WO2019144508A1 - 类志贺邻单胞菌o51血清型o抗原寡糖化学合成方法 - Google Patents

类志贺邻单胞菌o51血清型o抗原寡糖化学合成方法 Download PDF

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WO2019144508A1
WO2019144508A1 PCT/CN2018/082660 CN2018082660W WO2019144508A1 WO 2019144508 A1 WO2019144508 A1 WO 2019144508A1 CN 2018082660 W CN2018082660 W CN 2018082660W WO 2019144508 A1 WO2019144508 A1 WO 2019144508A1
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reaction
formula
group
acid
benzyl
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French (fr)
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尹健
胡静
彼特·泽贝格
秦春君
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江南大学
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Priority to JP2020540575A priority Critical patent/JP7085631B2/ja
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Publication of WO2019144508A1 publication Critical patent/WO2019144508A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/08—Deoxysugars; Unsaturated sugars; Osones
    • 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
    • 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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • 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

  • the invention relates to a chemical synthesis method of O. serovar O51 serotype O antigen oligosaccharide, in particular to a hetero-modified polyamino oligosaccharide capable of assembling an amino-linked arm at a reducing end, belonging to the chemical field.
  • Plesiomonas shigelloides is an active Gram-negative pathogen discovered in 1947 and is the most important pathogen causing severe diarrhea in international travelers.
  • Pseudosporium can cause a series of serious extraintestinal infections, especially those that are susceptible to infection in children and underlying diseases, including sepsis and meningitis caused by infection with P. Has a very high mortality rate (I.Stock, Rev. Med. Microbiol. 2004, 15, 129-139).
  • the treatment of this pathogenic infection relies on antibiotic treatment, and the emergence of drug resistance reports in clinical treatment makes the treatment of Shigella-like infections more difficult. What is worrying is that there are no vaccines listed on the world that are similar to Shigella. With the increasing international exchanges, it is particularly urgent to develop an effective vaccine against Pseudomonas streptococci.
  • the polysaccharide structure consists of a hetero-modified polyaminotriose repeat unit: [ ⁇ 4)- ⁇ -D-GlcpNAc3NHbA-(1 ⁇ 4)- ⁇ -L-FucpAm3OAc-(1 ⁇ 3)- ⁇ -D-QuipNAc- (1 ⁇ ] (as shown in Figure 1), comprising two aminododeoxyhexoses and one diaminododeoxyhexuronic acid structure, and the amino groups in the structure are respectively acetyl, acetamidino (Am) And D-3-hydroxybutyryl (Hb) modification (A.
  • the polysaccharide obtained by the extraction is limited by the cumbersome pathogenic bacteria cultivation and extraction and purification process, and the extracted polysaccharide has structural heterogeneity and the problem that the impurity is difficult to completely remove.
  • the chemically synthesized method to obtain a purely structured oligosaccharide-related product will provide an important basis for the study of pathogenicity of pathogenic bacteria, the development of new antibacterial drugs, and the development of new types of saccharide vaccines.
  • the aminododeoxyhexose and diaminohexuronic acid in the structure contain four amino groups and modify the rare acetyl group and hydroxybutyryl group in the saccharide compound, the stereochemistry of the glycosylation reaction Selectivity and orthogonal assembly of different modifying groups have an effect. Therefore, the overall synthesis of this structure requires a holistic route design, including the choice of protecting groups, the choice of timing for introducing groups, and the efficiency and selectivity of glycosylation reactions.
  • the present invention relates to an oligosaccharide fragment assembled with a tether of O. serovar O51 serotype O-antigen polysaccharide, and the chemical formula of the sugar chain can be expressed as Formula I:
  • the linking arm L may have a chain structure of 2 to 40 carbon atoms (including the number of carbon atoms in the side chain).
  • the chain when the length of the main chain of the linking arms is 4-8 atoms, the chain may contain 1, 2 or 3 hetero atoms (O, N and S). When the length of the backbone of the tether is 9-14 atoms, the chain may contain 1, 2, 3, 4, 5 or 6 heteroatoms (O, N and S).
  • the linker -L- may be substituted with all or part of fluorine.
  • the linking arm -L- may comprise a three-, four-, five- or six-membered saturated carbocyclic ring; it may also comprise a five-membered unsaturated carbocyclic ring (non-aromatic ring); it may also comprise a four-, five- or six-membered saturated oxygen heterocyclic ring; It may also contain a four, five or six membered saturated nitrogen heterocycle; it may also contain a six membered aromatic carbocyclic ring.
  • the linker -L- may also contain an amide bond and/or a urea group.
  • the tether -L- 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 sugar chain structure synthesized in the present invention contains a basic (ethinyl) group and an acidic (carboxyl group) which form a corresponding salt with an organic or inorganic acid or base.
  • Acids which can be used for salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, Maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzene Formic acid, methanesulfonic acid, ethanesulfonic acid
  • the sugar chain structure synthesized in the present invention may also contain a basic (acetyl) group and an acidic (carboxyl group), and may also be transferred to a basic group by intramolecular proton transfer, that is, proton transfer of an acidic group.
  • of formula I may contain -O - and -NH 3 + amphiphilic molecules.
  • connection between the monosaccharide blocks (U x , U x+1 , U x+2 ) is a monosaccharide block through the terminal base carbon (1 position carbon) and another monosaccharide A glycosidic bond formed by the corresponding hydroxyl oxygen of the block.
  • the serotype O-antigen saccharide chain of the genus Shigella oxysporum O51 referred to in the present invention can be expressed as the general formula II:
  • Formula II can be specifically represented by the formulae II-a, II-b and II-c:
  • the serotype O-antigen saccharide chain of the genus Shigella oxysporum O51 referred to in the present invention can be expressed as Formula III:
  • Formula III can be specifically represented by the formulae III-a, III-b and III-c:
  • the serotype O-antigen saccharide chain of the genus Shigella oxysporum O51 referred to in the present invention can be expressed as Formula IV:
  • Formula IV can be specifically represented by the formulae IV-a, IV-b and IV-c:
  • the present invention provides a chemical synthesis method of a tethered modified sugar chain (Formula I) of the genus O. serovar O51 serotype O-antigen polysaccharide, characterized in that three monosaccharide blocks are 1, 2, 3 And the connecting arm 4 is made of raw materials, as shown in Formula VI (1 ⁇ 4):
  • PG 1 , PG 3 , PG 4 are a hydroxy protecting group and may be selected from the group consisting of acetyl (Ac), levulinyl (Lev), benzoyl (Bz), chloroacetyl (ClAc), dichloro Acetyl (DCA), trichloroacetyl (TCA), pivaloyl (Piv), allyloxycarbonyl (Alloc), 2-naphthylmethyl (Nap), p-methoxybenzyl (PMB), tert-butyl Dimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triethylsilyl (TES), and the like.
  • PG 2 is a hydroxy protecting group and may be a benzyl group (Bn).
  • PG 5 is an amino protecting group and may be trichloroacetyl (TCA), dichloroacetyl (DCA), chloroacetyl (ClAc).
  • PG 6 is a hydroxy protecting group and may be benzyl (Bn), 2-naphthylmethyl (Nap), p-methoxybenzyl (PMB), levulinyl (Lev).
  • PG 7 is a carboxy protecting group and may be a benzyl group (Bn).
  • PG 9 , PG 10 is an amino protecting group and may be benzyl (Bn) or benzyloxycarbonyl (Cbz).
