(化合物9)。
在本发明的一种实施方式中，所述糖砌块1与糖砌块8的摩尔比为(1-2)：1
在本发明的一种实施方式中，所述有机溶剂为二氯甲烷、四氢呋喃、氯仿、乙腈中的一种或多种。
在本发明的一种实施方式中，所述有机溶剂优选二氯甲烷。
在本发明的一种实施方式中，所述二糖的合成方法具体包括：将糖砌块1与糖砌块8按照摩尔比溶于有机溶剂中，加入酸洗的分子筛，然后在路易斯酸催化，-10℃搅拌下2-4小时偶联完全，，制备出D-α-D-Hep-(1-2)连接的二糖化合物9。
在本发明的一种实施方式中，所述方法还包括合成三糖化合物，所述三糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000004
在本发明的一种实施方式中，利用二糖化合物9脱保护得到糖基受体二糖化合物10，然后以化合物2作为糖基供体，偶联得到D-α-D-Hep-(1-2)连接的三糖化合物11。
在本发明的一种实施方式中，二糖化合物10与化合物2的摩尔比为1：(1-2)。
在本发明的一种实施方式中，所述三糖化合物11的制备方法具体包括：二糖化合物9选择性的脱去二糖3的2-位保护基得到糖基受体10，再按照摩尔比将糖基供体2与糖基受体10在路易斯酸催化，在-10℃搅拌下偶联得到D-α-D-Hep-(1-2)连接的目标三糖化合物11。
在本发明的一种实施方式中，所述方法还包括合成四糖化合物，所述四糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000005
在本发明的一种实施方式中，以糖砌块3为糖基供体，三糖化合物12为糖基受体，在有机溶剂中偶联得到四糖化合物13。
在本发明的一种实施方式中，糖砌块3与三糖化合物12的的摩尔比为(1-2)：1。
在本发明的一种实施方式中，所述有机溶剂为二氯甲烷。
在本发明的一种实施方式中，所述四糖化合物13的合成方法具体包括：按照摩尔比，将糖砌块3，三糖化合物12溶于干燥的二氯甲烷中，加入酸洗的分子筛，然后在路易斯酸催化，在-10℃搅拌下偶联2-4小时，制备出目标四糖化合物13
在本发明的一种实施方式中，所述方法还包括合成五糖化合物，所述五糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000006
在本发明的一种实施方式中，以糖砌块3为糖基供体，三糖化合物14为糖基受体，在有机溶剂中偶联得到四糖化合物15；然后以四糖化合物15为糖基供体，三糖化合物12为糖基受体，在有机溶剂中偶联得到五糖化合物16。
在本发明的一种实施方式中，所述五糖化合物16的制备方法具体包括：
按照摩尔比(1-2)：1，将糖砌块3与糖砌块14溶于有机溶剂中，加入分子筛和路易斯酸，-10℃反应2-4小时，得到二糖供体15；然后与1-2倍摩尔当量的三糖化合物12偶联得到五糖化合物16。
在本发明的一种实施方式中，所述方法还包括合成八糖化合物，所述八糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000007
在本发明的一种实施方式中，以糖砌块3为糖基供体，二糖化合物17为糖基受体，在有机溶剂中偶联得到三糖化合物18；然后以三糖化合物18为糖基供体，五糖化合物19为糖基受体，在有机溶剂中偶联得到八糖化合物20。
在本发明的一种实施方式中，所述八糖化合物20的制备方法具体包括：
按照摩尔比(1-2)：1，将糖砌块3与二糖化合物17溶于有机溶剂中，加入分子筛和路易斯酸，-10℃反应2-4小时，得到三糖供体18；然后与1-2倍摩尔当量的五糖化合物19偶联得到八糖化合物20。
在本发明的一种实施方式中，所述方法还包括合成十三糖化合物，所述十三糖化合物的合成路线如下所示：
Figure PCTCN2019091198-appb-000008
在本发明的一种实施方式中，所述十三糖化合物的合成方法包括：
(1)以糖砌块6为糖基供体，糖砌块21为糖基受体，在有机溶剂中偶联得到二糖供体22；然后与糖基受体23偶联得到三糖片段24；
(2)三糖片段2作为，与糖基受体25在路易斯酸催化，在-10℃搅拌下偶联得到十一糖片段26，选择性脱去保护剂PG 21和PG 24，得到糖基受体27；再以糖砌块7为糖基供体，偶联反应制备得到十三糖化合物28。
在本发明的一种实施方式中，所述十三糖化合物28的制备方法具体包括：
(1)以1.5摩尔的糖砌块6为糖基供体，以1摩尔的糖砌块21为糖基受体，将糖基供体与糖基受体溶于干燥的二氯甲烷中，加入酸洗的
Figure PCTCN2019091198-appb-000009
分子筛，然后在路易斯酸催化，在0℃搅拌下偶联反应2-4小时，制备出目标二糖供体22，再使等摩尔比的糖基供体22与糖基受体23在路易斯酸催化，在-20℃搅拌下偶联得到三糖片段24；
(2)然后再使等摩尔比的糖基供体24与糖基受体25在路易斯酸催化，在-10℃搅拌下偶联得到十一糖片段26，选择性脱去两个临时保护剂PG 21和PG 24，得到糖基受体27；以4摩尔的糖砌块7为糖基供体，以1摩尔的十一糖27为糖基受体，将糖基供体与糖基受体溶于干燥的二氯甲烷/乙醚(1:1)中，加入酸洗的
Figure PCTCN2019091198-appb-000010
分子筛，然后在路易斯酸催化，在-40℃搅拌下偶联，反应2-4小时，制备出目标十三糖28。
本发明的第二个目的是利用上述方法合成得到一种组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物，所述化合物的结构如式I所示：
Figure PCTCN2019091198-appb-000011
其中，x为1、2或3；y为1、2或3；z为1、2或3；n 1，n 2，n 3，n 4，n 5为0～5之间的整数，其中n 1，n 2，n 3不同时为零；n；n 6，n 7为0或1；
其中，U 1、U 2、U 3、U 4、U 5、U 6、U 7的结构式如下所示：
Figure PCTCN2019091198-appb-000012
连接臂Linker包括氨基连接臂[-(CH 2) n-N-Y 1Y 2]；n代表连接臂可以为不同的碳链长度，n＝2～40；Y 1和Y 2为氨基的保护基，其中，Y 1为H或者为卞基(Bn)，Y 2为H或者卞甲氧羰基(Cbz)。
在本发明的一种实施方式，连接臂Linker可以全部或部分氟取代。
在本发明的一种实施方式，连接臂Linker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。
在本发明的一种实施方式，连接臂Linker也可以包含酰胺键和/或脲基。
在本发明的一种实施方式，连接臂Linker可以含有一个或多个取代基团，这些取代基可以包括：-F，-Cl，-CH 3，-C 2H 5，-C 3H 7，-C 5H 9，-C 6H 13，-OCH 3，-OC 2H 5，-CH 2F，-CHF 2，-CF 3，-C(O)-NH 2，-SCH 3，-SC 2H 5，-NHC(O)CH 3，-N(CH 3) 2和-N(C 2H 5) 2。
本发明的第三个目的是提供一种糖-蛋白缀合物的制备方法，所述方法是利用上述的组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物。
本发明的第四个目的是将上述的组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物应用于在开发或制备幽门螺旋杆菌疫苗或者治疗幽门螺旋杆菌感染导致的疾病的药物中。
本发明的有益效果：
本发明方法步骤简单、省时、省力且成本低廉。本发明是通过化学合成得到幽门螺旋杆菌O:6血清型O-抗原二糖，三糖、五糖、八塘、十三糖。本发明通过保护剂策略，温度效应，溶剂效应和添加剂效应，发现了一条选择性构建各糖砌块连接的合成路线方法，并且将此方法应用在幽门螺旋杆菌O:6血清型O-抗原二糖，三糖、五糖、八塘、十三糖的合成之中。合成得到的幽门螺旋杆菌O:6血清型O-抗原糖链片段的还原端均组装有氨基连接臂，可以与载体蛋白制成糖缀合物，用于免疫学研究，对发展预防和治疗幽门螺旋杆菌具有重要作用。
附图说明
图1：通式I中U 1，U 2，U 3，U 4，U 5，U 6，U 7所示化合物；
图2：单糖砌块1，2，3，4，5，6和7所示化合物；
图3：糖砌块6*和8*的合成；
图4：糖砌块11*和13*的合成；
图5：还原端三糖的合成；
图6：重复二糖和三糖的合成；
图7：还原端五糖和八糖的合成；
图8：幽门螺旋杆菌O:6血清型O-抗原十三糖的合成；
图9：实施例6中四糖、五塘供体的合成；
图10：实施例6中十三糖的不同合成方法[5+8]；
图11：实施例6中十三糖的不同合成方法[4+9]；
图12：幽门螺旋杆菌O:6血清型O-抗原十三糖化合物36*的核磁氢谱图；
图13：幽门螺旋杆菌O:6血清型O-抗原十三糖化合物36*的核磁碳谱；
图14：幽门螺旋杆菌O:6血清型O-抗原十三糖化合物36*的核磁氢氢相关谱；
图15：幽门螺旋杆菌O:6血清型O-抗原十三糖化合物36*的核磁碳氢相关谱。
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。
所有试剂除特殊说明外均为分析纯，且除特殊说明外未经进一步纯化。所有溶剂使用之前采用通用方法干燥和再蒸馏。所有的反应除另注明，都是利用磁力搅拌在烘干的玻璃器皿中惰性气体的保护下进行。薄层分析(TLC)所用硅胶薄板型号GF254，青岛海洋化工有限公司生产；TLC板通过紫外光(UV)和Hanessian溶液(硫酸铈和钼酸铵溶于硫酸溶液中)或5％的硫酸-乙醇溶液染色，可直观的进行检测。柱层析硅胶为青岛海洋化工公司生产，柱层析硅胶(300-400目)。1H NMR，13C NMR，1H-13C HSQC和1H-1H COSY谱由NVANCE III 400-MHz，600-MHz和700-MHz核磁共振仪测量，除特别指明外，均为CDCl3作溶剂，TMS作内标，环境温度下测定。峰型的表示方法：单峰(s),宽的单峰(br s),双峰(d),四重峰(dd),三重锋(t),多重峰(m)。所有NMR的化学位移(δ)单位记为ppm，耦合常数(J)单位记为Hz。质谱通过Thermo ScientificTSQ Quantum Ultra仪器测得，高分辨率质谱通过IonSpec Ultra仪器测得。
实施例1 糖砌块8*的合成：
合成路线如图3所示。
以2，3-O-丙叉基-4-O-卞基甘露乙硫糖苷为起始原料，经过swern氧化，6-位羟基被氧化成醛得到化合物1*。然后利用wittig反应延长6位的碳链，得到6位脱氧的烯烃化合物2*。烯烃化合物在锇酸钾(K 2OsO 4)、铁氰酸钾(K 3Fe(CN) 6)和碳酸钾(K 2CO 3)的共同作用下进行双羟基化，得到6，7-二-羟基化合物3*。在氢化钠(NaH)的作用下对6,7-二-羟基进行Bn保护得到化合物4*。在80％的醋酸作用下脱去丙叉基后得到化合物5*，然后在D(+)-10-樟脑磺酸(CSA)的作用下，对2,3-位羟基进行成环保护，在弱酸条件下开环，得到2-OBz保护的化合物6*。对2-OH用Lev保护后得到化合物7*，然后利用N-碘代丁二酰亚胺(NIS)和三氟甲磺酸(TfOH)对端基位乙硫基进行水解，最后与三氯乙腈反应得到三氯乙酰亚胺酯糖基供体8*。
具体试验操作和步骤：
化合物2*：将草酰氯(3.6mL,42.3mmol)溶于二氯甲烷(22mL)中,在-78℃条件下，将溶有DMSO(6.0mL,84.6mmol)的二氯甲烷溶液逐滴加入，搅拌15min之后，利用恒压滴液漏斗将溶有化合物2，3-O-丙叉基-4-O-卞基甘露乙硫糖苷(10.0g,28.2mmol)的二氯甲烷(115mL)溶液加入上述反应液中，在-78℃条件下反应1h之后，将Et 3N(15.7mL,112.8mmol)加入到上述溶液，反应温度升至室温，然后在室温下反应4h。TLC显示反应完成之后，加水将反应淬灭，反应液用二氯甲烷萃取，有机相依次用水和饱和食盐水洗涤，然后用无水Na 2SO 4干燥，浓缩有机相，真空干燥得到粗品醛，未经纯化直接用于下一步反应。在0℃条件下，将甲基三苯基溴化磷(24.2g,67.7mmol)溶于THF(90mL)中，然后加入n-BuLi(23.5mL,56.4mmol,2.5M in hexane)，反应搅拌1h，后将反应温度降到-78℃，将溶有上述粗醛产物的THF(28mL)逐滴加入，反应温度升至室温，继续反应12h。TLC检测反应完全之后，加入饱和的NH 4Cl淬灭反应，乙酸乙酯萃取(5×100mL)，无水Na 2SO 4干燥，浓缩有机相后利用柱层析进行纯化(石油醚/乙酸乙酯：100/1→50/1)得到化合物2*(5.5g,56％)。R f＝0.32,petroleum ether/EtOAc＝15:1.[α] 25 D＝+129.3(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.40–7.23(m,5H,arom.H),5.99(ddd,J＝16.7,10.6,5.5Hz,1H,6-H),5.58(s,1H,1-H),5.41(dt,J＝17.3,1.7Hz,1H,7-H),5.25(dt,J＝10.7,1.6Hz,1H,7-H’),4.85(d,J＝11.5Hz,1H,Ph-CH 2),4.63(d,J＝11.5Hz,1H,Ph-CH 2),4.42(dd,J＝10.0,5.5Hz,1H,5-H),4.29(dd,J＝7.2,5.7Hz,1H,3-H),4.19(d,J＝5.6Hz,1H,2-H),3.38(dd,J＝10.0,7.2Hz,1H,4-H),2.58(ddq,J＝52.5,13.1,7.4Hz,2H,SCH 2),1.49(s,3H,CH 3),1.36(s,3H,CH 3),1.28(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ138.2,135.0,128.2,128.0,127.6,117.3,109.4,80.1,79.6,78.5,76.7,73.2,69.5,28.0,26.4,24.4,14.6.IR(film):ν＝2985,2931,1454,1380,1242,1219,1162,1124,1090,1066,996,872,748,697cm -1.HRMS(ESI)m/z calcd for C 19H 26O 4SNa[M+Na] +373.1449,found 373.1445.
化合物3*：将铁氰化钾(K 3Fe(CN) 6,46.2mmol,15.2g)，二水合锇酸钾(K 2O SO 4·2H 2O,0.385mmol,142mg)和碳酸钾(K 2CO 3,50.8mmol,7.0g)加入到叔丁醇(77mL)和水(77mL)溶液中，然后在0℃下，将溶有化合物2*(5.4g,15.4mmol)的甲苯(30mL)溶液逐滴加入到反应溶液中，反应混合液在0℃下反应36h。TLC检测反应完全之后，加入亚硫酸钠(Na 2SO 3，25g)淬灭反应，搅拌15min之后，乙酸乙酯萃取，有机相用1M KOH洗涤，无水Na 2SO 4干燥，过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯：5/1→4/1)得到化合物3*(4.3g,73％)。R f＝0.36,petroleum ether/EtOAc＝1:1.[α] 25 D＝+173.6(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.41–7.28(m,5H,arom.H),5.55(s,1H,1-H),4.98(d,J＝11.3Hz,1H,Ph-CH 2),4.64(d,J＝11.3Hz,1H,Ph-CH 2),4.32(dd,J＝6.9,5.7Hz,1H,3-H),4.20(dd,J＝5.7,0.7Hz,1H,2-H),4.06(dd,J＝9.9,6.3Hz,1H,5-H),3.89(ddt,J＝6.8,4.8,2.2Hz,1H,6-H),3.69(dd,J＝10.0,7.0Hz,1H,4-H),3.66–3.63(m,2H,7-H/7-H’),3.54(d,J＝2.8Hz,1H,OH),2.63(ddq,J＝51.1,12.9,7.4Hz,2H,SCH 2),1.54(s,3H,CH 3),1.37(s,3H,CH 3),1.29(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ137.2,109.6,79.7,79.5,78.3,76.5,73.2,72.8,67.7,62.8,28.1,26.4,24.1,14.2.IR(film):ν＝3446,2984,2931,1454,1380,1242,1219,1162, 1066,872,750,699cm -1.HRMS(ESI)m/z calcd for C 19H 28O 6SNa[M+Na] +407.1504,found 407.1507.
化合物4*：将化合物3*(1.7g,4.4mmol)溶于DMF(22mL))中，加入氢化钠(0.7g,17.7mmol)(60％分散在矿物油中)，将反应温度降至0℃，然后加入BnBr(2.1mL,17.7mmol)，反应在室温下搅拌3h，TLC检测反应完全之后，加入适量的甲醇淬灭，二氯甲烷萃取，有机相依次用水、饱和食盐水洗涤，无水Na 2SO 4干燥之后减压浓缩。粗品利用柱层析分离纯化(石油醚/乙酸乙酯:100/1→50/1)得到化合物4*(2.2g,89％)。R f＝0.27,petroleum ether/EtOAc＝20:1.[α] 25 D＝+117.7(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.40–7.17(m,15H,arom.H),5.54(s,1H,1-H),4.83(d,J＝11.4Hz,1H,)Ph-CH 2,4.72(d,J＝11.8Hz,1H,Ph-CH 2),4.67(d,J＝11.8Hz,1H,Ph-CH 2),4.53(d,J＝11.4Hz,1H,Ph-CH 2),4.49(d,J＝12.0Hz,1H,Ph-CH 2),4.43(d,J＝12.0Hz,1H,Ph-CH 2),4.32–4.24(m,2H,3-H/5-H),4.15(d,J＝5.7Hz,1H,2-H),4.04(td,J＝5.7,1.5Hz,1H,6-H),3.68(d,J＝5.7Hz,2H,7-H/7-H’),3.68(dd,J＝10.0,7.0Hz,1H,4-H),2.59(ddq,J＝67.5,12.8,7.4Hz,2H,SCH 2),1.46(s,3H,CH 3),1.35(s,3H,CH 3),1.23(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ138.7,138.5,138.2,128.3,128.2,128.2,128.0,127.7,127.5,127.5,127.4,127.3,109.3,79.5,79.0,77.9,76.4,76.3,73.2,72.7,72.5,70.5,69.3,28.0,26.5,23.8,14.2.IR(film):ν＝2929,1453,1380,1218,1093,1066,1027,870,734,696cm -1.HRMS(ESI)m/z calcd for C 33H 40O 6SNa[M+Na] +587.2443,found 587.2429.
化合物5*：将化合物4*(2.2g,3.8mmol)溶于80％醋酸溶液(40mL)中，反应混合物在60℃下反应5h，TLC检测反应完全之后，旋转蒸发浓缩反应液，加入适量DCM溶解，然后依次用饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩，柱层析分离纯化(石油醚/乙酸乙酯：4/1)得到化合物5*(2g,quan.)。R f＝0.33,petroleum ether/EtOAc＝2:1.[α] 25 D＝+119.6(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.45–7.14(m,15H,arom.H),5.25(d,J＝1.8Hz,1H,1-H),4.74(d,J＝11.9Hz,1H,Ph-CH 2),4.69(d,J＝11.8Hz,1H,Ph-CH 2),4.68(d,J＝11.7Hz,1H,Ph-CH 2),4.63(d,J＝11.5Hz,1H,Ph-CH 2),4.53(d,J＝12.0Hz,1H,Ph-CH 2),4.48(d,J＝11.9Hz,1H,Ph-CH 2),4.28(dd,J＝9.5,1.6Hz,1H,5-H),4.00(ddd,J＝6.7,5.1,1.6Hz,1H,6-H),3.93(ddd,J＝7.4,3.9,2.2Hz,1H,2-H),3.89(dq,J＝5.7,3.4,2.8Hz,1H,3-H),3.81(d,J＝5.2Hz,0H),3.79(d,J＝10.0,5.2Hz,1H,7-H),3.77(dd,J＝10.0,7.0Hz,1H,4-H),3.70(dd,J＝10.2,6.7Hz,1H,7-H’),2.71–2.50(m,2H,SCH 2),2.47(d,J＝4.6Hz,1H,2-OH),2.28(d,J＝5.7Hz,1H,3-OH),1.25(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ138.6,138.3,138.2,128.6,128.4,128.3,127.9,127.9,127.8,127.7,127.6,127.5,83.4,77.8,76.4,74.2,73.4,72.6,72.5,72.2,71.7,70.6,24.7,14.7.IR(film):ν＝3420,2924,1453,1075,1027,792,733,696cm -1.HRMS(ESI)m/z calcd for C 30H 36O 6SNa[M+Na] +547.2130,found 547.2118.
化合物6*：将化合物5*(1.06g,2.0mmol)溶于无水的DCM(20mL)中，加入原苯甲酸三乙酯(0.7mL,3.0mmol)和CSA(23mg,0.1mmol)，在室温下搅拌反应1h，TLC检测原料完全转化为中间体后，加入水(70μL,～4.0mmol)，在室温下搅拌反应1h，TLC检测反应完全后，加入适量DCM稀释，用饱和的NaHCO 3洗涤，水层用DCM萃取一次，合并有机相并用饱和的食盐水洗，无水Na 2SO 4干燥,过滤浓缩后柱层析分离纯化(石油醚/乙酸乙酯:15/1→10/1)得到化合物6*(1.0g,80％)。R f＝0.43,petroleum ether/EtOAc＝4:1.[α] 25 D＝+47.5(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.31–7.12(m,20H,arom.H),5.42(dd,J＝3.3,1.7Hz,1H,2-H),5.39(d,J＝1.6Hz,1H,1-H),4.82(t,J＝11.4Hz,2H,Ph-CH 2),4.71(dd,J＝12.8,11.5Hz,2H,,Ph-CH 2),4.53(s,2H,Ph-CH 2),4.38–4.32(m,1H,5-H),4.20(ddd,J＝8.9,5.4,3.3Hz,1H,3-H),4.04(t,J＝9.4Hz,1H,4-H),4.04(m,1H,6-H),3.83(dd,J＝10.3,5.0Hz,1H,7-H),3.74(dd,J＝10.3,6.8Hz,1H,7-H’),2.76–2.54(m,2H,SCH 2),2.08(d,J＝5.4Hz,1H,3-OH),1.27(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ166.1,138.8,138.4, 138.2,133.3,129.8,129.7,128.5,128.3,128.3,128.0,127.8,127.5,127.5,127.4,82.1,78.8,76.4,74.8,74.7,73.3,72.7,72.2,71.8,71.1,25.4,14.9.IR(film):ν＝3435,3030,2870,1719,1452,1267,1089,1026,902,735,711,697cm -1.HRMS(ESI)m/z calcd for C 37H 40O 7SNa[M+Na] +651.2392,found 651.2383.
化合物7*：将化合物6*(1.6g,2.5mmol)溶于无水DCM(20mL)中，然后依次加入乙酰丙酸(0.4mL,3.8mmol)，N,N-二环己基二酰亚胺(0.79g,3.8mmol)和4-二甲氨基吡啶(0.47g,3.8mmol)，反应在室温下搅拌1h。TLC检测反应完全后，加入适量DCM稀释，有机层依次用饱和NaHCO 3和饱和食盐水洗，无水Na 2SO 4干燥，过滤浓缩后，柱层析分离纯化(石油醚/乙酸乙酯:10/1→8/1)得到化合物7*(1.5g,82％)。R f＝0.43,petroleum ether/EtOAc＝3:1.[α] 25 D＝+27.9(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.15–7.10(m,20H,arom.H),5.58(dd,J＝3.2,1.7Hz,1H,2-H),5.41(dd,J＝9.6,3.2Hz,1H,3-H),5.37(d,J＝1.6Hz,1H,1-H),4.87(d,J＝12.0Hz,1H,Ph-CH 2),4.75(d,J＝11.9Hz,1H,Ph-CH 2),4.68(d,J＝11.1Hz,1H,Ph-CH 2),4.62(d,J＝11.1Hz,1H,Ph-CH 2),4.51(s,2H,Ph-CH 2),4.45(d,J＝9.9Hz,1H,5-H),4.24(t,J＝9.7Hz,1H,4-H),4.04(ddd,J＝6.4,5.0,1.2Hz,1H,6-H),3.80(dd,J＝10.3,5.0Hz,1H,7-H),3.72(dd,J＝10.3,6.8Hz,1H,7-H’),2.77–2.53(m,4H,SCH 2/CH 2),2.51–2.33(m,2H,CH 2),2.07(s,3H,Ac),1.27(t,J＝7.4Hz,3H,SCH 2CH 3). 13C NMR(101MHz,Chloroform-d)δ206.1,171.7,165.4,138.7,138.3,138.0,133.4,129.8,129.6,128.5,128.3,128.3,127.8,127.6,127.5,127.5,127.4,127.4,82.0,78.9,74.6,73.5,73.4,73.3,72.8,72.4,72.1,71.1,37.8,29.7,27.9,25.2,14.8.IR(film):ν＝2928,1719,1452,1265,1151,1089,1026,736,711,697cm -1.HRMS(ESI)m/z calcd for C 42H 46O 9SNa[M+Na] +749.2760,found 749.2763.
化合物8*：将化合物7*(1.34g,1.84mmol)溶于CH 2Cl 2(18mL)中，然后加入水(0.33mL,18.4mmol)搅拌，在0℃条件下加入NIS(0.62g,2.76mmol)和TfOH(36μL,0.41mmol)，然后在0℃下搅拌1.5h，TLC检测反应完全后加入Et 3N终止反应，加适量DCM稀释，后用10％的Na 2S 2O 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩后柱层析分离纯化(石油醚/乙酸乙酯:3/1→2/1)得到相应的半缩醛(1.33g,quan.)。R f＝0.36,petroleum ether/EtOAc＝1:1.
将上面得到的半缩醛(238mg,0.35mmol)溶于CH 2Cl 2(4mL)中，在0℃下加入CCl 3CN(107μL,1.07mmol)和DBU(7μL,0.046mmol)，反应在室温下搅拌45min。TLC检测反应完全之后，在30℃下浓缩反应液，后经硅胶柱层析分离纯化(petroleum ether/EtOAc:6/1→4/1)得到化合物8*(266mg,92％)。R f＝0.33,petroleum ether/EtOAc＝3:1. 1H NMR(400MHz,Chloroform-d)δ8.72(s,1H,arom.H),8.13–7.93(m,2H,arom.H),7.71–7.53(m,1H,arom.H),7.44–7.14(m,16H,arom.H),6.40(d,J＝2.1Hz,1H,1-H),5.70(dd,J＝3.3,2.1Hz,1H,2-H),5.54(dd,J＝9.4,3.3Hz,1H,3-H),4.89(d,J＝11.9Hz,1H,Ph-CH 2),4.76(d,J＝11.9Hz,1H,Ph-CH 2),4.72(d,J＝10.9Hz,1H,Ph-CH 2),4.64(d,J＝10.9Hz,1H,Ph-CH 2),4.47(d,J＝1.8Hz,2H,Ph-CH 2),4.37(t,J＝9.7Hz,1H,4-H),4.30(d,J＝10.0Hz,1H,5-H),4.10(t,J＝6.4Hz,1H,6-H),3.76(dd,J＝10.2,5.7Hz,1H,7-H),3.71(dd,J＝10.1,6.8Hz,1H,7’-H),2.74(dt,J＝18.5,7.2Hz,1H,CH 2),2.62(dt,J＝18.5,6.4Hz,1H,CH 2),2.54–2.36(m,2H,CH 2),2.09(s,3H,CH 3CO). 13C NMR(101MHz,Chloroform-d)δ206.0,171.8,165.2,160.1,138.7,138.2,137.7,133.6,129.8,129.2,128.6,128.3,128.3,128.0,127.7,127.6,127.5,127.4,127.4,94.9,90.7,78.8,74.9,74.7,73.3,73.1,72.6,72.6,70.8,68.7,37.8,29.7,27.9.