  • LG is a leaving group for the glycosylation reaction, which may be ethylthio, p-toluylthio, phenylthio, bromo, fluoro, trichloroacetimidate, N-phenyl trifluoroacetimidate , dibutyl phosphate.
  • the method for chemical synthesis of P. stipitis O51 oligosaccharide provided by the invention is characterized in that the compound 49 is used as a raw material, and the synthesis of diaminoglucose 52 is completed by the second transposition of the 3 position (see FIG. 7). Show).
  • the steps of compounds 49 to 50 are carried out by a Lattrell-Dax reaction, and the reagent R 1 NO 2 may be potassium nitrite (KNO 2 ), sodium nitrite (NaNO 2 ), tetrabutylammonium nitrite (TBANO 2 ), etc.
  • the temperature can be between room temperature and 80 °C.
  • the synthesis of 52 is carried out by nucleophilic substitution of compound 51 by an azide group.
  • R 2 N 3 may be sodium azide (NaN 3 ), trimethylsilyl azide (TMSN 3 ), etc., and the reaction temperature may be room temperature to 60 ° C. between.
  • PG 11 is allyloxy (OAll), ethylthio (SEt), phenylthio (SPh), p-tolylthio (STol), selenylphenyl (SePh), tert-butyldimethylsiloxy ( OTBDMS) and so on.
  • PG 12 , PG 13 is a hydroxy protecting group, which may be benzylidene (PhCH), propylidene ((CH 3 ) 2 CH), benzoyl (Bz), acetyl (Ac), tert-butyldimethyl Silyl group (TBDMS), tert-butyldiphenylsilyl (TBDPS), triethylsilyl (TES), and the like.
  • the method for chemical synthesis of P. oryzae O51 oligosaccharide comprises the following reaction module:
  • Reaction module A glycosidation reaction
  • the activator in the glycosidation reaction may be selected from methyl trifluoromethanesulfonate (TfOMe), dimethylmethylsulfide.
  • TfOMe trifluoromethanesulfonate
  • DMTST dimethylmethylsulfide.
  • TfOH trifluoromethanesulfonic acid
  • NIS N-iodosuccinimide
  • TMSOTf trimethylsilyl trifluoromethanesulfonate
  • N- iodosuccinimide N-iodosuccinimide
  • the reaction temperature may be between -40 ° C and room temperature;
  • the activator in the glycosidation reaction may be silver perchlorate (AgClO 4 ), titanium tetrafluoride (TiF 4 ), trifluoromethanesulfonic anhydride (Tf 2 O), etc., and the reaction temperature may be Between -40 ° C and room temperature;
  • the activator in the glycosidation reaction may be silver perchlorate (AgClO 4 ), silver trifluoromethanesulfonate (AgOTf), and the reaction temperature may be between -40 ° C and room temperature;
  • the activator in the glycosidation reaction may be a boron trifluoride diethyl ether complex (BF 3 ) OEt 2 ), trimethylsilyl trifluoromethanesulfonate (TMSOTf), silver triflate (AgOTf), and the reaction temperature may be between -40 ° C and room temperature.
  • the molecular sieve used for removing water in the glycosidation reaction may be Molecular sieve or Molecular sieves.
  • Reaction module B azide reduction acetylation
  • Azide-reductive acetylation can be accomplished directly by the thioacetic acid (AcSH)/pyridine method or by reduction to an amino group via an azide group followed by acetylation.
  • the method for reducing an azide group to an amino group may be trimethylphosphine (PMe 3 )/water, triphenylphosphine (PPh 3 )/water, 1,3-propanedithiol/triethylamine, sodium borohydride (NaBH).
  • NiCl 2 nickel dichloride
  • SnCl 2 tin dichloride
  • PhSH benzene mercaptan
  • the aminoacetylation method may be acetic anhydride (Ac 2 O) / methanol, acetic anhydride / pyridine, acetyl chloride (AcCl) / triethylamine, and the like.
  • Reaction module C Monosaccharide block 1 (quinose amine) 3 position deprotection
  • hydroxy protecting group is acetyl (Ac), benzoyl (Bz), chloroacetyl (ClAc), dichloroacetyl (DCA), trichloroacetyl (TCA), pivaloyl (Piv)
  • the deprotection conditions used may be sodium methoxide/methanol, potassium hydroxide/methanol, sodium hydroxide/methanol, and the like;
  • the deprotecting conditions used may be cerium acetate/pyridine or the like;
  • the deprotection conditions used may be palladium diacetate (Pd(OAc) 2 ) / diethylamine, etc.;
  • the deprotection conditions used may be 2,3-dichloro-5,6-dicyano-1,4- P-benzoquinone (DDQ) / water, ammonium cerium nitrate (CAN) / water, etc.;
  • the deprotection conditions used may be tetrabutyl fluoride.
  • Reaction module D azide reduction modification 2,2,2-trichloroethoxycarbonyl (Troc)
  • the method for reducing an azide group to an amino group may be trimethylphosphine (PMe 3 )/water, triphenylphosphine (PPh 3 )/water, 1,3-propanedithiol/triethylamine, sodium borohydride (NaBH). 4 ) / nickel dichloride (NiCl 2 ), tin dichloride (SnCl 2 ) / benzene mercaptan (PhSH) / triethylamine, zinc / copper / acetic acid, Lindlar catalyst / hydrogen;
  • Reaction module E Monosaccharide block 2 (fucosylamine) 3,4 position selective naphthylmethyl protection
  • the molybdenum methyl group protection at the 3,4 position of fucose specifically refers to the selective protection of the hydroxyl group at the 3 position and the hydroxyl group at the 3 position, while retaining the hydroxyl group at the 4 position, the method may be
  • the raw material was treated with dibutyltin oxide at 110 ° C for 1 hour, and then treated with 2-bromomethylnaphthalene and tetrabutylammonium bromide (TBAB) to complete the reaction between room temperature and 110 ° C.
  • TBAB 2-bromomethylnaphthalene and tetrabutylammonium bromide
  • Reaction module F monosaccharide block 2 (fucosylamine) 3 position acetylation
  • the acetylation of the fucose 3 position is accomplished by selective removal of the naphthylmethyl group and introduction of the acetyl group.
  • the selective removal method of naphthylmethyl group may be a 2,3-dichloro-5,6-dicyano-1,4-p-benzoquinone (DDQ)/water method;
  • the method of introducing the acetyl group may be acetic anhydride/pyridine, acetyl chloride/pyridine, acetyl chloride/triethylamine or the like.
  • the method for reducing an azide group to an amino group may be trimethylphosphine (PMe 3 )/water, triphenylphosphine (PPh 3 )/water, 1,3-propanedithiol/triethylamine, sodium borohydride (NaBH). 4 ) / nickel dichloride (NiCl 2 ), tin dichloride (SnCl 2 ) / benzene mercaptan (PhSH) / triethylamine, zinc / copper / acetic acid, Lindlar catalyst / hydrogen;
  • the amino group-modified butyryl group may be carried out by using (R)-3-O-PG 8 -butyric anhydride (as shown in FIG. 8 ) as a raw material, with or without alkali treatment, and the base used may be pyridine or triethylamine. Wait;
  • the amino group-modified butyryl group may be carried out by using (R)-3-O-PG 8 -butyryl halide (acid chloride, acid bromide or acid fluoride) (as shown in FIG. 8 ) as a raw material, and performing alkali treatment. It may be pyridine, triethylamine, sodium carbonate, sodium hydrogencarbonate, potassium carbonate, etc.;
  • the amino group-modified butyryl group may be a raw material of (R)-3-O-PG 8 -butyric acid (as shown in FIG. 8 ), and the condensation condition may be dicyclohexylcarbodiimide (DCC)/4- N,N-lutidine (DMAP), dicyclohexylcarbodiimide (DCC) / 1-hydroxybenzotriazole (HOBt), diisopropylcarbodiimide (DIC) / 4-N, N-lutidine (DMAP), diisopropylcarbodiimide (DIC) / 1-hydroxybenzotriazole (HOBt), 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimine (EDCI) / 4-N,N-lutidine (DMAP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) / 1-hydroxybenzo Triazole (HOBt), 2-(7-ox
  • the hydroxy protecting group PG 8 in the (R)-3-hydroxybutanoyl group may be a benzyl group (Bn) or the like.