实施例2 糖砌块13*的合成：
合成路线如图4所示。
如图2，以化合物3*为起始原料，利用二丁基氧化锡(Bu 2SnO)选择性的7-OH进行Bn保护得到化合物9*，然后6-OH用Lev保护得到化合物10*。在80％的醋酸作用下脱去化合物10*的丙叉基后，然后对2,3-OH进行乙酰基保护得到糖砌块11*。
糖砌块13*的合成，首先利用先前制备的中间体化合物3,4位起始原料，在二丁基氧化锡(Bu 2SnO)的作用下，选择性对化合物5*的3-OH进行Bn保护得到化合物12*，最后对2-OH进行乙酰基保护得到庚糖砌块13*。
具体试验操作和步骤：
化合物9*：将化合物3*(0.77g,2mmol)和Bu 2SnO(0.75g,3mmol)溶于干燥的甲苯(10mL)中，反应回流4h，在此过程中，用Dean-Stark装置除去甲苯-水共沸混合物(～5mL)，然后将反应体系降至室温，浓缩并利用真空干燥。将上述残余物溶于CH 3CN(5mL)中，然后加入CsF(456mg,3mmol)和BnBr(360μL,3mmol)，反应在70℃下搅拌10h，TLC检测反应完全后，用硅藻土过滤反应混合物并浓缩，粗品利用硅胶柱层析分离纯化(石油醚/乙酸乙酯:8/1)得到化合物9*(0.63g,66％)。R f＝0.56,petroleum ether/EtOAc＝3:1.[α] 25 D＝+124.6(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.48–7.21(m,10H,arom.H),5.52(s,1H,1-H),4.92(d,J＝11.4Hz,1H,Ph-CH 2),4.59(d,J＝11.3Hz,1H,Ph-CH 2),4.53(d,J＝12.0Hz,1H,Ph-CH 2),4.49(d,J＝12.0Hz,1H,Ph-CH 2),4.34–4.27(m,1H,3-H),4.18(dd,J＝5.7,0.8Hz,1H,2-H),4.13–4.04(m,2H,6-H/5-H),3.69(dd,J＝9.4,6.9Hz,1H,4-H),3.58(dd,J＝10.3,6.5Hz,1H,7-H),3.54(dd,J＝10.4,3.5Hz,1H,7-H’),3.00(d,J＝2.7Hz,1H,6-OH),2.57(ddq,J＝57.7,12.8,7.4Hz,2H,SCH 2),1.52(s,3H,CH 3),1.36(s,3H,CH 3),1.24(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ138.2,137.7,128.4,128.3,128.2,127.8,127.7,127.6,109.5,79.6,78.6,77.9,76.5,73.5,72.8,72.3,70.9,68.5,28.0,26.4,24.1,14.2.IR(film):ν＝3482,2984,2930,1454,1380,1241,1219,1162,1068,1027,870,736,698cm -1.HRMS(ESI)m/z calcd for C 26H 34O 6SNa[M+Na] +497.1974,found 497.1969.
化合物10*：将化合物9*(567mg,1.2mmol)溶于干燥的CH 2Cl 2(23mL)，然后加入LevOH(185μL,1.8mmol),DCC(370mg,1.8mmol)和DMAP(220mg,1.86mmol)，反应在室温下搅拌1h。TLC检测反应完全后加入适量DCM稀释，反应液用饱和的NaHCO 3和饱和的食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩，硅胶柱层析纯化(石油醚/乙酸乙酯:8/1→4/1)得到化合物10*(707mg,quan.)。R f＝0.32,petroleum ether/EtOAc＝4:1.[α] 25 D＝+105.8(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.45–7.16(m,10H,arom.H),5.57–5.52(m,1H,6-H),5.52(s,1H,1-H),4.87(d,J＝11.6Hz,1H,Ph-CH 2),4.55(d,J＝11.6Hz,1H,Ph-CH 2),4.47(d,J＝12.0Hz,1H,Ph-CH 2),4.43(d,J＝12.0Hz,1H,Ph-CH 2),4.26(t,J＝6.3Hz,1H,3-H),4.19(dd,J＝10.3,2.2Hz,1H,5-H),4.13(dd,J＝5.7,0.7Hz,1H,2-H),3.68–3.58(m,3H,4-H/7-H/7-H’),2.75–2.63(m,3H,CH 2),2.62–2.44(m,3H,SCH 2/CH 2),2.16(s,3H,OAc),1.46(s,3H,CH 3),1.34(s,3H,CH 3),1.28(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ206.4,171.8,138.1,138.1,128.3,128.0,127.6,127.5,127.5,109.4,79.5,78.8,76.4,76.2,73.0,72.5,71.6,68.9,68.0,37.9,29.9,28.0,28.0,26.4,23.9,14.4.IR(film):ν＝2984,2931,1739,1719,1361,1218,1159,1096,1066,871,748,698cm -1.HRMS(ESI)m/z calcd for C 31H 40O 8SNa[M+Na] +595.2342,found 595.2331.
化合物11*：将化合物10*(652mg,1.14mmol)溶于80％醋酸溶液(11mL)中，反应混合物在60℃下反应5h，TLC检测反应完全之后，旋转蒸发浓缩反应液，加入适量DCM溶解，然后依次用饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩，真空干燥。将上述残余物溶于吡啶(4mL)中，然后加入Ac 2O(1.1mL,11.4mmol)和DMAP(cat.)，反应在室温下搅拌3h，TLC检测反应完全后，蒸干反应混合物，加入适量DCM稀释，并分别用1M HCl(aq)、饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，浓缩后利用硅胶柱层析分离纯化(石油醚/乙酸乙酯:10/1→6/1)得到化合物11*(605mg,86％)。R f＝0.35,petroleum ether/EtOAc＝3:1.[α] 25 D＝+79.9(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.37–7.20(m,10H,arom.H),5.51(td,J＝6.2,2.1Hz,1H,6-H),5.30(dd,J＝3.3,1.8Hz,1H,2-H),5.25(dd,J＝9.3,3.3Hz,1H,3-H),5.21(d,J＝1.7Hz,1H,1-H),4.64(s,2H,Ph-CH 2),4.51(d,J＝2.6Hz,2H,Ph-CH 2),4.33(dd,J＝9.9,2.1Hz,1H,5-H),4.01(t,J＝9.6Hz,1H,4-H),3.77 (dd,J＝10.3,5.8Hz,1H,7-H),3.62(dd,J＝10.2,6.5Hz,1H,7-H’),2.74(t,J＝6.7Hz,2H,CH 2),2.69–2.52(m,4H,SCH 2/CH 2),2.17(s,3H,OAc),2.11(s,3H,OAc),1.92(s,3H,OAc),1.27(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ206.3,171.7,169.9,169.6,138.0,137.9,128.4,128.4,127.7,127.6,127.6,127.5,81.8,74.3,73.8,73.2,72.3,71.6,71.5,68.3,37.9,29.8,28.0,25.1,20.9,20.8,14.8.IR(film):ν＝2922,1744,1719,1365,1236,1157,1093,914,734,698cm -1.HRMS(ESI)m/z calcd for C 32H 40O 10SNa[M+Na] +639.2240,found 639.2229.
化合物12*：将化合物5*(600mg,1.14mmol)和Bu 2SnO(426mg,1.71mmol)溶于干燥的甲苯(5.7mL)中，反应回流4h，在此过程中，用Dean-Stark装置除去甲苯-水共沸混合物(～3mL)，然后将反应体系降至室温，浓缩并利用真空干燥。将上述残余物溶于CH 3CN(3mL)中，然后加入CsF(260mg,1.71mmol)和BnBr(200μL,1.71mmol)，反应在70℃下搅拌10h，TLC检测反应完全后，用硅藻土过滤反应混合物并浓缩，粗品利用硅胶柱层析分离纯化(石油醚/乙酸乙酯:6/1)得到化合物12*(540mg,77％)。R f＝0.38,petroleum ether/EtOAc＝3:1.[α] 25 D＝+95.5(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.43–7.12(m,20H,arom.H),5.33(d,J＝1.5Hz,1H,1-H),4.79(d,J＝10.9Hz,1H,Ph-CH 2),4.75(d,J＝12.0Hz,1H,Ph-CH 2),4.68(d,J＝12.0Hz,1H,Ph-CH 2),4.65–4.63(m,2H,Ph-CH 2),4.59(d,J＝10.9Hz,1H,Ph-CH 2),4.50(d,J＝12.0Hz,1H,Ph-CH 2),4.45(d,J＝12.1Hz,1H,Ph-CH 2),4.30(dd,J＝9.5,1.3Hz,1H,5-H),4.05(dt,J＝3.4,1.7Hz,1H,2-H),3.99(ddd,J＝6.4,4.7,1.5Hz,1H,6-H),3.90(t,J＝9.2Hz,1H,4-H),3.85(dd,J＝8.8,3.1Hz,1H,3-H),3.76(dd,J＝10.4,4.6Hz,1H,7-H),3.69(dd,J＝10.5,6.9Hz,1H,7-H’),2.72–2.47(m,2H,SCH 2),2.58(d,J＝2.2Hz,1H,2-OH),1.24(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ138.7,138.4,138.3,137.6,128.6,128.3,128.3,128.2,128.1,128.0,127.8,127.7,127.6,127.6,127.4,127.3,83.0,80.9,78.1,74.7,74.6,73.2,72.3,72.1,71.9,70.8,69.6,29.7,24.6,14.7.IR(film):ν＝2917,2849,1453,1088,1027,790,733,696cm -1.HRMS(ESI)m/z calcd for C 37H 42O 6SNa[M+Na] +637.2600,found 637.2585.
化合物13*：将化合物12*(1.2g,2mmol)溶于吡啶(6mL)中，然后加入Ac 2O(1.1mL,11.4mmol)和DMAP(cat.)，反应在室温下搅拌3h，TLC检测反应完全后，蒸干反应混合物，加入适量DCM稀释，并分别用1M HCl(aq)、饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，浓缩后利用硅胶柱层析分离纯化(石油醚/乙酸乙酯:10/1→5/1)得到化合物13*(1.2g,93％)。R f＝0.36,petroleum ether/EtOAc＝3:1.[α] 25 D＝+67.4(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.48–7.11(m,20H,arom.H),5.39(dd,J＝3.0,1.7Hz,1H,2-H),5.25(d,J＝1.7Hz,1H,1-H),5.25(d,J＝1.7Hz,1H,1-H,Ph-CH 2),4.84(d,J＝10.8Hz,1H,Ph-CH 2),4.77(d,J＝11.9Hz,1H,Ph-CH 2),4.68(d,J＝11.90Hz,1H,Ph-CH 2),4.66(d,J＝11.19Hz,1H,Ph-CH 2),4.57(d,J＝10.8Hz,1H,Ph-CH 2),4.50(d,J＝11.8Hz,1H,Ph-CH 2),4.50(d,J＝11.1Hz,1H,Ph-CH 2),4.46(d,J＝12.1Hz,1H,Ph-CH 2),4.29(dd,J＝9.3,1.2Hz,1H,5-H),3.99(ddd,J＝6.4,4.7,1.5Hz,1H,6-H),3.97(t,J＝9.3Hz,1H,4-H),3.92(dd,J＝9.1,3.0Hz,1H,3-H),3.75(dd,J＝10.4,4.6Hz,1H,7-H),3.68(dd,J＝10.4,6.9Hz,1H,7-H’),2.72–2.50(m,2H,SCH 2),2.10(s,3H,OAc),1.25(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ170.2,138.8,138.8,138.4,137.6,128.4,128.3,128.2,128.2,127.8,127.8,127.5,127.5,127.4,82.1,79.0,78.6,74.8,74.6,73.3,72.4,72.3,71.8,71.1,70.4,25.3,21.1,14.8.IR(film):ν＝3029,2869,1742,1453,1369,1230,1091,1027,734,696cm -1.HRMS(ESI)m/z calcd for C 39H 44O 7SNa[M+Na] +679.2705,found 679.2694.
实施例3 还原端三糖的合成：
还原端三糖的合成路线如图5所示。
3.1对图5所示的还原端三糖中糖基化反应条件进行优化(表1)，确定较优的糖基化反应的条件如下：糖基供体和受体在甲苯中共蒸三次；加入无水的DCM，反应浓度为0.1M，活化的
Figure PCTCN2019091198-appb-000013
或者
Figure PCTCN2019091198-appb-000014
分子筛；混合液冷却至-10℃下搅拌15min后，加入活化试剂 TMSOTf(0.12eq)和NIS(1.2eq)，反应时间为3h。反应结束后用三乙胺(Et 3N)终止反应。反应液过滤，DCM稀释后用饱和的NaHCO 3洗涤，无水Na 2SO 4干燥，浓缩后经硅胶柱层析分离纯化。
表1 糖基化反应条件优化
Figure PCTCN2019091198-appb-000015
Figure PCTCN2019091198-appb-000016
3.2脱去乙酰基条件为：将起始原料溶解在MeOH/THF(v/v，1：1)中，反应浓度为0.05M，加入0.5当量MeONa(5M in MeOH)。反应温度为室温，TLC检测反应结束后用Amerlite IR 120(H +)树脂中和反应液pH达到7。过滤，浓缩后利用硅胶柱层析分离纯化。
具体试验操作和步骤：
化合物14*：根据反应条件3.1，糖基供体13*(980mg,1.49mmol)和糖基受体linker(1.04g,3.43mmol)反应得到14*(1.13g,85％)。R f＝0.33,petroleum ether/EtOAc＝4:1.[α] 25 D＝+14.8(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.46–7.11(m,30H,arom.H),5.27(s,1H,2-H),5.17(s,2H,Ph-CH 2),4.84(d,J＝10.7Hz,1H,Ph-CH 2),4.78–4.59(m,3H,Ph-CH 2),4.68(s,1H,1-H),4.56(d,J＝10.8Hz,1H,Ph-CH 2),4.53–4.37(m,5H),3.98(t,J＝5.8Hz,1H,6-H),3.94–3.78(m,3H,3-H/4-H/-5-H),3.73(dd,J＝10.4,4.7Hz,1H,7-H),3.67(dd,J＝10.4,6.7Hz,1H,7-H’),3.74–3.56(m,1H,CH 2),3.44–3.20(m,3H,CH 2),2.09(s,3H,CH 3CO),1.89–1.64(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ170.2,156.4(d,J＝52.1Hz),138.7,138.4(d,J＝3.1Hz),137.8,137.8,136.8,128.6,128.4,128.3,128.2,128.1,127.9,127.9,127.7,127.5,127.4,97.5,78.5,78.4,74.8,74.3,73.3,72.5,72.2,71.8,70.8,68.7,67.2,65.3(d,J＝22.4Hz),50.7(d,J＝24.7Hz),44.1(d,J＝91.4Hz),27.9(d,J＝48.9Hz),21.0.IR(film):ν＝3030,2919,1744,1698,1453,1368,12321090,1027,734,696cm -1.HRMS(ESI)m/z calcd for C 55H 59O 10NNa[M+Na] +916.4037,found 916.4020.
化合物15*：根据反应条件3.2，化合物14*(960mg,1.12mmol)脱去酯基得到化合物15*(908mg,quan.)。R f＝0.34,petroleum ether/EtOAc＝2:1.[α] 25 D＝+26.8(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.46–7.08(m,30H,arom.H),5.23–5.11(m,2H,Ph-CH 2)4.79(d,J＝10.8Hz,1H,Ph-CH 2),4.75–4.60(m,3H,Ph-CH 2),4.66(s,1H,1-H),4.58(d,J＝10.8Hz,1H,Ph-CH 2),4.51–4.39(m,5H,Ph-CH 2),4.02–3.93(m,1H,6-H),3.89(s,1H,2-H),3.88–3.77(m,3H,3-H/4-H/5-H),3.74(dd,J＝10.4,4.7Hz,1H,7-H),3.68(dd,J＝10.4,6.7Hz,1H,7-H’),3.74–3.58(m,1H,CH 2),3.45–3.11(m,3H,CH 2),2.35(d,J＝13.5Hz,1H,2-OH),1.88–1.62(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ156.4(d,J＝47.1Hz),138.7,138.4,138.4,137.9,137.8,136.8,128.6,128.5,128.5,128.3,128.3,128.2,127.9,127.9,127.8,127.7,127.6,127.4,127.4,99.0,80.7,78.0,74.7,74.3,73.2,72.5,72.0,71.9,70.6,68.2,67.2,65.0,50.7,44.2(d,J＝86.6Hz),27.9(d,J＝40.6Hz).IR(film):ν＝3482,3030,2920,1698,1453,1217,1092,1053,1027,734,696cm -1.HRMS(ESI)m/z calcd for C 53H 57O 9NNa[M+Na] +874.3931,found 874.3916.
化合物16*：根据反应条件3.1，糖基供体13*(472mg,0.72mmol)和糖基受体15*(908mg,1.06mmol)反应得到16*(793mg,76％)。R f＝0.51,petroleum ether/EtOAc＝3:1.[α] 25 D＝+11.7(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.72–6.78(m,50H,arom.H),5.45(t,J＝2.3Hz,1H,2-H),5.14(d,J＝11.4Hz,2H,Ph-CH 2),4.89(s,1H,1’-H),4.88(s,1H,1-H),4.88–4.30(m,14H,Ph-CH 2),4.25(d,J＝11.0Hz,1H,Ph-CH 2),4.15–4.09(m,1H),3.99(dt,J＝12.5,5.8Hz,2H),3.94–3.80(m,3H),3.77(dd,J＝10.3,4.9Hz,2H),3.71(dd,J＝5.8,2.6Hz,2H),3.67(dd,J＝10.4,6.5Hz,1H,7-H),3.50–3.27(m,1H,CH 2),3.27–3.05(m,2H,CH 2),3.05–2.83(m,1H,CH 2),2.07(s,3H,CH 3CO),1.72–1.43(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ169.9,156.3,156.3,138.8,138.5,138.4,138.4,138.3,137.8,128.5,128.4,128.3,128.3,128.3,128.2,128.2,128.0,128.0,127.9,127.7,127.7,127.6,127.6,127.6,127.5,127.5,127.4,127.3,127.2,99.9(C-1),98.2(C-1’),79.9,78.7,78.5,77.6,76.3,74.9,74.5,74.4,73.3,73.1,72.5,72.4,72.3,72.2,72.0,71.7,71.2,70.3,68.6,67.1,64.8,60.4,50.6,44.0(d,J＝83.2Hz),27.9(d,J＝38.4Hz),21.0.IR(film):ν＝3030,2922,1744,1699,1454,1368,1234,1095,1028,736,697cm -1.HRMS(ESI)m/z calcd for C 90H 95O 16NNa[M+Na] +1468.6549,found 1468.6521.
化合物17*：根据反应条件3.2，化合物16*(753mg,0.52mmol)脱去乙酰基得到化合物17*(657mg,90％)。R f＝0.32,petroleum ether/EtOAc＝3:1.[α] 25 D＝+13.5(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.50–6.97(m,36H,arom.H),5.14(d,J＝11.3Hz,2H),4.94(s,1H,1’-H),4.93(s,1H,1-H),4.87(d,J＝10.7Hz,1H),4.77(d,J＝11.0Hz,1H),4.68(s,1H),4.66–4.53(m,4H),4.50–4.34(m,7H),4.10(d,J＝9.6Hz,1H),4.06(s,1H),3.99(q,J＝6.1Hz,2H),3.92(d,J＝9.5Hz,1H),3.85–3.65(m,7H),3.35(d,J＝37.4Hz,1H),3.14(d,J＝34.3Hz,2H),2.93(d,J＝42.3Hz,1H),2.33(d,J＝2.3Hz,1H),1.79–1.44(m,2H). 13C NMR(101MHz,Chloroform-d)δ156.6,156.0,138.9,138.5,138.5,138.4,138.4,138.3,137.8,136.9,128.5,128.4,128.4,128.3,128.2,128.2,128.2,127.9,127.9,127.8,127.8,127.7,127.7,127.5,127.5,127.4,127.4,127.4,127.3,127.2,101.6,98.3,80.5,80.0,78.9,76.3,74.8,74.6,74.4,73.2,73.1,72.3,72.3,72.2,71.9,71.3,70.2,68.3,67.1,64.8,50.6,50.4,44.4,43.6,29.7,28.1,27.7.IR(film):ν＝3030,2918,1698,1453,1216,1054,1027,734,696cm -1.HRMS(ESI)m/z calcd for C 88H 93O 15NNa[M+Na] +1426.6443,found 1426.6480.
化合物18*：根据反应条件3.1，糖基供体11*(258mg,0.42mmol)和糖基受体17*(487mg,0.42mmol)反应得到18*(441mg,66％)。R f＝0.37,toluene/EtOAc＝9:1.[α] 25 D＝+16.8(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.48–6.99(m,60H,arom.H),5.61–5.54(m,1H),5.38(d,J＝3.3Hz,1H),5.36(s,1H),5.13(d,J＝9.9Hz,2H),5.11(s,1H,1”-H),4.93(s,1H,1’-H),4.82(t,J＝10.8Hz,3H),4.75(s,1H,1-H),4.72–4.57(m,7H),4.53–4.28(m,12H),4.10(dd,J＝10.1,5.5Hz,2H),3.98(q,J＝6.9,4.7Hz,3H),3.93–3.63(m,12H),3.54(dd,J＝10.1,6.6Hz,1H),3.44–3.21(m,1H),3.20–2.96(m,2H),2.98–2.71(m,1H),2.61(qd,J＝10.9,6.6,5.4Hz,1H),2.50(t,J＝6.4Hz,2H),2.37(m,1H),2.06(s,3H),1.98(s,3H),1.94(s,3H),1.64–1.47(m,2H). 13C NMR(101MHz,Chloroform-d)δ206.2,171.8,169.6,169.4,156.2,156.2,138.9,138.8,138.7,138.5,138.5,138.0,137.9,136.8,128.5,128.3,128.3,128.3,128.2,128.2,128.1,128.0,127.9,127.8,127.7,127.7,127.6,127.6,127.6,127.5,127.4,127.4,127.3,127.3,127.1,101.2,99.5,98.4,79.9,79.6,78.9,74.9,74.8,74.7,74.5,74.4,73.5,73.1,73.1,72.9,72.4,72.2,72.1,71.7,71.3,70.2,70.0,68.1,67.1,65.0,65.0,60.4,50.7,50.4,44.5,43.7,37.7,29.7,28.2,27.8,20.8,20.8,14.2.IR(film):ν＝3030,2919,1749,1698,1453,1365,1238,1216,1070,1027,735,696cm -1.HRMS(ESI)m/z calcd for C 118H 131O 25N 2[M+NH 4] +1975.9035,found 1975.9043.
化合物19*：将化合物18*(550mg,0.28mmol)溶于CH 2Cl 2/MeOH(20/1)(3.3mL)中，加入醋酸肼(40mg,0.42mmol)，反应在室温下搅拌3h。TLC检测反应完全后，加入适量DCM稀释，然后反应混合物用饱和NaHCO 3和食盐水洗，无水Na 2SO 4干燥，浓缩后硅胶柱 层析分离纯化(石油醚/乙酸乙酯:6/1→4/1)得到化合物19*(494mg,95％)。R f＝0.35,petroleum ether/EtOAc＝3:1.[α] 25 D＝+25.2(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.49–7.02(m,60H,arom.H),5.39(m,2H),5.15(s,2H,1 c-H),5.10(s,1H),4.97(s,1H,1 a-H),4.88(s,1H,1 b-H),4.78(d,J＝11.0Hz,2H),4.74–4.26(m,19H),4.12(m,2H),4.05–3.67(m,15H),3.57(dd,J＝9.9,4.4Hz,1H),3.48(dd,J＝9.8,7.4Hz,1H),3.42–3.19(m,1H),3.16–3.01(m,2H),3.01–2.77(m,1H),2.66(s,1H),2.07(s,3H,OAc),1.93(s,3H,OAc),1.69–1.41(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ169.8,169.6,156.8,156.2,139.1,139.0,138.9,138.7,138.7,138.6,138.6,138.0,128.7,128.6,128.6,128.5,128.4,128.4,128.3,128.2,128.1,128.0,127.9,127.8,127.8,127.6,127.5,127.4,127.4,127.3,101.3,99.3,98.4,80.2,79.7,79.2,76.7,75.5,75.1,75.0,74.8,74.6,73.7,73.6,73.4,73.3,73.3,73.1,72.5,72.4,72.3,71.6,70.9,70.6,70.2,67.3,65.1,28.0,21.1.IR(film):ν＝3030,2920,2360,1750,1698,1453,1366,1239,1218,1072,1028,913,735,696cm -1.HRMS(ESI)m/z calcd for C 113H 125O 23N 2[M+NH 4] +1877.8668,found 1877.8698.
实施例4 重复二糖和三糖的合成：
合成路线如图6所示。
具体试验操作和步骤：
4.1此发明中如未说明，脱去Lev基条件为：将起始原料溶解于CH 2Cl 2/MeOH(20/1，0.1M)中，加入醋酸肼(2eq)，反应在室温下搅拌3h。TLC检测反应完全后，加入适量DCM稀释，然后反应混合物用饱和NaHCO 3和食盐水洗，无水Na 2SO 4干燥，浓缩后硅胶柱层析分离纯化得到脱Lev受体。
化合物20*：根据反应条件3.1，活化试剂只加TMSOTf(0.12eq)，糖基供体8*(674mg,0.817mmol)和糖基受体6*(428mg,0.68mmol)反应得到20*(670mg,75％)。R f＝0.36,petroleum ether/EtOAc＝3:1.[α] 25 D＝+18.3(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.17–6.93(m,40H,arom.H),5.58–5.53(m,2H),5.40(d,J＝1.7Hz,2H,1-H/2-H),5.21(d,J＝2.0Hz,1H,1-H),4.94(d,J＝12.1Hz,1H),4.88(d,J＝10.6Hz,1H),4.80(d,J＝12.1Hz,1H),4.73(d,J＝10.7Hz,1H),4.69(d,J＝12.3Hz,1H),4.58(d,J＝11.0Hz,1H),4.55–4.45(m,3H),4.39–4.35(m,2H),4.30(dd,J＝9.5,3.3Hz,1H),4.27(d,J＝7.2Hz,1H),4.20(t,J＝9.6Hz,1H),4.06–3.94(m,3H),3.78(dd,J＝10.2,5.0Hz,1H),3.70(dd,J＝10.2,6.7Hz,1H),3.52(dd,J＝10.5,7.8Hz,1H),3.29(dd,J＝10.5,3.6Hz,1H),2.71–2.46(m,5H),2.43–2.27(m,2H,CH 2),2.03(s,3H,CH 3CO),1.25(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ206.1,171.6,165.7,165.2,139.3,138.6(d,J＝2.7Hz),138.3,138.0,137.8,133.3,129.9,129.7,129.7,129.4,128.6,128.5,128.3,128.3,128.2,128.2,127.5,127.4,127.4,127.4,127.3,127.3,127.1,99.7,82.0,79.7,79.4,78.5,75.3,75.0,74.2,74.0,73.6,73.5,73.3,72.8,72.6,72.5,72.5,71.8,70.9,70.3,37.8,29.7,28.0,25.5,14.9.IR(film):ν＝3030,2870,1720,1452,1264,1148,1093,1026,736,712,697cm -1.HRMS(ESI)m/z calcd for C 77H 84O 16SN[M+NH 4] +1310.5505,found 1310.5540.