  • Reaction module H removal of Troc modified acetamidine
  • the method for removing Troc may be zinc powder/acetic acid, the content of acetic acid may be between 50% and 100%, and the reaction temperature may be between room temperature and 65 ° C;
  • the method of modifying an amino group to an acetamyl group can be carried out by a thioacetimidin aryl ester halide or a thioacetimidate alkyl ester halide (as shown in FIG. 9) under an alkali treatment, and an aryl group.
  • the alkyl group may be a methyl group or an ethyl group
  • the halogen acid may be hydrochloric acid or hydrobromic acid, etc.
  • the base used may be pyridine, triethylamine, diisopropylethylamine, 1 , 8-diazabicycloundec-7-ene (DBU), potassium carbonate, sodium carbonate, etc.;
  • the method of modifying an amino group to an acetamyl group can be carried out by an alkali treatment of an acetimidinyl alkyl ester halide (as shown in FIG. 10), and the alkyl group may be an ethyl group, a trifluoroethyl group, a trichloroethyl group or the like.
  • the halogen acid may be hydrochloric acid or hydrobromic acid, etc.
  • the base used may be pyridine, triethylamine, diisopropylethylamine, 1,8-diazabicycloundec-7-ene (DBU), carbonic acid. Potassium, sodium carbonate, etc.
  • the catalytic hydrogenation complete deprotection can be carried out by introducing hydrogen gas under catalytic conditions at room temperature.
  • the catalyst for catalytic hydrogenation may be a 10% palladium carbon catalyst or palladium hydroxide.
  • the solvent used for the reaction may be a water/methanol/dichloromethane/acetic acid mixture, a water/tert-butanol/methylene chloride mixture or the like.
  • the deprotection method may be 2,3-dichloro-5,6-dicyano-1,4-p-benzene. ⁇ (DDQ) / water method, etc.;
  • the deprotecting method may be cerium acetate/pyridine or the like.
  • Reaction module K azide reduction modification acetamido
  • the method for reducing an azide group to an amino group may be trimethylphosphine (PMe 3 )/water, triphenylphosphine (PPh 3 )/water, 1,3-propanedithiol/triethylamine, sodium borohydride (NaBH). 4 ) / nickel dichloride (NiCl 2 ), tin dichloride (SnCl 2 ) / benzene mercaptan (PhSH) / triethylamine, zinc / copper / acetic acid, Lindlar catalyst / hydrogen;
  • the method of modifying an amino group to an acetamyl group can be carried out by a thioacetimidin aryl ester halide or a thioacetimidate alkyl ester halide (as shown in FIG. 9) under an alkali treatment, and an aryl group.
  • the alkyl group may be a methyl group or an ethyl group
  • the halogen acid may be hydrochloric acid or hydrobromic acid, etc.
  • the base used may be pyridine, triethylamine, diisopropylethylamine, 1 , 8-diazabicycloundec-7-ene (DBU), potassium carbonate, sodium carbonate, etc.;
  • the method of modifying an amino group to an acetamyl group can be carried out by an alkali treatment of an acetimidinyl alkyl ester halide (as shown in FIG. 10), and the alkyl group may be an ethyl group, a trifluoroethyl group, a trichloroethyl group or the like.
  • the halogen acid may be hydrochloric acid or hydrobromic acid, etc.
  • the base used may be pyridine, triethylamine, diisopropylethylamine, 1,8-diazabicycloundec-7-ene (DBU), carbonic acid. Potassium, sodium carbonate, etc.
  • the method for chemical synthesis of P. oryzae O51 oligosaccharide provided by the invention comprises the following reaction steps:
  • the synthesis step of II-a includes (as shown in FIG. 11): monosaccharide block 1 is assembled by a glycosidation reaction, and is obtained by reaction module B, C.
  • the monosaccharide receptor 5 is glycosylated with a monosaccharide block 2 to give a disaccharide 6.
  • Disaccharide 6 is obtained by reaction module D, E to obtain disaccharide receptor 7, and glycosidation reaction with monosaccharide block 3 to obtain trisaccharide 8.
  • Trisaccharide 8 is modified by fucose acetylation via reaction module F.
  • the amino-modified group is assembled by reaction module G and H to obtain trisaccharide 11, which is catalytically hydrogenated by reaction module I to obtain trisaccharide II-a.
  • the synthesis step of II-b includes (as shown in FIG. 12): monosaccharide block 3 is assembled by glycosidation, and monosaccharide 12 is passed through reaction module G. J gives a monosaccharide receptor 13. Monosaccharide 13 is reacted with monosaccharide block 1 by glycosidation to obtain disaccharide 14, and disaccharide acceptor 15 is obtained via reaction module B, C.
  • the disaccharide receptor 15 and the monosaccharide block 2 are subjected to glycosidation reaction to obtain a trisaccharide 16, and the ethylene group is assembled by the reaction module K to obtain a trisaccharide 17, which is further subjected to catalytic hydrogenation through the reaction module I to obtain a trisaccharide II-b.
  • the synthesis step of II-c includes (as shown in FIG. 13): monosaccharide block 2 is assembled by a glycosidation reaction, and the obtained compound 18 is subjected to a reaction module. D, E produced a monosaccharide receptor 19. The monosaccharide receptor 19 is reacted with the monosaccharide block 3 to obtain a disaccharide, and the disaccharide receptor 21 is obtained through the reaction modules F, G, and J. The disaccharide receptor 21 is glycosidically reacted with the monosaccharide block 1 to obtain a trisaccharide 22, and the amino modification group is assembled via the reaction module B, H to obtain a trisaccharide 24. Trisaccharide 24 is catalytically hydrogenated by reaction module I to obtain trisaccharide II-c.
  • the synthesis step of III-a includes (as shown in FIG. 14): trisaccharide 16 is subjected to reaction module D, E to obtain trisaccharide receptor 25.
  • the trisaccharide receptor 25 and the monosaccharide block 3 are subjected to glycosidation reaction to obtain tetrasaccharide 26, and the fucose acetyl group is modified by the reaction module F, and the amino group is assembled by the reaction module G and H to obtain the tetrasaccharide 29.
  • Tetrasaccharide 29 is catalytically hydrogenated by reaction module I to give tetrasaccharide III-a.
  • the synthesis step of III-b includes (as shown in FIG. 15): trisaccharide 23 is deprotected by reaction module C to obtain trisaccharide receptor 30.
  • the trisaccharide receptor 30 is reacted with the monosaccharide block 2 to obtain the tetrasaccharide 31, and the ethylene group is assembled through the reaction module D and H to prepare the tetrasaccharide 32.
  • Tetrasaccharide 32 is catalytically hydrogenated by reaction module I to obtain tetrasaccharide III-b.
  • the synthesis step of III-c includes (as shown in FIG. 16): trisaccharide 10 is deprotected by reaction module J to obtain trisaccharide receptor 33.
  • the acceptor 33 and the monosaccharide block 1 are subjected to glycosidation to prepare tetrasaccharide 34, and further assembly of the amino-modified group is carried out through the reaction module B, H to obtain tetrasaccharide 36.
  • Tetrasaccharide 36 is catalytically hydrogenated by reaction module I to obtain tetrasaccharide III-c.
  • the synthesis step of IV-a includes (as shown in FIG. 17): tetrasaccharide 31 is prepared by reaction module D, E to obtain tetrasaccharide receptor 37, further with single Glycosidyl 3 glycosidation gives pentasaccharide 38.
  • the pentasaccharide 38 is modified by the reaction module F to complete the fucose acetyl group, and the amino group is assembled by the reaction module G and H to obtain the pentasaccharide 41, and further subjected to catalytic hydrogenation of the reaction module I to obtain the pentasaccharide IV-a.
  • the synthesis step of IV-b includes (as shown in FIG. 18): tetrasaccharide 35 is deprotected by reaction module C to obtain tetrasaccharide receptor 42, further with monosaccharide Block 2 glycosidation reaction to obtain pentasaccharide 43. 43 through the reaction module D, H to complete the oxime modification, to obtain pentasaccharide 44, further catalytically hydrogenated by reaction module I to obtain pentasaccharide IV-b.
  • the synthesis step of IV-c includes (as shown in FIG. 19): tetrasaccharide 28 is deprotected by reaction module J to obtain tetrasaccharide receptor 45, further with monosaccharide Block 1 glycosidation reaction produces pentasaccharide 46.
  • the pentasaccharide 46 is assembled by the reaction module B, H to obtain the amino-modified group to obtain the pentasaccharide 48, and further subjected to catalytic hydrogenation of the reaction module I to obtain the pentasaccharide IV-c.
  • the chemical synthesis method of the O. lucidum O51 oligosaccharide provided by the invention when the sugar block 1 (quinose) is used as a glycosyl donor, the amino group at the 2 position can be protected by an azide group, Conducive to the formation of 1,2-cis- ⁇ -glucosidic bonds.
  • the chemical synthesis method of the O. gingivalus O51 oligosaccharide provided by the invention when the sugar block 2 (fucose) is used as a glycosyl donor, the amino group at the 2 position can be protected by an azide group, Conducive to the formation of 1,2-cis- ⁇ -glucosidic bonds.
  • the chemical synthesis method of the O. oxysporum O51 oligosaccharide provided by the present invention when the hydroxyl group at the 4 position of the sugar block 2 (fucose) is used as a glycosylation reaction acceptor, in order to increase the hydroxyl group at the 4th position Nucleophilic, the amino group at position 2 of the sugar building block 2 (fucose) can be protected by Troc, and the hydroxyl group at the 3 position can be protected by naphthylmethyl.
  • O. lucidum O51 oligosaccharide provided by the invention, when the sugar block 3 (glucuronic acid) is used as a glycosyl donor, the amino group at the 3 position in the structure can be protected by an azide group. Base to avoid hindrance of the amide protecting group to the glycosylation reaction.
  • the chemical synthesis method of the O. gingivalus O51 oligosaccharide provided by the invention when the sugar block 3 (glucuronic acid) is used as a glycosyl donor, the amino acid at the 2 position in the structure can be protected by trichloroacetyl group To facilitate the formation of ⁇ -configuration glycosidic bonds, and can be converted to acetamido by catalytic hydrogenation.
  • sugar block 3 glucuronic acid
  • the chemical synthesis method of the P. stipitis O51 oligosaccharide provided by the invention the acetylamino group at the 2 position in the sugar block 1 (quinose) structure can complete the sugar of the sugar block 1 (quinose)
  • the basic reaction is introduced immediately afterwards to avoid hindrance to the introduction of the post-sugar block.
  • the chemical synthesis method of O. lucidum O51 oligosaccharide provided by the invention the naphthylmethyl group at the 3 position of the sugar block 2 (fucose) can be converted into acetyl after completing the glycosylation reaction at the 4 position
  • the base is modified to avoid hindrance to the introduction of the post sugar block.
  • the chemical synthesis method of the O. lucidum O51 oligosaccharide provided by the present invention the assembly of the butanamide group at the 3 position in the sugar block 3 (glucuronic acid) structure should be before the deprotection of the glucuronic acid 4 position Completed to avoid obstruction of the introduction of the sugar block.
  • the present invention also provides three orthogonal protective intermediates for the preparation of hetero-modified polyamino sugars, which are compounds of the formula 8, formula 16, and formula 22, respectively.
  • the assembly of different amino modifying groups can be accomplished with these three compounds, and can be further used for the extension of oligosaccharide chains.
  • the present invention is the first to complete the chemical synthesis of the O-antigen sugar structure of the genus O. serovar O51.
  • it provides a basis for the synthesis of the oligosaccharide structure to couple the carrier molecule or immobilization to the corresponding substrate, and can be used for the biological action of the O-antigen glycostructure of O. serovar O51.
  • Figure 1 O. serovar O51 serotype O-antigen trisaccharide repeat unit.
  • Figure 2 Compounds of the general formula I, U x , U x+1 , U x+2 .
  • Figure 3 Compounds of the formulae II-a, II-b and II-c.
  • Figure 4 Compounds of the formulae III-a, III-b and III-c.
  • Figure 5 Compounds of the formulae IV-a, IV-b and IV-c.
  • Figure 6 Compounds shown in monosaccharide blocks 1, 2, 3 and tether 4.
  • Figure 8 Butyryl compounds used for aminobutyrylation modification.
  • Figure 9 Thioacetylimidate aryl (alkyl) ester halide.
  • Figure 10 Acetylimidate alkyl ester halide.
  • Figure 11 Synthetic reaction formula of P. oryzae O51 trisaccharide II-a.
  • Figure 12 Synthetic reaction formula of P. oryzae O51 trisaccharide II-b.
  • Figure 13 Synthetic reaction formula of P. oryzae O51 trisaccharide II-c.
  • Figure 14 Synthetic reaction formula of P. oryzae O51 tetrasaccharide III-a.
  • Figure 17 Synthetic reaction formula of P. oryzae O51 pentasaccharide IV-a.
  • Figure 18 Synthetic reaction formula of P. oryzae O51 pentasaccharide IV-b.
  • Figure 21 Chemical synthesis reaction formula of compounds 12* and 13*.
  • the anhydrous solvent used in the reaction was prepared by a MBraun MB-SPS Model 800 solvent drying system.
  • the solvents used in the silica gel column chromatography were all analytically pure and used after distillation under reduced pressure.
  • the silica gel plate used for thin layer chromatography (TLC) was a glass-based or aluminum foil-based silica gel plate prepared from 60-F254 silica gel.
  • the thin layer chromatography color developing reagent is a sugar developer (0.1% (v/v) 3-methoxyphenol, 2.5% (v/v) sulfuric acid ethanol solution), or a CAM developer (5% (w/) v) ammonium molybdate, 1% (w/v) barium (II) sulfate and 10% (v/v) aqueous sulfuric acid), or ninhydrin developer (1.5% (w/v) ninhydrin and 3 % (v/v) n-butanol acetate solution).
  • the silica gel used for normal phase silica gel column chromatography was 200-300 mesh silica gel.
  • the residue used in size exclusion chromatography is a dextran gel LH-20 (GE Healthcare).
  • the yield of each reaction step was calculated separately, and the yield was calculated as: (amount of target product substance / amount of raw material) ⁇ 100%.