化合物21*：根据反应条件4.1，化合物20*(440mg,0.34mmol)脱去Lev基得到化合物21*(405mg,quan.)。R f＝0.45,petroleum ether/EtOAc＝3:1.[α] 25 D＝+18.3(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.20–6.99(m,40H,arom.H),5.56(dd,J＝3.1,1.7Hz,1H),5.36(d,J＝1.6Hz,1H),5.34(dd,J＝3.2,1.8Hz,1H),5.25(d,J＝1.7Hz,1H),4.92(d,J＝12.1Hz,1H),4.85–4.75(m,3H),4.70(dd,J＝11.3,7.8Hz,3H),4.60(d,J＝11.4Hz,1H),4.49(d,J＝1.2Hz,2H),4.46(d,J＝12.2Hz,1H),4.41–4.36(m,2H),4.32(dd,J＝9.3,3.1Hz,1H),4.22(t,J＝9.6Hz,1H),4.09(pd,J＝6.6,6.0,3.5Hz,1H),4.01(dq,J＝5.5,2.6,1.9Hz,4H),3.79(dd,J＝10.2,5.1Hz,1H),3.70(dd,J＝10.2,6.7Hz,1H),3.63(dd,J＝10.4,7.5Hz,1H),3.52(dd,J＝10.3,4.3Hz,1H),2.72–2.46(m,2H,CH 2),1.91(d,J＝5.0Hz,1H,OH),1.23(t,J＝7.4Hz,3H, CH 3). 13C NMR(101MHz,Chloroform-d)δ165.8,165.4,139.2,138.7,138.6,138.3,138.2,137.7,133.3,133.2,129.8,129.8,129.5,128.6,128.4,128.3,128.3,128.2,128.2,128.0,127.8,127.6,127.5,127.5,127.4,127.4,127.3,127.2,99.4,82.1,79.2,78.6,77.7,75.6,75.4,75.1,74.2,73.8,73.3,73.2,73.0,72.8,72.5,72.5,71.4,71.0,70.4,25.4,14.8.IR(film):ν＝3050,2926,1720,1452,1265,1093,1070,1026,825,736,711,698cm -1.HRMS(ESI)m/z calcd for C 72H 78O 14SN[M+NH 4] +1212.5138,found 1212.5189.
化合物22*：根据反应条件3.1，活化试剂只加TMSOTf(0.12eq)，糖基供体8*(239mg,0.29mmol)和糖基受体21*(288mg,0.24mmol)反应得到22*(285mg,64％)。R f＝0.23,petroleum ether/EtOAc＝3:1.[α] 25 D＝+10.2(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.24–6.85(m,60H,arom.H),5.57(s,1H),5.56–5.52(m,1H),5.49–5.42(m,1H),5.39–5.36(m,1H),5.37(d,J＝1.8Hz,1H,1 c-H),5.33(d,J＝1.9Hz,1H,1 b-H),4.97(d,J＝1.9Hz,1H,1 a-H),4.93(d,J＝10.6Hz,1H),4.87(dd,J＝11.6,5.7Hz,2H),4.78(d,J＝12.1Hz,1H),4.75–4.62(m,4H),4.58–4.50(m,2H),4.49–4.41(m,2H),4.41–4.31(m,3H),4.28(d,J＝12.3Hz,1H),4.17(t,J＝9.8Hz,1H),4.10(d,J＝12.4Hz,1H),3.97(ddd,J＝11.9,7.3,4.2Hz,3H),3.87(d,J＝9.7Hz,1H),3.82(dd,J＝8.5,2.9Hz,1H),3.72(dd,J＝10.3,4.8Hz,1H),3.65(dd,J＝10.3,6.9Hz,1H),3.57(dd,J＝10.4,7.6Hz,1H),3.40–3.30(m,2H),3.03(dd,J＝10.5,3.0Hz,1H),2.71–2.42(m,4H,CH 2),2.40–2.23(m,2H,CH 2),2.01(s,3H,CH 3CO),1.22(t,J＝7.4Hz,3H,CH 3). 13C NMR(101MHz,Chloroform-d)δ206.1,171.6,165.5,165.5,165.1,139.4,139.2,138.7,138.7,138.3,137.9,137.9,137.7,133.2,133.2,129.9,129.8,129.8,129.7,129.4,129.4,128.5,128.4,128.3,128.2,128.2,128.1,128.1,128.1,127.5,127.5,127.4,127.4,127.4,127.3,127.2,127.2,127.2,127.1,127.0,127.0,99.8,98.7,82.0,80.3,79.2,78.6,78.2,75.4,75.3,74.9,74.2,74.0,73.7,73.6,73.2,73.2,72.9,72.6,72.5,72.4,72.2,72.1,71.6,71.0,70.2,37.8,29.7,27.9,25.4,14.9.IR(film):ν＝3030,2868,1721,1452,1263,1149,1092,1026,735,711,696cm -1.HRMS(ESI)m/z calcd for C 112H 118O 23SN[M+NH 4] +1876.7810,found 1876.7877.
还原端五糖和八糖的合成路线如图7所示。
化合物24*：根据反应条件3.1，糖基供体20*(417mg,0.30mmol,1.2equiv)和糖基受体23*(452mg,0.243mmol,1equiv)反应得到24*(488mg,65％)。R f＝0.18,petroleum ether/EtOAc＝2:1.[α] 25 D＝+14.8(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.04(d,J＝7.7Hz,2H,arom.H),7.93(d,J＝7.6Hz,2H,arom.H),7.69–6.82(m,96H,arom.H),5.73(t,J＝2.4Hz,1H),5.61(t,J＝2.5Hz,1H),5.49(d,J＝1.8Hz,1H,1-H),5.45(dd,J＝9.8,3.2Hz,1H),5.41(dd,J＝9.0,2.9Hz,1H),5.37(d,J＝2.3Hz,1H),5.22(s,1H,1-H),5.10(d,J＝4.5Hz,2H),4.94(s,1H,1-H),4.91(d,J＝2.5Hz,1H,1-H),4.90–4.81(m,3H),4.80(d,J＝1.4Hz,1H,1-H),4.79–4.70(m,4H),4.70–4.49(m,7H),4.49–4.08(m,23H),4.04(d,J＝9.8Hz,1H),4.00–3.64(m,16H),3.62–3.50(m,2H),3.45(tq,J＝7.5,4.2,3.2Hz,3H),3.39–3.15(m,2H),3.16–2.93(m,2H),2.94–2.70(m,1H),2.61(dt,J＝18.2,7.3Hz,1H),2.50(dt,J＝18.2,6.4Hz,1H),2.35(q,J＝6.8Hz,2H),2.08(s,3H,OAc),2.02(s,3H,OAc),1.77(s,3H,CH 3CO),1.61–1.41(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ206.0,171.6,170.2,169.5,165.5,165.2,139.1,139.1,139.0,138.9,138.8,138.7,138.6,138.6,138.2,138.2,138.1,138.0,137.9,137.8,136.9,133.3,129.9,129.7,129.7,129.5,128.6,128.5,128.4,128.3,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.1,128.0,127.9,127.8,127.7,127.7,127.7,127.5,127.5,127.4,127.3,127.3,127.3,127.2,127.2,127.1,127.1,101.4,100.3,99.1,98.3,96.0,80.1,79.6,79.3,79.1,78.9,77.5,76.0,75.9,75.3,75.2,75.0,74.8,74.7,74.6,74.2,73.4,73.4,73.3,73.1,73.1,73.0,73.0,72.9,72.9,72.7,72.7,72.4,72.3,72.3,72.3,72.2,72.1,72.1,71.6,71.5,71.4,70.5,70.4,69.8,67.1,65.0,50.6,43.7,37.8,29.6,28.5,28.0,20.6,20.6,7.9.IR(film):ν＝3030,2917,1748,1722,1453,1365,1265,1240,1071,1026,988,734,696cm -1.HRMS(ESI)m/z calcd for C 188H 195NO 39Na 2[M+2Na] 2+1568.1545,found 1568.1525.
化合物25*：根据反应条件4.1，化合物24*(393mg,0.127mmol)脱去Lev基得到化合物25*(353mg,93％)。R f＝0.48,petroleum ether/EtOAc＝2:1.[α] 25 D＝+19.2(c 1.0,CH 3Cl). 1H NMR(600MHz,Chloroform-d)δ8.03(d,J＝7.7Hz,2H,arom.H),7.92(d,J＝7.7Hz,2H,arom.H),7.69–6.87(m,94H,arom.H),5.77(s,1H),5.53(s,1H),5.47(dd,J＝9.9,3.1Hz,1H),5.44(d,J＝3.2Hz,1H),5.38(d,J＝3.1Hz,1H),5.31(s,1H),5.13(d,J＝9.9Hz,2H),4.98(d,J＝22.1Hz,1H),4.94–4.85(m,5H),4.84–4.59(m,14H),4.59–4.47(m,5H),4.47–4.10(m,24H),4.06(d,J＝9.8Hz,1H),4.02–3.93(m,7H),3.90(t,J＝9.7Hz,1H),3.87–3.68(m,8H),3.62(t,J＝9.3Hz,1H),3.58–3.47(m,4H),3.44(dd,J＝10.4,4.2Hz,1H),3.37–3.21(m,1H),3.18–2.96(m,3H),2.98–2.72(m,1H),2.08(s,3H,CH 3CO),1.97(d,J＝5.0Hz,1H,3-OH),1.76(s,3H,CH 3CO),1.64–1.46(m,2H,CH 2). 13C NMR(101MHz,Chloroform-d)δ170.1,169.5,165.9,165.4,139.2,139.1,139.0,138.9,138.8,138.7,138.6,138.2,138.2,138.1,138.0,137.9,136.9,133.3,133.2,129.8,129.8,129.6,128.6,128.5,128.4,128.4,128.3,128.3,128.3,128.1,128.0,127.9,127.9,127.8,127.8,127.7,127.6,127.6,127.5,127.4,127.4,127.3,127.2,127.2,127.2,127.1,101.4,100.0,99.1,98.3,96.0,80.1,79.5,79.3,78.9,78.6,78.4,77.5,76.0,75.7,75.5,75.2,75.0,74.8,74.6,74.1,73.4,73.3,73.2,73.2,73.1,73.0,73.0,72.8,72.4,72.4,72.3,72.2,72.1,72.1,71.6,71.6,71.3,70.8,70.5,69.8,67.1,65.0,60.4,50.7,31.6,29.1,22.6,20.6,20.6,14.1,14.1.IR(film):ν＝3030,2918,1749,1722,1453,1266,1071,1027,734,696cm -1.HRMS(ESI)m/z calcd for C 183H 189NO 37Na 2[M+2Na] 2+1519.1361,found 1519.1348.
化合物26*：根据反应条件3.1，糖基供体22*(228mg,0.126mmol)和糖基受体25*(314mg,0.105mmol)反应得到26*(306mg,61％)。R f＝0.29,petroleum ether/EtOAc＝2:1.[α] 25 D＝+4.6(c 0.5,CH 3Cl). 1H NMR(600MHz,Chloroform-d)δ8.02(d,J＝7.7Hz,2H,arom.H),7.98(d,J＝7.7Hz,2H,arom.H),7.88(dd,J＝12.9,7.8Hz,4H,arom.H),7.82(d,J＝7.7Hz,2H,arom.H),7.62–6.72(m,150H),5.67(s,1H),5.61(s,1H),5.46(s,3H),5.41(d,J＝9.8Hz,1H),5.37(s,1H),5.33(s,2H),5.09(d,J＝8.0Hz,3H),5.03(s,1H),4.99(d,J＝10.7Hz,1H),4.93(d,J＝21.1Hz,1H),4.85(s,3H),4.77(s,1H),4.68(ddd,J＝16.6,11.0,6.0Hz,4H),4.63–4.55(m,5H),4.55–4.42(m,8H),4.34(tdd,J＝28.2,17.9,10.5Hz,11H),4.25–4.10(m,10H),4.10–3.92(m,9H),3.92–3.82(m,4H),3.82–3.64(m,15H),3.60–3.22(m,9H),3.17(d,J＝10.1Hz,1H),3.12–3.02(m,1H),2.97(s,1H),2.92–2.79(m,3H),2.75(d,J＝8.8Hz,1H),2.55(dt,J＝18.2,7.3Hz,1H),2.45(dt,J＝18.2,6.5Hz,1H),2.30(tt,J＝18.1,8.8Hz,3H,CH 3CO),2.05(s,3H,CH 3CO),1.98(s,3H,CH 3CO),1.70(s,3H,CH 3CO),1.54(tt,J＝14.6,6.7Hz,1H,CH 2),1.48–1.38(m,1H,CH 2). 13C NMR(101MHz,Chloroform-d)δ206.0,171.6,170.3,169.6,165.4,165.4,165.2,165.1,139.5,139.5,139.2,139.2,138.9,138.9,138.8,138.8,138.7,138.6,138.6,138.2,138.1,137.9,137.8,137.6,137.6,136.9,133.2,129.9,129.9,129.7,129.5,129.5,129.3,128.6,128.6,128.5,128.4,128.3,128.3,128.2,128.1,128.0,127.9,127.8,127.7,127.6,127.5,127.4,127.3,127.3,127.2,127.2,127.1,127.1,126.9,126.8,101.4,99.9,99.6,99.1,99.0,98.3,96.0,80.6,80.5,80.1,79.6,79.2,78.9,75.9,75.5,75.1,75.0,74.6,74.1,73.9,73.8,73.6,73.6,73.5,73.3,73.1,72.9,72.9,72.8,72.7,72.6,72.6,72.5,72.3,72.2,72.1,71.8,71.7,71.5,71.4,70.5,70.3,69.8,67.1,65.0,60.4,50.6,37.7,29.6,27.9,21.0,20.6.IR(film):ν＝3030,2922,2360,1722,1452,1365,1264,1092,1026,733,696cm -1.HRMS(ESI)m/z calcd for C 293H 305N 3O 60[M+2NH 4] 2+2412.5448,found 2412.5464.