  • the structure of the product was identified by nuclear magnetic spectroscopy, infrared spectroscopy, optical rotation, and high-resolution mass spectrometry.
  • the purity of the product was analyzed by nuclear magnetic spectroscopy and high performance liquid chromatography.
  • the hydrogen spectrum, carbon spectrum, and two-dimensional nuclear magnetic spectrum were measured by a Bruker Ascend 400 mega nuclear magnetic resonance apparatus, or a Bruker Ultrashield Plus 400 mega nuclear magnetic resonance apparatus, or a Bruker AVIII 700 mega nuclear magnetic resonance apparatus at 25 °C.
  • High resolution mass spectra were measured on an Agilent 6220 electrospray ion source-time of flight mass spectrometer.
  • the optical rotation was measured by a Schmidt & Haensch UniPol L 1000 fully automatic polarimeter at 589 nm and the concentration (c) was determined to be g/100 mL.
  • the infrared spectrum was measured by a Thermo Fisher Scientific Nicolet iS5 infrared meter.
  • Analytical high performance liquid chromatography was performed on an Agilent 1200 series liquid chromatography quadrupole electrospray mass spectrometer 6130 using a Thermo Scientific Hypercarb column (150 x 4.6 mm).
  • Preparative high performance liquid chromatography was performed on an Agilent 1200 series liquid chromatography quadrupole electrospray mass spectrometer 6130 using a Thermo Scientific Hypercarb column (150 x 10 mm).
  • the trihydroxy sugar (71 g, 0.195 mol) was further dissolved in 300 mL of anhydrous DMF, and benzaldehyde dimethyl acetal (35 mL, 0.234 mol) and p-toluenesulfonic acid (4.45 g, 0.023 mol) were added.
  • the reaction solution was reacted at 60 ° C for 24 hours, the methanol formed in the reaction was distilled off under reduced pressure by a rotary evaporator, and the reaction was further carried out at 60 ° C. This operation was repeated, and DMF was distilled off under reduced pressure until the reaction was completed.
  • the obtained crude product was dissolved in ethyl acetate.
  • EtOAc EtOAc (EtOAc m.
  • the obtained organic phase was subjected to removal of water over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure at a low temperature.
  • the crude product was purified on silica gel column ( petroleum ether: ethyl acetate, 10: 1-5: 1-2:1, v/v). The product was obtained as a yellow syrup 5* (160 mg, 0.275 mmol, 70%).
  • Phenyl 4,6-O-benzylidene-3-O-acetyl-2-azido-2-deoxy-1-selenium- ⁇ -D-glucopyranose (F. Santoyo-González et al., Synlett, 1994, 6, 454-456) (1 g, 2.1 mmol) was azeotroped three times with toluene and dissolved in 18 mL of anhydrous dichloromethane under argon.
  • the compound 17* (4.81 g, 10.5 mmol) was dissolved in a THF/water mixture (25 mL, 1:1, v/v), and bromosuccinimide (4.45 g, 25.0 mmol) was added. Stir at room temperature for 6 hours. After the reaction was completed, the reaction mixture was diluted with dichloromethane, and the organic phase was extracted with a mixture of 10% Na 2 S 2 O 3 /1M NaHCO 3 (1:1, v/v).
  • trichloroacetamidite sugar donor 19* (2.48g, 5.325mmol) and N-benzyl-N-benzyloxycarbonyl-3-aminopropan-1-ol (H.Ishida et al. , Org. Biomol. Chem. 2015, 13, 7762-7771) (1.91 g, 6.390 mmol) dissolved in anhydrous diethyl ether / anhydrous dichloromethane mixture (130 mL, 3:1, v/v), added to activated Molecular sieves (type Aw-300) were stirred for 30 minutes.
  • a trifluoroimidate sugar donor 13* (159 mg, 0.358 mmol) and a receptor 22* (207 mg, 0.358 mmol) were dissolved in a dry dichloromethane/anhydro ether mixture (12 mL, 1/ In 3, v/v), after adding thiophene (344 ⁇ L, 4.296 mmol) and activated molecular sieve (AW-300 type), the resulting reaction solution was stirred at room temperature for 30 minutes. After cooling to -40 ° C, trimethylsilyl triflate (6.5 ⁇ L, 0.036 mmol) was added, after which the reaction temperature was gradually increased to room temperature.
  • the trifluoroacetimide ester sugar donor 11* (69 mg, 0.096 mmol) and the disaccharide acceptor 27* (20 mg, 0.019 mmol) were dissolved in 3.2 mL of anhydrous dichloromethane and added to the activated molecular sieve ( After the Aw-300 type), it was stirred at room temperature for 30 minutes. After trimethylsilyl trifluoromethanesulfonate (1.8 ⁇ L, 0.01 mmol) was added at room temperature, the reaction mixture was stirred at room temperature. After completion of the reaction, the reaction was quenched by the addition of 4 drops of pyridine at 0 ° C, and the obtained organic phase was filtered and evaporated.
  • an amino compound 33* (12.5 mg, 9.35 ⁇ mol) was dissolved in 2 mL of anhydrous pyridine, and after cooling to 0 ° C, benzyl thioacetimidate hydrochloride (3.8 mg, 18.7 ⁇ mol) was added. The reaction mixture was stirred at 0<0>C for 5 hr., EtOAc (EtOAc:EtOAc:EtOAc %).
  • Trisaccharide 34* (4.3 mg, 3.1 ⁇ mol) was dissolved in a t-butanol/water/dichloromethane mixture (3 mL, 5:2:1, v/v/v), and after 10 N of the reaction system was added, 10% was added. The palladium carbon hydrogenation catalyst was continuously replaced with nitrogen for 5 minutes.