化合物27*：根据反应条件4.1，化合物26*(300mg,0.063mmol)脱去Lev基得到化合物27*(264mg,90％)。R f＝0.46,petroleum ether/EtOAc＝2:1.[α] 25 D＝+25.6(c 1.0,CH 3Cl). 1H NMR(600MHz,Chloroform-d)δ8.02(d,J＝7.7Hz,2H,arom.H),7.98(d,J＝7.7Hz,2H,arom.H),7.91(d,J＝7.6Hz,2H,arom.H),7.86(d,J＝7.7Hz,2H,arom.H),7.81(d,J＝7.7Hz,2H,arom.H),7.51(t,J＝7.5Hz,2H,arom.H),7.43(q,J＝7.9Hz,3H,arom.H),7.37–6.79(m,145H,arom.H),5.68(s,1H),5.61(s,1H),5.48–5.45(m,2H),5.44(s,1H),5.41(dd,J＝10.0,2.9 Hz,1H),5.33(s,2H),5.13–5.07(m,5H),5.03(s,1H),4.99(d,J＝10.6Hz,1H),4.95(s,0H),4.90(s,1H),4.88–4.80(m,5H),4.80–4.73(m,3H),4.72–4.64(m,5H),4.63–4.55(m,5H),4.55–4.48(m,6H),4.48–4.25(m,16H),4.24–4.11(m,10H),4.11–3.82(m,15H),3.81–3.63(m,18H),3.53(t,J＝9.1Hz,1H),3.47(t,J＝9.0Hz,1H),3.44–3.37(m,1H),3.35(dd,J＝10.5,3.6Hz,1H),3.28(dq,J＝20.0,9.8Hz,3H),3.17(d,J＝10.2Hz,1H),3.07(s,1H),3.01–2.93(m,2H),2.87(d,J＝10.1Hz,3H),2.75(s,1H),2.05(s,3H,CH 3CO),1.76(d,J＝5.3Hz,1H,3-OH),1.69(s,3H,CH 3CO),1.47–1.40(m,1H,CH 2). 13C NMR(101MHz,Chloroform-d)δ170.4,169.7,165.7,165.4,165.3,165.3,165.2,156.5,139.5,139.4,139.2,139.1,138.9,138.8,138.7,138.7,138.6,138.5,138.2,138.1,138.0,137.8,137.7,137.6,137.5,136.8,133.3,129.9,129.9,129.8,129.6,129.4,129.3,128.7,128.6,128.5,128.4,128.3,128.3,128.3,128.2,128.2,128.2,128.1,128.1,128.0,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.4,127.4,127.4,127.3,127.2,127.2,127.1,127.1,127.0,126.9,126.9,101.4,99.5,99.0,98.2,95.9,80.6,80.4,80.2,80.0,79.7,79.6,79.2,78.9,75.7,75.5,75.1,75.0,74.7,74.5,74.1,73.8,73.6,73.6,73.5,73.2,73.1,72.9,72.8,72.8,72.6,72.6,72.5,72.4,72.3,72.2,72.1,71.8,71.7,71.4,70.5,69.7,67.1,50.6,50.4,44.4,43.6,28.2,27.7,20.7,20.7.IR(film):ν＝3033,2932,1726,1498,1455,1267,1096,737,698cm -1.HRMS(ESI)m/z calcd for C 288H 299N 3O 60[M+2NH 4] 2+2363.5264,found 2363.5398.
实施例5 幽门螺旋杆菌O:6血清型O-抗原十三糖的合成：
合成路线如图8所示。
化合物30*：根据反应条件3.1，活化试剂为TfOH(0.2eq)和NIS(1.2eq)，糖基供体29*(39mg,0.036mmol)和糖基受体28*(22mg,0.043mmol)反应得到30*(41mg,70％)。R f＝0.35,Hexane/EtOAc＝3:2.[α] 25 D＝-12.0(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.00–7.94(m,2H,arom.H),7.65–7.10(m,33H,arom.H),6.36(d,J＝9.3Hz,1H,NH),5.49(dd,J＝10.0,7.6Hz,1H),5.11(dd,J＝10.0,7.9Hz,1H),5.00(d,J＝11.6Hz,1H,Ph-CH 2),4.84(d,J＝11.4Hz,1H,Ph-CH 2),4.67(d,J＝7.8Hz,1H,1-H),4.64–4.56(m,3H),4.48–4.39(m,6H),4.37(d,J＝11.7Hz,1H,Ph-CH 2),4.31(d,J＝7.9Hz,1H,1-H),4.03(dd,J＝10.2,3.1Hz,1H),4.01–3.88(m,5H),3.81(dd,J＝11.1,3.5Hz,1H),3.73(dd,J＝11.1,1.8Hz,1H),3.65(t,J＝6.1Hz,1H),3.61–3.48(m,5H),3.38(dd,J＝8.3,5.1Hz,1H),3.33(dd,J＝10.1,2.8Hz,1H),2.77(ddd,J＝18.3,8.0,5.7Hz,1H,CH 2),2.57(dt,J＝18.3,6.0Hz,1H,CH 2),2.52–2.41(m,2H,CH 2),2.40–2.26(m,4H,CH 2),2.15(s,3H,CH 3CO),1.88(s,3H,CH 3CO),0.69(s,9H,CH 3),0.01(s,3H,SiCH 3),-0.07(s,3H,SiCH 3). 13C NMR(101MHz,Chloroform-d)δ206.5,206.4,172.5,171.1,164.7,162.0,138.6,138.5,138.1,138.0,137.9,137.7,133.2,130.0,129.7,128.9,128.6,128.5,128.5,128.4,128.4,128.2,128.1,128.1,128.0,128.0,127.9,127.8,127.7,127.5,127.5,127.4,101.2,100.5,96.7,91.9,80.2,78.9,75.9,75.0,74.8,74.6,74.2,74.1,74.0,73.5,73.1,72.3,72.2,71.9,71.7,69.2,67.8,67.7,55.9,37.8,37.7,30.0,29.6,27.9,27.8,25.4,17.7,-4.1,-5.4.IR(film):ν＝1720,1071,838,700cm -1.
化合物31*：将化合物30*(40mg,0.025mmol)溶于THF(1mL)中，然后加入醋酸(14μL,0.25mmol)搅拌，在0℃条件下加入TBAF/THF(1M,0.25mL)，然后在反应在室温下搅拌4h，TLC检测反应完全后，加入适量DCM稀释，后用饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩后柱层析分离纯化得到相应的半缩醛。
将上面得到的半缩醛溶于CH 2Cl 2(2mL)中，在0℃下加入CCl 3CN(18μL,0.125mmol)和DBU(11μL,0.075mmol)，反应在室温下搅拌1.5min。TLC检测反应完全之后，在30℃下浓缩反应液，后经硅胶柱层析分离纯化(正己烷/乙酸乙酯:2/1→1/1)得到化合物31*(37mg,87％)。R f＝0.44,Hexane/EtOAc＝1:1.[α] 25 D＝7.3(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ8.03(d,J＝7.8Hz,2H,arom.H),7.72–6.96(m,38H,arom.H),6.57(d,J＝7.7Hz,2H),6.41(d,J＝9.3Hz,1H),5.91(s,1H),5.77(t,J＝9.1Hz,1H),5.21–5.01(m,2H),5.00–4.77(m,2H),4.77–4.56(m,4H),4.55–4.26(m,9H),4.26–3.88(m,5H),3.90–3.69(m,2H), 3.70–3.49(m,6H),3.37(ddd,J＝22.6,9.1,4.0Hz,2H),2.79(ddd,J＝18.3,8.3,5.4Hz,1H,CH 2),2.67–2.26(m,7H,CH 2),2.17(s,3H,CH 3CO),1.90(s,3H,CH 3CO). 13C NMR(101MHz,Chloroform-d)δ206.5,206.3,172.6,171.1,164.6,162.0,143.2,138.5,138.3,137.9,137.8,137.7,137.6,133.7,129.8,129.2,128.9,128.8,128.6,128.5,128.4,128.2,128.0,128.0,127.9,127.8,127.8,127.6,127.6,127.4,124.2,119.2,101.4,100.5,95.4,91.9,80.2,78.6,75.4,75.1,75.0,74.6,74.0,73.5,73.1,72.3,72.0,71.9,71.8,71.6,67.7,55.9,37.7,37.7,30.0,29.6,27.9,27.8.IR(film):ν＝2873,1719,1456,12661210,1164,1096,820,737,698cm -1.
化合物32*：根据反应条件3.1，活化试剂只加TMSOTf(0.15eq)，糖基供体31*(37mg,0.0223mmol，1.5eq)和糖基受体27*(70mg,0.0149mmol，1eq)反应得到32*(59mg,65％)。R f＝0.26,Hexane/EtOAc＝3:2.[α] 25 D＝+11.8(c 1.0,CH 3Cl). 1H NMR(700MHz,Chloroform-d)δ8.04(d,J＝7.6Hz,2H,arom.H),7.98(d,J＝7.6Hz,2H,arom.H),7.93(d,J＝7.7Hz,2H,arom.H),7.85(d,J＝7.6Hz,2H,arom.H),7.62–6.56(m,187H,arom.H),6.06(d,J＝9.2Hz,1H,NH),5.69(s,1H),5.62(d,J＝3.1Hz,1H),5.48(s,1H),5.47(s,1H),5.43(d,J＝12.4Hz,2H),5.39–5.32(m,3H),5.18–5.09(m,4H),5.09–5.04(m,2H),5.00(d,J＝10.6Hz,1H),4.97–4.92(m,2H),4.91–4.57(m,27H),4.56–4.29(m,33H),4.27–4.08(m,17H),4.05–3.85(m,18H),3.84–3.65(m,15H),3.63(d,J＝8.8Hz,1H),3.60–3.47(m,7H),3.46–3.33(m,7H),3.30(t,J＝9.5Hz,1H),3.25(t,J＝9.6Hz,1H),3.18(d,J＝9.8Hz,1H),3.10(dd,J＝8.9,5.0Hz,2H),2.98(q,J＝9.3,6.7Hz,2H),2.87(d,J＝9.7Hz,1H),2.84–2.75(m,1H),2.73(d,J＝9.9Hz,1H),2.65–2.57(m,2H),2.50(ddd,J＝14.2,8.2,5.4Hz,1H),2.44(dd,J＝16.4,7.5Hz,1H),2.41–2.26(m,4H,CH 2),2.18(s,3H,CH 3CO),2.07(s,3H,CH 3CO),1.87(s,3H,CH 3CO),1.72(s,3H,CH 3CO),1.50–1.41(m,1H). 13C NMR(176MHz,Chloroform-d)δ206.2,206.1,172.4,171.1,170.3,169.6,165.3,165.2,165.0,164.0,161.8,139.6,139.5,139.5,139.2,139.1,139.0,138.9,138.9,138.8,138.8,138.7,138.6,138.6,138.5,138.5,138.1,138.1,138.0,138.0,137.9,137.9,137.8,137.7,137.4,132.5,129.9,129.9,129.6,129.4,129.3,129.2,129.1,129.0,128.8,128.6,128.6,128.5,128.5,128.4,128.3,128.3,128.2,128.2,128.1,128.1,128.1,128.0,128.0,128.0,127.9,127.9,127.8,127.8,127.8,127.7,127.7,127.6,127.6,127.5,127.5,127.4,127.4,127.3,127.3,127.3,127.2,127.1,127.1,127.1,127.0,127.0,127.0,126.9,126.9,126.8,126.7,101.3,101.1,100.5,99.5,99.0,98.8,91.7,80.8,80.3,80.0,79.2,78.5,75.9,75.8,75.1,75.0,75.0,74.7,74.5,74.5,74.3,74.0,73.9,73.7,73.5,73.5,73.2,73.1,73.1,72.9,72.8,72.7,72.6,72.4,72.4,72.3,72.2,72.2,72.1,72.0,71.8,71.7,71.3,70.5,69.7,69.0,67.7,67.5,67.0,55.7,37.7,29.9,29.5,27.8,20.6,20.6.IR(film):ν＝2928,1724,1455,1267,1097,737,698cm -1.