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Abstract

本发明公开了类志贺邻单胞菌 O51 血清型 O 抗原寡糖化学合成方法，属于化学领域。本发明利用来源丰富的 D-葡萄糖，L-岩藻糖和 D-葡萄糖胺等为原料，制备得到三种糖基化砌块，设计了由 11 个反应模块组成的合成路线，通过保护基团的优化，修饰基团引入时机的优化，成功完成了目标寡糖链的制备。本发明制备的寡糖链原料廉价易得、制备方法简单易重复，将会在类志贺邻单胞菌的新型药物和疫苗开发等方面具有良好的应用前景。

Description

类志贺邻单胞菌O51血清型O抗原寡糖化学合成方法 技术领域
本发明涉及类志贺邻单胞菌O51血清型O抗原寡糖化学合成方法，尤其是一种还原端组装氨基连接臂的杂修饰多氨基寡糖，属于化学领域。
背景技术
类志贺邻单胞菌(Plesiomonas shigelloides)是一种发现于1947年的活动性革兰氏阴性致病菌，是导致国际旅行者重度腹泻的最主要致病菌。此外，类志贺邻单胞菌还可引起一系列严重的肠道外感染，尤其易于感染儿童和有潜在疾病的成年人，其中由类志贺邻单胞菌感染引起的脓毒症和脑膜炎具有极高的致死率(I.Stock,Rev.Med.Microbiol.2004,15,129-139)。目前，对于该致病菌感染的治疗手段依赖于抗生素治疗，而临床治疗中不断出现的耐药性报告使得类志贺邻单胞菌感染的治疗愈加困难。令人担忧的是，全球范围内尚无类志贺邻单胞菌的疫苗注册上市。随着国际化交流日益密切，开发有效的类志贺邻单胞菌疫苗就显得尤为紧迫。
类志贺邻单胞菌的血清型研究显示其有102种菌体抗原(O-抗原)和51种鞭毛抗原(H-抗原)(E.Aldova et al.,Folia Microbiol.2000,45,301-304)。由于类志贺邻单胞菌为不产荚膜细菌，其细胞表面被脂多糖层所包裹。鉴于细菌脂多糖(lipopolysaccharide，LPS)的O-抗原部分具有高度的结构特异性，且在致病菌的感染和宿主的免疫应答等生物过程中均发挥重要作用(J.Lukasiewicz et al.,Biochemistry 2006,45,10434-10447)，类志贺邻单胞菌的O-抗原是开发类志贺邻单胞菌疫苗的重要目标分子。
在诸多已报道结构的类志贺邻单胞菌O-抗原中，O51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。该多糖结构由一个杂修饰多氨基三糖重复单元组成：[→4)-β-D-GlcpNAc3NHbA-(1→4)-α-L-FucpAm3OAc-(1→3)-α-D-QuipNAc-(1→](如图1所示)，包含两个氨基二脱氧己糖和一个二氨基二脱氧己糖醛酸结构，而结构中的氨基分别被乙酰基，乙脒基(acetamidino，Am)和D-3-羟基丁酰基(D-3-hydroxybutyryl，Hb)所修饰(A.Maciejewska et al.,Carbohydr.Res.2009,344,894-900)。虽然自然界中糖结构的高度多样性已广为人知，具有与类志贺邻单胞菌O51多糖结构中类似的二酰氨基糖醛酸以及稀有基团乙脒基和3-羟基丁酰基的糖分子仍极为稀少。已报道的相似结构均是来自一些重要致病菌的细胞表面组分，如创伤弧菌YJ016菌株(S.N.Senchenkova et al.,Carbohydr.Res.2009,344,1009-1013)和CECT5198菌株(A.S.Shashkov et al.,Carbohydr.Res.2009,344,2005-2009)，铜绿假单胞菌O5血 清型(A.Larkin et al.,Biochemistry 2009,48,5446-5455)，鲍氏不动杆菌ATCC 17961菌株(E.Fregolino et al.,Carbohydr.Res.2011,346,973-977)等，表明此类糖结构可能与病原菌的致病性存在着关联，而进一步验证糖类结构的相关生物学作用，依赖于得到纯品物质用于相关研究。通过提取得到多糖受到繁琐的致病菌培养和提取纯化过程的限制，而且提取所得多糖存在结构不均一，且杂质难以完全去除的问题。综上所述，通过化学合成方法得到结构明确的相关寡糖纯品将为致病菌致病性的研究，新型抗菌药物的开发，新型糖类疫苗的研发提供重要基础。
虽然在过去几十年中，合成化学家们不断发展着合成糖化学，如保护基团策略的发展，新型保护基团的开发，糖基化方法的开发，以及自动化合成技术等。但是以类志贺邻单胞菌O51多糖为代表的杂修饰多氨基糖结构仍被合成化学家认为是极具挑战性的目标分子。目前，包括类志贺邻单胞菌O51多糖在内的类似杂修饰多氨基糖结构的化学合成尚未有报道。由于其结构中的氨基二脱氧己糖和二氨基己糖醛酸含有4个氨基类基团，且修饰着糖类化合物中稀有的乙脒基和羟基丁酰基，将对糖基化反应的立体选择性以及不同修饰基团的正交组装造成影响。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
本发明涉及类志贺邻单胞菌O51血清型O-抗原多糖的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I：
V*-[U x+2-U x+1-U x] n-V-O-L-NH 2   式I
其中，x为1，2，3；n为1，2，3；-V-表示：一根化学键，-U x+2-，或者-U x+2-U x+1-；V*-表示：H-，H-U x-，或者H-U x+1-U x-；L表示连接臂；U x，U x+1，U x+2如式Ⅴ(图2)所示。
Figure PCTCN2018082660-appb-000001
本发明中连接臂L可以是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(O，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。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。
本发明中连接臂-L-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。
本发明中所合成糖链结构中含有碱性(乙脒基)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。可以用于成盐的酸有盐酸，氢溴酸，硫酸，磷酸，乙酸，柠檬酸，草酸，丙二酸，水杨酸，对氨基水杨酸，苹果酸，延胡索酸，丁二酸，抗坏血酸，马来酸，磺酸，膦酸，高氯酸，硝酸，甲酸，丙酸，葡萄糖酸，乳酸，酒石酸，羟基马来酸，丙酮酸，苯乙酸，苯甲酸，对氨基苯甲酸，对羟基苯甲酸，甲磺酸，乙磺酸，亚硝酸，羟基乙磺酸，乙烯磺酸，对甲苯磺酸，萘磺酸，对氨基苯磺酸，樟脑磺酸，扁桃酸，邻甲基扁桃酸，羟基苯磺酸，苦味酸，己二酸，邻甲苯酒石酸，丙醇二酸，氨基萘磺酸，以及其他矿物酸或羧酸类物质。可以用于成盐的无机或有机碱有氢氧化钠，氢氧化钾，氨水，四烷基氢氧化铵，赖氨酸，精氨酸等。
本发明中所合成糖链结构中因同时含有碱性(乙脒基)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I可以是含有-O -和-NH 3 +的两性分子。
本发明中单糖砌块(U x，U x+1，U x+2)之间的连接方式均为一单糖砌块通过端基位碳(1号位碳)与另一单糖砌块的相应羟基氧所形成的糖苷键。