化合物33*：将化合物32*(20mg,0.0032mmol)溶于吡啶(0.5mL)中，加入醋酸肼(2mg,0.016mmol)，反应在室温下搅拌3h。TLC检测反应完全后，浓缩除去吡啶，加入适量DCM稀释，然后反应混合物分别用1M HCl、饱和NaHCO 3和食盐水洗，无水Na 2SO 4干燥，浓缩后硅胶柱层析分离纯化(石油醚/乙酸乙酯:2/1→3/2)得到化合物33*(17mg,89％)。R f＝0.34,Hexane/EtOAc＝3:2.[α] 25 D＝+13.4(c 1.0,CH 3Cl). 1H NMR(700MHz,Chloroform-d)δ8.04(d,J＝7.6Hz,2H,arom.H),7.98(d,J＝7.7Hz,2H,arom.H),7.93(d,J＝7.7Hz,2H,arom.H),7.86(d,J＝7.5Hz,2H,arom.H),7.53–6.55(m,187H,arom.H),6.04(d,J＝8.6Hz,1H,NH),5.69(s,1H),5.63(s,1H),5.48(s,1H),5.47(s,2H),5.42(q,J＝10.5Hz,3H),5.34(d,J＝7.8Hz,2H),5.12(d,J＝10.4Hz,3H),5.08(s,2H),5.01(d,J＝10.7Hz,1H),4.98–4.90(m,3H),4.90–4.45(m,40H),4.37(ddt,J＝45.4,24.8,10.6Hz,13H),4.28–4.15(m,11H),4.15–4.07(m,6H),4.01(dt,J＝20.8,9.3Hz,10H),3.94–3.84(m,6H),3.83–3.65(m,16H),3.62(d,J＝9.1Hz,2H),3.52(tq,J＝28.2,9.6,8.4Hz,7H),3.37(dtt,J＝50.0,18.4,10.2Hz,7H),3.25(t,J＝9.7Hz,1H),3.19(t,J＝13.6Hz,1H),3.10(d,J＝6.6Hz,2H),2.99(q,J＝8.3,6.7Hz,2H),2.88(t,J＝15.7Hz,1H),2.81–2.75(m,1H),2.72(d,J＝10.0Hz,1H),2.62(d,J＝10.3Hz,1H),2.07(s,3H,CH 3CO),1.72(s,3H,CH 3CO),1.50–1.41(m,1H). 13C NMR(176MHz,Chloroform-d)δ 170.3,169.6,165.3,165.3,165.2,165.2,165.1,164.1,161.8,139.6,139.5,139.5,139.2,139.1,139.0,139.0,138.8,138.8,138.7,138.6,138.6,138.5,138.4,138.1,138.1,138.1,137.9,137.9,137.8,137.8,137.8,137.7,137.4,137.4,137.3,133.2,133.1,132.8,132.6,129.9,129.9,129.6,129.4,129.3,129.2,129.2,129.1,129.0,128.7,128.6,128.6,128.5,128.5,128.3,128.3,128.3,128.2,128.2,128.2,128.2,128.1,128.1,128.0,128.0,128.0,127.9,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.6,127.4,127.4,127.3,127.3,127.2,127.1,127.1,127.1,127.0,127.0,127.0,126.9,126.9,126.8,126.7,104.2,101.3,100.3,99.5,99.0,98.8,97.7,95.9,92.1,82.7,81.9,81.2,81.1,80.8,80.5,80.0,79.6,79.2,78.9,77.9,75.8,75.5,75.1,75.0,74.7,74.5,74.5,74.4,74.0,74.0,73.9,73.8,73.7,73.6,73.4,73.2,73.2,73.1,72.9,72.8,72.8,72.6,72.5,72.4,72.3,72.3,72.2,72.1,71.9,71.8,71.7,71.4,71.3,71.0,70.5,69.7,69.0,68.9,68.4,67.5,67.1,57.3,50.6,45.3,29.7,20.6,20.6.IR(film):ν＝3432,3066,3033,2926,2869,1725,1603,1498,1455,1367,1267,1218,1096,1028,912,820,736,698cm -1.
化合物35*：将糖基供体34*(7mg，0.0107mmol)和糖基受体33*(15mg，0.00268mmol，1eq)混合，溶于甲苯中共蒸两次。加入干燥的DCM/Et 2O(v/v，1：1)(0.2mL)中，加入活化的
Figure PCTCN2019091198-appb-000017
或者
Figure PCTCN2019091198-appb-000018
分子筛。加入10eq的噻酚，将反应温度降至-40℃，搅拌15min，然后加入活化试剂TMSOTf(0.4eq)，反应在-40℃下搅拌3h。TLC检测反应完全后，加入适量Et 3N终止反应。反应液过滤，用DCM稀释后用饱和NaHCO 3和饱和的食盐水洗涤，无水Na 2SO 4干燥，浓缩后利用硅胶柱层析分离纯化(正己烷/乙酸乙酯:2/1→3/2)得到化合物35*(9mg，52％)。R f＝0.48,Hexane/EtOAc＝3:2.[α] 25 D＝-1.5(c1.0,CH 3Cl). 1H NMR(700MHz,Chloroform-d)δ8.02(d,J＝7.7Hz,2H,arom.H),7.96(d,J＝7.6Hz,2H,arom.H),7.90(d,J＝7.5Hz,2H,arom.H),7.83(d,J＝7.5Hz,2H,arom.H),7.60–6.52(m,217H,arom.H),6.35(d,J＝7.6Hz,1H,NH),5.68–5.66(m,1H),5.65(d,J＝4.0Hz,1H),5.62–5.57(m,1H),5.46(s,1H),5.45–5.39(m,4H),5.35–5.30(m,1H),5.32(s,2H),5.12–5.08(m,3H),5.05(s,1H),5.01–4.92(m,5H),4.89(d,J＝4.2Hz,1H),4.87–4.81(m,5H),4.80–4.74(m,7H),4.67(ddd,J＝13.6,11.0,6.7Hz,5H),4.62–4.42(m,23H),4.41–4.25(m,16H),4.25–4.04(m,22H),4.03–3.95(m,9H),3.95–3.88(m,3H),3.87–3.56(m,23H),3.55–3.45(m,5H),3.44–3.30(m,4H),3.30–3.25(m,2H),3.22(td,J＝9.8,5.1Hz,2H),3.16(d,J＝10.2Hz,1H),3.12(d,J＝2.5Hz,1H),3.03(tq,J＝32.8,11.5,9.2Hz,3H),2.84(d,J＝9.6Hz,1H),2.79–2.73(m,1H),2.73–2.66(m,1H),2.62(d,J＝9.9Hz,1H),2.05(s,3H,CH 3CO),1.70(s,3H,CH 3CO),1.45(ddd,J＝15.8,7.5,4.0Hz,1H),1.12(d,J＝6.3Hz,3H,CH 3-Fucose),1.02(d,J＝6.3Hz,3H,CH 3-Fucose). 13C NMR(176MHz,Chloroform-d)δ170.4,169.7,165.5,165.5,165.3,165.2,164.6,161.2,156.4,156.4,139.7,139.7,139.6,139.4,139.2,139.1,139.1,139.0,139.0,138.9,138.8,138.8,138.7,138.6,138.3,138.3,138.2,138.1,138.0,138.0,137.9,137.9,137.8,137.6,137.5,133.4,133.2,132.8,130.1,130.0,129.8,129.6,129.5,129.4,129.3,129.1,129.0,128.8,128.7,128.7,128.6,128.6,128.6,128.5,128.5,128.5,128.5,128.4,128.3,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.0,128.0,128.0,127.9,127.9,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.6,127.5,127.5,127.4,127.4,127.4,127.3,127.3,127.2,127.2,127.2,127.1,127.1,127.1,127.0,126.9,126.8,126.2,101.5,100.3,99.6,99.5,99.1,99.0,98.4,98.1,97.7,97.5,96.0,91.8,84.0,81.3,81.2,81.0,80.6,80.1,79.8,79.7,79.4,79.3,79.2,79.1,78.6,78.4,78.3,76.7,76.3,75.9,75.8,75.7,75.6,75.4,75.2,75.1,75.0,75.0,74.9,74.8,74.7,74.6,74.3,74.2,74.0,73.9,73.9,73.8,73.7,73.7,73.5,73.5,73.3,73.3,73.2,73.2,73.1,73.0,73.0,72.9,72.8,72.7,72.6,72.6,72.5,72.4,72.4,72.3,72.3,72.2,72.2,72.1,72.0,71.9,71.8,71.6,71.5,71.2,70.6,70.2,69.8,68.8,68.1,67.8,67.6,67.2,66.9,66.9,66.6,66.3,65.0,50.8,50.5,44.5,43.8,28.3,27.8,20.8,20.8,16.5,16.3.IR(film):ν＝1725,1266,1096,734,698cm -1.
化合物36*：将化合物35*(9mg，0.0014mmol，52％)溶于AcOH(1mL)中，加入新活化的锌粉(100mg)，反应在室温下搅拌12h，TLC检测原料消失后，溶液过滤并减 压浓缩，然后加入适量的DCM稀释，饱和NaHCO 3洗涤，无水Na 2SO 4干燥，溶液过滤后减压浓缩，真空干燥得到NAc中间产物。将上述中间产物溶于THF/MeOH(1:1，1mL)中，加入MeONa(30mg)，室温下搅拌15min，然后加入NaOH(aq，1M，100μL)，反应在室温下搅拌12h，TLC检测反应完全后，加入Amerlite IR 120(H +)树脂中和反应液pH达到7，溶液过滤并减压浓缩，硅胶柱层析(二氯甲烷/甲醇:50/1)分离纯化得到中间产物。将上述脱酰基化合物溶于MeOH/THF/H 2O/AcOH(10:5:4:1，12mL)中，加入10％Pd/C(50mg)，反应混合物在氢气(1bar)条件下搅拌24h，经飞行时间质谱检测反应完成后，溶液过滤并浓缩，真空干燥，后经Sephadex LH20凝胶柱分离纯化后得到化合物36*。 1H NMR(700MHz,Deuterium Oxide)δ5.20(d,J＝2.8Hz,1H,1-H),5.13(s,1H,1-H),5.10(s,1H,1-H),5.09(s,1H,1-H),5.06–5.01(m,5H,1-H),4.95(s,1H,1-H),4.82–4.78(m,1H),4.65(d,J＝8.6Hz,1H,1-H),4.44(dd,J＝7.8,4.1Hz,2H),4.21–4.14(m,6H),4.12(s,1H),4.06(s,1H),3.99(t,J＝9.7Hz,12H),3.95–3.86(m,10H),3.86–3.68(m,44H),3.64(dt,J＝21.4,9.9Hz,15H),3.60–3.50(m,6H),3.41–3.36(m,1H),3.11–3.01(m,2H,CH 2),1.95(s,3H,CH 3CO),1.94–1.89(m,2H,CH 2),1.19(d,J＝6.5Hz,3H,Fucose-CH 3),1.18–1.13(d,J＝6.5Hz,3H,Fucose-CH 3). 13C NMR(176MHz,Deuterium Oxide)δ158.8,102.1,102.0,101.9,101.7,101.2,100.5,100.2,99.4,98.5,98.1,82.1,79.7,79.0,78.6,78.6,78.2,77.9,77.8,76.3,75.4,74.8,74.8,74.0,73.9,73.8,73.7,73.5,73.2,73.1,72.1,71.9,71.7,71.5,71.4,71.4,71.3,70.6,70.4,70.1,69.9,69.7,69.5,69.4,69.1,68.7,68.4,68.3,67.7,67.7,67.5,67.5,66.9,66.8,66.8,66.7,66.0,65.7,64.9,62.1,62.0,61.7,61.6,61.5,61.5,,61.0,59.8,37.5,26.6,22.3,15.4(d,J＝3.2Hz).
实施例6 不同的十三糖合成策略：
发明人在实验过程中，也曾尝试其他不同的[5+8]和[4+9]合成策略方法，分别以下述的四塘、五糖作为供体来构建十三糖，合成路线分别如图10、11所示。
(1)糖基化供体四糖和五糖的合成：
合成路线如图9所示。
该方法是以单糖砌块37*为受体，与糖基化供体38*在活化试剂TMSOTf的作用下反应得到二糖39*，然后利用CH 3ONa脱去酰基Bz和Lev得到二糖受体40*，后与4当量的糖基供体34*反应得到全保护的Le y四糖。为了进一步得到五糖供体，利用化合物28*作为糖基化受体与供体Le y四糖反应得到五糖42*，由于供体上的氨基保护基Phth钝化了供体的活性，所以导致此反应产率只有21％。后经TBAF脱去还原端的TBS，进而转化为NPh基三氟乙酰亚胺酯供体43*用于后续的糖基化反应。
为了优化五糖的合成，我们决定替换氨基保护基Phth为Troc，用以提高四糖供体的活性，首先利用乙二胺加热的条件下脱去邻苯二甲酰基，然后对氨基进行Troc保护得到化合物44*，同样在TfOH和NIS的作用下与受体28*反应得到化合物45*。由于在用TBAF脱除TBS时会脱掉部分氨基保护基Troc，因此我们选择HF来脱除TBS，虽然我们成功脱除了TBS，但是仍然得到了氨基裸露的副产物，混合物经分离纯化之后转化为NPh基三氟乙酰亚胺酯供体43*用于后续的糖基化反应。
具体试验操作和步骤：
化合物39*：将化合物37*(270mg,0.50mmol)和38*(435mg,0.60mmol)溶于无水的CH 2Cl 2(10mL)中，加入新活化的
Figure PCTCN2019091198-appb-000019
分子筛，降低温度至-10℃，然后在氩气保护下逐滴加入TMSOTf(11μL,0.06mmol)。在-10℃条件下反应3h，TLC检测反应完全之后，加入适量的Et 3N淬灭反应，反应液经过滤浓缩后，利用硅胶柱层析纯化(石油醚/乙酸乙酯:20/1→8/1)得到二糖化合物39*(330mg,63％)。
化合物40*：将化合物39*(214mg,0.20mmol)溶于THF/MeOH(1/1,v/v,2mL)中，室温下加入甲醇钠(20mg,0.40mmol)。在室温下反应过夜，TLC检测原料反应完全之后，加入甲醇稀释，利用树脂中和过量的甲醇钠，调节pH至0后溶液过滤浓缩，真空干燥。将上述粗 品化合物溶于吡啶(20mL)中，加入10％AcOH(2mL)，加热回流16h。TLC检测反应完成之后浓缩干燥，经硅胶柱层析纯化(石油醚/乙酸乙酯:5/1→2/1)得到化合物40*(150mg,86％)。
化合物41*：将化合物34*(412mg,0.68mmol,4eq)和化合物40*(150mg,0.17mmol,1eq)溶于无水的CH 2Cl 2/Et 2O(1/1,v/v,3mL)中，加入
Figure PCTCN2019091198-appb-000020
分子筛，将反应温度降至-40℃,然后在氩气保护下逐滴加入TMSOTf(12μL)。在-40℃条件下反应2.5h。TLC检测反应完成之后，加入适量Et 3N淬灭反应，溶液过滤浓缩，硅胶柱层析纯化(石油醚/乙酸乙酯:4/1)得到化合物41*(215mg,74％)。R f＝0.40,Petroleum ether/EtOAc＝2:1.[α] 25 D＝-51.2(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.91–7.65(m,4H,arom.H),7.48–6.81(m,50H,arom.H),5.73(d,J＝3.8Hz,1H,1-H),5.13(d,J＝10.5Hz,1H,1-H),5.00(d,J＝11.3Hz,1H,Ph-CH 2),4.84–4.71(m,6H,Ph-CH 2),4.68(d,J＝4.2Hz,1H,1-H),4.62(d,J＝8.3Hz,1H,1-H),4.60–4.42(m,9H),4.42–4.33(m,2H),4.29(d,J＝11.6Hz,2H),4.23(t,J＝9.5Hz,1H),4.16–4.07(m,2H),4.02(d,J＝2.9Hz,1H),3.92(dd,J＝11.6,3.6Hz,1H),3.89–3.82(m,2H),3.81–3.65(m,6H),3.62(dd,J＝9.6,3.0Hz,1H),3.41(ddd,J＝21.0,9.6,4.0Hz,2H),3.11(d,J＝2.4Hz,1H),2.83–2.63(m,2H,SCH 2),1.43(d,J＝6.5Hz,3H,CH 3-Fucose,),1.22(t,J＝7.4Hz,3H,CH 3),1.14(d,J＝6.4Hz,3H,CH 3-Fucose). 13C NMR(101MHz,Chloroform-d)δ168.4,167.1,139.2,138.8,138.8,138.7,138.5,138.4,138.1,138.0,137.7,134.3,134.1,131.8,131.6,128.7,128.6,128.4,128.4,128.3,128.2,128.1,128.1,128.0,127.8,127.8,127.6,127.5,127.5,127.4,127.4,127.3,127.3,127.2,127.0,126.9,126.1,123.8,123.5,100.0,98.1,97.9,83.9,81.5,80.6,79.7,79.1,78.3,78.1,75.6,75.4,74.9,74.7,73.9,73.6,73.3,73.2,73.1,72.9,72.8,72.5,72.4,71.7,70.9,68.2,67.7,67.0,66.8,55.6,23.5,16.4,16.2,14.9.IR(film):ν＝3032,2870,2351,1778,1716,1498,1455,1385,1365,1208,1095,1044,1028,912,815,735,697cm -1.HRMS(ESI)m/z calcd for C 104H 109NO 19SNa[M+Na] +1730.7207,found 1730.7238.