本发明中涉及的类志贺邻单胞菌O51血清型O-抗原糖链可以表示为通式II：
V*-[U x+2-U x+1-U x] n-O-L-NH 2    式II，
其中x，n，L，U x，U x+1，U x+2和V*与通式I一致。通式II可以具体表示为通式II-a，II-b和II-c：
Figure PCTCN2018082660-appb-000002
本发明中涉及的类志贺邻单胞菌O51血清型O-抗原糖链可以表示为通式III：
V*-[U x+2-U x+1-U x] n-U x+2-O-L-NH 2      式III，
其中x，n，L，U x，U x+1，U x+2和V*与通式I一致。通式III可以具体表示为通式III-a，III-b和III-c：
Figure PCTCN2018082660-appb-000003
本发明中涉及的类志贺邻单胞菌O51血清型O-抗原糖链可以表示为通式IV：
V*-[U x+2-U x+1-U x]n-U x+2-U x+1-O-L-NH 2    式IV，
其中x，n，L，U x，U x+1，U x+2和V*与通式I一致。通式IV可以具体表示为通式IV-a，IV-b和IV-c：
Figure PCTCN2018082660-appb-000004
本发明提供了类志贺邻单胞菌O51血清型O-抗原多糖的连接臂修饰糖链(通式I)的化学合成方法，其特征在于，以三个单糖砌块1，2，3和连接臂4为原料，如式Ⅵ(1～4)所示：
Figure PCTCN2018082660-appb-000005
其中：
PG 1，PG 3，PG 4为羟基保护基团，可以选自如下基团：乙酰基(Ac)，乙酰丙酰基(Lev)，苯甲酰基(Bz)，氯乙酰基(ClAc)，二氯乙酰基(DCA)，三氯乙酰基(TCA)，新戊酰基(Piv)，烯丙氧羰酰基(Alloc)，2-萘甲基(Nap)，对甲氧苄基(PMB)，叔丁基二甲基硅烷基(TBDMS)，叔丁基二苯基硅烷基(TBDPS)，三乙基硅烷基(TES)等。
PG 2为羟基保护基团，可以是苄基(Bn)。
PG 5为氨基保护基团，可以是三氯乙酰基(TCA)，二氯乙酰基(DCA)，氯乙酰基(ClAc)。
PG 6为羟基保护基团，可以是苄基(Bn)，2-萘甲基(Nap)，对甲氧苄基(PMB)，乙酰丙酰基(Lev)。
PG 7为羧基保护基团，可以是苄基(Bn)。
PG 9，PG 10为氨基保护基团，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，可以是乙硫基，对甲苯硫基，苯硫基，溴，氟，三氯乙酰亚氨酯，N-苯基三氟乙酰亚胺酯，二丁基磷酸酯。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，其特征在于，以化合物49为原料，通过3号位二次转位完成了二氨基葡萄糖52的合成(如图7所示)。化合物49至50的步骤通过Lattrell-Dax反应完成，所用试剂R 1NO 2可以是亚硝酸钾(KNO 2)，亚硝酸钠(NaNO 2)，四丁基亚硝酸铵(TBANO 2)等，反应温度可以是室温至80℃之间。由化合物51经叠氮基亲核取代完成52的合成，R 2N 3可以是叠氮化钠(NaN 3)，三甲基硅叠氮(TMSN 3)等，反应温度可以是室温至60℃之间。PG 11为烯丙氧基(OAll)，乙硫基(SEt)，苯硫基(SPh)，对甲苯硫基(STol)，硒苯基(SePh)，叔丁基二甲基硅烷氧基(OTBDMS)等。PG 12，PG 13为羟基保护基，可以是苄叉基(PhCH)，丙叉基((CH 3) 2CH)，苯甲酰基(Bz)，乙酰基(Ac)，叔丁基二甲基硅烷基(TBDMS)，叔丁基二苯基硅烷基(TBDPS)，三乙基硅烷基(TES)等。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，其特征包括如下反应模块：
1)反应模块A：糖苷化反应
当糖苷化反应中所用离去基团为乙硫基，对甲苯硫基，苯硫基时，糖苷化反应中活化剂可以选自三氟甲磺酸甲酯(TfOMe)，二甲基甲硫基硫鎓三氟甲磺酸盐(DMTST)，三氟甲磺酸(TfOH)/N-碘代丁二酰亚胺(NIS)，三氟甲磺酸三甲基硅酯(TMSOTf)/N-碘代丁二酰亚胺(NIS)，反应温度可以是-40℃至室温之间；
当离去基是氟时，糖苷化反应中活化剂可以是高氯酸银(AgClO 4)，四氟化钛(TiF 4)，三氟甲磺酸酐(Tf 2O)等，反应温度可以是-40℃至室温之间；
当离去基是溴时，糖苷化反应中活化剂可以是高氯酸银(AgClO 4)，三氟甲磺酸银(AgOTf)，反应温度可以是-40℃至室温之间；
当离去基团是三氯乙酰亚胺酯，N-苯基三氟乙酰亚胺酯，二丁基磷酸酯时，糖苷化反应中活化剂可以是三氟化硼乙醚络合物(BF 3·OEt 2)，三氟甲磺酸三甲基硅酯(TMSOTf)，三氟甲磺酸银(AgOTf)，反应温度可以是-40℃至室温之间。
糖苷化反应中用于除水的分子筛可以是
Figure PCTCN2018082660-appb-000006
分子筛或
Figure PCTCN2018082660-appb-000007
分子筛。
2)反应模块B：叠氮基还原乙酰化
叠氮基还原乙酰化可以通过硫代乙酸(AcSH)/吡啶方法直接完成，也可以经叠氮基还原为氨基，再乙酰化完成。叠氮基还原为氨基的方法可以是三甲基膦(PMe 3)/水，三苯基膦 (PPh 3)/水，1,3-丙二硫醇/三乙胺，硼氢化钠(NaBH 4)/二氯化镍(NiCl 2)，二氯化锡(SnCl 2)/苯硫醇(PhSH)/三乙胺，锌/铜/醋酸，Lindlar催化剂/氢气等；
氨基乙酰化方法可以是醋酸酐(Ac 2O)/甲醇，醋酸酐/吡啶，乙酰氯(AcCl)/三乙胺等。
3)反应模块C：单糖砌块1(奎诺糖胺)3号位脱保护
当羟基保护基团为乙酰基(Ac)，苯甲酰基(Bz)，氯乙酰基(ClAc)，二氯乙酰基(DCA)，三氯乙酰基(TCA)，新戊酰基(Piv)时，所用脱保护条件可以是甲醇钠/甲醇，氢氧化钾/甲醇，氢氧化钠/甲醇等；
当羟基保护基团为乙酰丙酰基(Lev)时，所用脱保护条件可以是醋酸肼/吡啶等；
当羟基保护基团为烯丙氧羰酰基(Alloc)时，所用脱保护条件可以是二乙酸钯(Pd(OAc) 2)/二乙胺等；
当羟基保护基团为2-萘甲基(Nap)，对甲氧苄基(PMB)时，所用脱保护条件可以是2,3-二氯-5,6-二氰基-1,4-对苯醌(DDQ)/水，硝酸铈铵(CAN)/水等；
当羟基保护基团为叔丁基二甲基硅烷基(TBDMS)，叔丁基二苯基硅烷基(TBDPS)，三乙基硅烷基(TES)时，所用脱保护条件可以是四丁基氟化铵(TBAF)，氢氟酸等。
4)反应模块D：叠氮基还原修饰2,2,2-三氯乙氧羰基(Troc)
叠氮基还原为氨基的方法可以是三甲基膦(PMe 3)/水，三苯基膦(PPh 3)/水，1,3-丙二硫醇/三乙胺，硼氢化钠(NaBH 4)/二氯化镍(NiCl 2)，二氯化锡(SnCl 2)/苯硫醇(PhSH)/三乙胺，锌/铜/醋酸，Lindlar催化剂/氢气等；
氨基修饰2,2,2-三氯乙氧羰基(Troc)的方法可以通过2,2,2-三氯乙氧羰酰氯(TrocCl)在一种弱碱如碳酸氢钠，吡啶，三乙胺等反应条件下完成。
5)反应模块E:单糖砌块2(岩藻糖胺)3,4号位选择性萘甲基保护