化合物42*：将供体四糖41*(35mg,0.019mmol)和受体28*(21mg,0.038mmol)溶于无水的CH 2Cl 2(1mL)中，加入
Figure PCTCN2019091198-appb-000021
分子筛和NIS(5mg,0.023mmol)，将反应温度降至-20℃,然后在氩气保护下逐滴加入TfOH(0.34μL,0.0038mmol)。在-20℃条件下反应3h。TLC检测反应完成之后，加入适量Et 3N淬灭反应，溶液过滤，用10％Na 2S 2O 3溶液洗至无色，然后依次用饱和的NaHCO 3、饱和的食盐水洗，无水Na 2SO 4干燥，溶液浓缩后经硅胶柱层析纯化(正己烷/乙酸乙酯:4/1)得到化合物42*(5.5mg,13％)。
化合物43*：将化合物42*(11mg,0.005mmol)溶于THF(0.5mL)中，然后加入醋酸(3μL,0.05mmol)搅拌，在0℃条件下加入TBAF/THF(1M,0.05mL)，然后在反应在室温下搅拌4h，TLC检测反应完全后，加入适量DCM稀释，后用饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩后柱层析分离纯化得到相应的半缩醛。
将上面得到的半缩醛溶于CH 2Cl 2(0.5mL)中，在0℃下加入CCl 3CN(4μL,0.025mmol)和DBU(3μL,0.019mmol)，反应在室温下搅拌1.5h。TLC检测反应完全之后，在30℃下浓缩反应液，后经硅胶柱层析分离纯化(正己烷/乙酸乙酯:5/1→3/1)得到化合物43*(8mg,71％)。
化合物44*：将化合物41*(164mg,0.096mmol)溶于n-BuOH(5mL)中，加入乙二胺(5mL)，在95℃条件下反应6h，减压浓缩，利用甲苯共沸蒸发两次得到粗品化合物。将上述粗品溶于吡啶(2mL)中,然后加入TrocCl(33μL,0.24mmol)，反应在室温下搅拌过夜，TLC检测反应完成后，在0℃下加入适量甲醇淬灭,溶液浓缩后经硅胶柱层析纯化(正己烷/乙酸乙酯:5:1→3:1)得到化合物44*(128mg,76％)。R f＝0.50,Hexane/EtOAc＝2:1.[α] 22 D＝-45.3(c 1.0,CH 3Cl). 1H NMR(400MHz,Chloroform-d)δ7.47–6.96(m,50H,arom.H),5.67(d,J＝3.9Hz,1H,1-H),5.33(d,J＝6.9Hz,1H,NH),5.13(d,J＝10.2Hz,1H,1-H),4.98(d,J＝3.9Hz,1H,1-H),4.85–4.71(m,5H),4.70–4.63(m,4H),4.64–4.55(m,5H),4.55–4.45(m,6H),4.50(d,J＝8.3Hz,1H,1-H),4.41(d,J＝2.7Hz,2H),4.36(d,J＝10.6Hz,1H),4.26(q,J＝6.4Hz,1H),4.10–4.02(m,3H),4.01–3.94(m,3H),3.89(d,J＝11.3Hz,1H),3.85–3.73(m,3H),3.73– 3.67(m,1H),3.66–3.61(m,2H),3.61–3.58(m,1H),3.55(dd,J＝9.8,3.1Hz,1H),3.32(dd,J＝8.9,4.9Hz,1H),3.22(dd,J＝10.2,3.0Hz,1H),3.16(d,J＝2.4Hz,1H),3.04(q,J＝9.2Hz,1H),2.74–2.53(m,2H),1.32–1.18(m,6H,CH 3/CH 3-Fucose),1.11(d,J＝6.4Hz,3H,CH 3-Fucose). 13C NMR(101MHz,Chloroform-d)δ153.3,139.3,138.8,138.8,138.8,138.7,138.5,138.4,138.3,138.1,137.7,128.8,128.6,128.5,128.5,128.4,128.4,128.3,128.2,128.1,128.0,127.8,127.7,127.5,127.5,127.4,127.3,127.2,127.2,127.1,126.1,100.0,98.5,97.9,95.3,83.9,82.3,80.2,80.1,79.0,78.2,78.2,75.8,75.6,75.5,75.4,74.8,74.4,74.3,73.5,73.5,73.2,72.9,72.7,72.4,72.4,72.3,71.9,70.9,68.1,67.7,59.1,24.3,16.4,16.2,15.3.IR(film):ν＝2871,1741,1498,1455,1364,1096,822,736,697cm -1.HRMS(ESI)m/z calcd for C 99H 112Cl 3NO 19S[M+NH 4] +1769.6640,found 1769.6631.
化合物45*：将供体四糖44*(20mg,0.0114mmol)和受体28*(12mg,0.0228mmol)溶于无水的CH 2Cl 2(1.1mL)中，加入
Figure PCTCN2019091198-appb-000022
分子筛和NIS(3mg,0.0137mmol)，将反应温度降至-15℃,然后在氩气保护下逐滴加入TfOH(0.11μL,0.0011mmol)。在-20℃条件下反应3h。TLC检测反应完成之后，加入适量Et 3N淬灭反应，溶液过滤，用10％Na 2S 2O 3溶液洗至无色，然后依次用饱和的NaHCO 3、饱和的食盐水洗，无水Na 2SO 4干燥，溶液浓缩后经硅胶柱层析纯化(正己烷/乙酸乙酯:6/1→4/1)得到化合物45*(18mg,70％)。
化合物46*：将化合物30*(50mg,0.022mmol)溶于吡啶(1mL)中，在0℃条件下加入HF/pyridine(1M,0.22mL)，然后在反应在室温下搅拌5h，TLC检测反应完全后，加入适量DCM稀释，后用饱和NaHCO 3和饱和食盐水洗涤，无水Na 2SO 4干燥，过滤浓缩后柱层析分离纯化得到相应的半缩醛。
将上面得到的半缩醛溶于CH 2Cl 2(2mL)中，在0℃下加入CCl 3CN(16μL,0.11mmol)和DBU(10μL,0.066mmol)，反应在室温下搅拌2h。TLC检测反应完全之后，在30℃下浓缩反应液，后经硅胶柱层析分离纯化(正己烷/乙酸乙酯:5/1→3/1)得到化合物46*(21mg,42％)。
(2)目标十三糖的合成：
合成路线如图10、11所示。
对于[5+8]合成策略的实施，是以1.2当量的糖基供体43*或46*分别与八糖受体27*在活化试剂TMSOTf的作用下进行，但是很遗憾并未得到目标分子，合成策略[5+8]失败。因为考虑到全保护的Le y四糖与半乳糖受体28*的反应可以成功进行，考虑采用[4+9]的合成策略，利用1.2当量的糖基供体41*或44*分别与九糖受体49反应，活化试剂为TfOH和NIS，尝试反应之后仍然没有得到目标分子十三糖。由此，推测上述反应策略失败的原因，发明人得出两条重要的结论：一是糖基化供体的支链结构增大了反应的空间位阻从而导致反应失败；二是糖基化供体与受体之间的活性不匹配，供体活性相对较高，而受体活性相对较低，从而导致反应失败。因此主要的解决策略是减小反应的空间位阻和降低供体的反应活性。
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 (12)

  1. 一种合成幽门螺旋杆菌O:6血清型O-抗原糖链片段的方法，其特征在于，所述方法是利用七个单糖砌块构建幽门螺旋杆菌O:6血清型O-抗原糖链片段，所述七个单糖砌块分别为式1～7所示化合物：
    Figure PCTCN2019091198-appb-100001
    其中，PG 1，PG 2，PG 3，PG 4，PG 5，PG 6，PG 7，PG 8，PG 9，PG 10，PG 11，PG 12，PG 13，PG 14，PG 15，PG 17，PG 18，PG 19，PG 21，PG 22，PG 23，PG 25，PG 26，PG 27，PG 28，PG 29和PG 30分别独立的选自氢、酰基、2-萘甲基及其衍生物、苄基及其衍生物、烯丙基和硅烷基中任意一种；
    PG 16和PG 24分别独立的选自氢、酰基、烷氧羰基和烷氧羰酰基中任意一种；
    PG 20选自烷酰基、二甲酰基、苄氧羰基及其衍生物中任意一种；
    式1～7结构中的离去基团LG分别独立的选自卤素、亚胺酯基、硫基和膦酸基中任意一种。
  2. 根据权利要求1所述的方法，其特征在于，所述LG选自卤素、三氯乙酰亚胺酯、N-苯基三氟乙酰亚胺酯糖苷、乙硫基、苯硫基、对甲苯硫基以及二丁基膦酸基任意一种。
  3. 根据权利要求1或2所述的方法，其特征在于，所述PG 20选自三氯乙酰基、三氯乙酰氧羰基、邻苯二甲酰基、苄氧羰基中任意一种。
  4. 根据权利要求1所述的方法，其特征在于，所述PG 16、PG 24选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰、乙酰丙酰基、9-芴甲氧羰基、烯丙氧羰酰基中任意一种。
  5. 根据权利要求1所述的方法，其特征在于，所述PG 1，PG 9，PG 12，PG 17，PG 21，PG 22和PG 24分别独立选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰(ClAc)、乙酰丙酰基、9-芴甲氧羰基、烯丙氧羰酰基、2-萘甲基、对甲氧基苄基以及烯丙基中任意一种。
  6. 根据权利要求3所述的方法，其特征在于，所述PG 1，PG 9，PG 12，PG 17，PG 21，PG 22和PG 24分别独立选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰(ClAc)、乙酰丙酰基、9-芴甲氧羰基、烯丙氧羰酰基、2-萘甲基、对甲氧基苄基以及烯丙基中任意一种。
  7. 根据权利要求1～6任一所述的方法，其特征在于，所述PG 2，PG 3，PG 4，PG 6，PG 7，PG 8，PG 11，PG 13，PG 14，PG 18，PG 25，PG 26，PG 29和PG 30分别独立选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰、烯丙氧羰酰基、苄基、2-萘甲基、对甲氧基苄基以及烯丙基中任意一种。
  8. 根据权利要求1～6任一所述的方法，其特征在于，所述PG 5，PG 10，PG 15，PG 19，PG 23，PG 27和PG 28选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰、烯丙氧羰酰基、苄基、2-萘甲基、对甲氧基苄基、烯丙基、叔丁基二甲基硅烷基、叔丁基二苯基硅烷基和三乙基硅烷基中任意一种。
  9. 根据权利要求7所述的方法，其特征在于，所述PG 5，PG 10，PG 15，PG 19，PG 23，PG 27和PG 28选自氢、乙酰基、苯甲酰、新戊酰基、氯乙酰、烯丙氧羰酰基、苄基、2-萘甲基、 对甲氧基苄基、烯丙基、叔丁基二甲基硅烷基、叔丁基二苯基硅烷基和三乙基硅烷基中任意一种。
  10. 一种组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物，其特征在于，所述化合物式是利用权利要求1～6任一所述方法制备得到的，所述化合物的结构如式I所示：
    Figure PCTCN2019091198-appb-100002
    x为1、2或3；y为1、2或3；z为1、2或3；n 1，n 2，n 3，n 4，n 5为0～5之间的整数，其中n 1，n 2，n 3不同时为零；n；n 6，n 7为0或1；
    其中，U 1、U 2、U 3、U 4、U 5、U 6、U 7的结构式如下所示：
    Figure PCTCN2019091198-appb-100003
    连接臂Linker包括氨基连接臂[-(CH 2) n-N-Y 1Y 2]；n代表连接臂可以为不同的碳链长度，n＝2～40；Y 1和Y 2为氨基的保护基，其中，Y 1为H或者为卞基(Bn)，Y 2为H或者卞甲氧羰基(Cbz)。
  11. 一种糖-蛋白缀合物的制备方法，其特征在于，所述方法是利用权利要求10所述的组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物与蛋白进行缀合。
  12. 权利要求10所述的组装有连接臂的幽门螺旋杆菌O:6血清型O-抗原寡类糖化合物在开发或制备幽门螺旋杆菌疫苗或者治疗幽门螺旋杆菌感染导致的疾病的药物中的应用。
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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
CN109776632A (zh) * 2019-03-01 2019-05-21 江南大学 一种幽门螺旋杆菌o：6血清型o-抗原糖链的合成方法

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