岩藻糖3,4号位选择性萘甲基保护具体指对3,4号位羟基选择性进行3号位羟基的萘甲基保护，而保留4号位羟基不变，所用方法可以是在110℃下二丁基氧化锡处理原料1小时，再经室温至110℃反应温度之间，以2-溴甲基萘和四丁基溴化铵(TBAB)处理反应液完成。
6)反应模块F：单糖砌块2(岩藻糖胺)3号位乙酰化
对于岩藻糖3号位进行乙酰化需要通过选择性脱去萘甲基和引入乙酰基完成。萘甲基的选择性脱去方法可以是2,3-二氯-5,6-二氰基-1,4-对苯醌(DDQ)/水方法等；
乙酰基的引入方法可以是醋酸酐/吡啶，乙酰氯/吡啶，乙酰氯/三乙胺等。
7)反应模块G：叠氮基还原丁酰化
叠氮基还原为氨基的方法可以是三甲基膦(PMe 3)/水，三苯基膦(PPh 3)/水，1,3-丙二硫醇/三乙胺，硼氢化钠(NaBH 4)/二氯化镍(NiCl 2)，二氯化锡(SnCl 2)/苯硫醇(PhSH)/三乙胺，锌/铜/醋酸，Lindlar催化剂/氢气等；
氨基修饰丁酰基的方法可以是以(R)-3-O-PG 8-丁酸酐(如图8所示)为原料，在有或无碱处理下进行，所用碱可以是吡啶，三乙胺等；
氨基修饰丁酰基的方法可以是以(R)-3-O-PG 8-丁酰卤(酰氯，酰溴或酰氟)(如图8所示)为原料，在碱处理下进行，所用碱可以是吡啶，三乙胺，碳酸钠，碳酸氢钠，碳酸钾等；
氨基修饰丁酰基的方法可以是以(R)-3-O-PG 8-丁酸(如图8所示)为原料，所用缩合条件可以是二环己基碳二亚胺(DCC)/4-N,N-二甲基吡啶(DMAP)，二环己基碳二亚胺(DCC)/1-羟基苯并三唑(HOBt)，二异丙基碳二亚胺(DIC)/4-N,N-二甲基吡啶(DMAP)，二异丙基碳二亚胺(DIC)/1-羟基苯并三唑(HOBt)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI)/4-N,N-二甲基吡啶(DMAP)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI)/1-羟基苯并三唑(HOBt)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)，6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯(HCTU)，二苯基磷酰氯(DPP-Cl)，氰代磷酸二乙酯(DECP)等。
(R)-3-羟基丁酰基中羟基保护基团PG 8可以是苄基(Bn)等。
8)反应模块H：脱去Troc修饰乙脒基
脱除Troc的方法可以是锌粉/醋酸，醋酸的含量可以是50％至100％之间，反应温度可以是室温至65℃之间；
氨基修饰为乙脒基的方法可以由硫代乙酰亚氨酸芳基酯卤酸盐或硫代乙酰亚氨酸烷基酯卤酸盐(如图9所示)在碱处理下进行，芳基可以是苄基或萘甲基等，烷基可以是甲基或乙基等，卤酸可以是盐酸或氢溴酸等，所用碱可以是吡啶，三乙胺，二异丙基乙胺，1,8-二氮杂二环十一碳-7-烯(DBU)，碳酸钾，碳酸钠等；
氨基修饰为乙脒基的方法可以由乙酰亚氨酸烷基酯卤酸盐(如图10所示)在碱处理下进行，烷基可以是乙基，三氟乙基，三氯乙基等，卤酸可以是盐酸或氢溴酸等，所用碱可以是吡啶，三乙胺，二异丙基乙胺，1,8-二氮杂二环十一碳-7-烯(DBU)，碳酸钾，碳酸钠等。
9)反应模块I：催化加氢全脱保护
催化加氢全脱保可通过催化条件下通入氢气在室温下进行反应。催化加氢所用催化剂可以是10％钯碳催化剂或氢氧化钯等，反应所用溶剂可以是水/甲醇/二氯甲烷/醋酸混合液，水/叔丁醇/二氯甲烷混合液等。
10)反应模块J：单糖砌块3(葡萄糖醛酸)4号位脱保护
当羟基保护基为2-萘甲基(Nap)或对甲氧苄基(PMB)时，脱保护方法可以是2,3-二氯-5,6-二氰基-1,4-对苯醌(DDQ)/水方法等；
当羟基保护基为乙酰丙酰基(Lev)时，脱保护方法可以是醋酸肼/吡啶等。
11)反应模块K：叠氮基还原修饰乙脒基
叠氮基还原为氨基的方法可以是三甲基膦(PMe 3)/水，三苯基膦(PPh 3)/水，1,3-丙二硫醇/三乙胺，硼氢化钠(NaBH 4)/二氯化镍(NiCl 2)，二氯化锡(SnCl 2)/苯硫醇(PhSH)/三乙胺，锌/铜/醋酸，Lindlar催化剂/氢气等；
氨基修饰为乙脒基的方法可以由硫代乙酰亚氨酸芳基酯卤酸盐或硫代乙酰亚氨酸烷基酯卤酸盐(如图9所示)在碱处理下进行，芳基可以是苄基或萘甲基等，烷基可以是甲基或乙基等，卤酸可以是盐酸或氢溴酸等，所用碱可以是吡啶，三乙胺，二异丙基乙胺，1,8-二氮杂二环十一碳-7-烯(DBU)，碳酸钾，碳酸钠等；
氨基修饰为乙脒基的方法可以由乙酰亚氨酸烷基酯卤酸盐(如图10所示)在碱处理下进行，烷基可以是乙基，三氟乙基，三氯乙基等，卤酸可以是盐酸或氢溴酸等，所用碱可以是吡啶，三乙胺，二异丙基乙胺，1,8-二氮杂二环十一碳-7-烯(DBU)，碳酸钾，碳酸钠等。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，其特征包括如下反应步骤：
1)当n为1，V*-为H-时，II-a的合成步骤包括(如图11所示)：单糖砌块1经糖苷化反应组装连接臂，经反应模块B，C得到单糖受体5，与单糖砌块2糖苷化反应得到二糖6。二糖6经反应模块D，E得到二糖受体7，与单糖砌块3糖苷化反应得到三糖8。三糖8经反应模块F完成岩藻糖乙酰基修饰，经反应模块G，H完成氨基修饰基团组装得到三糖11，经反应模块I催化加氢得到三糖II-a。
2)当n为1，V*-为H-时，II-b的合成步骤包括(如图12所示)：单糖砌块3经糖苷化组装连接臂，单糖12经反应模块G，J得到单糖受体13。单糖13与单糖砌块1经糖苷化反应得到二糖14，经反应模块B，C得到二糖受体15。二糖受体15与单糖砌块2经糖苷化反应得到三糖16，经反应模块K完成乙脒基组装，得到三糖17，进一步经反应模块I催化氢化得到三糖II-b。
3)当n为1，V*-为H-时，II-c的合成步骤包括(如图13所示)：单糖砌块2经糖苷化反应组装连接臂，得到的化合物18经反应模块D，E制得单糖受体19。单糖受体19与单糖砌块3糖苷化反应得到二糖，经反应模块F，G，J制得二糖受体21。二糖受体21与单 糖砌块1糖苷化反应得到三糖22，经反应模块B，H完成氨基修饰基团组装，得到三糖24。三糖24经反应模块I催化氢化制得三糖II-c。
4)当n为1，V*-为H-时，III-a的合成步骤包括(如图14所示)：三糖16经反应模块D，E得到三糖受体25。三糖受体25与单糖砌块3经糖苷化反应得到四糖26，经反应模块F完成岩藻糖乙酰基修饰，经反应模块G，H完成氨基修饰基团组装，得到四糖29。四糖29经反应模块I催化氢化得到四糖III-a。
5)当n为1，V*-为H-时，III-b的合成步骤包括(如图15所示)：三糖23经反应模块C脱保护得到三糖受体30。三糖受体30与单糖砌块2糖苷化反应制得四糖31，经反应模块D，H完成乙脒基组装，制得四糖32。四糖32经反应模块I催化氢化制得四糖III-b。
6)当n为1，V*-为H-时，III-c的合成步骤包括(如图16所示)：三糖10经反应模块J脱保得到三糖受体33。受体33与单糖砌块1经糖苷化反应制得四糖34，进一步通过反应模块B，H完成氨基修饰基团的组装，得到四糖36。四糖36经反应模块I催化氢化制得四糖III-c。
7)当n为1，V*-为H-时，IV-a的合成步骤包括(如图17所示)：四糖31经反应模块D，E制得四糖受体37，进一步与单糖砌块3糖苷化反应得到五糖38。五糖38经反应模块F完成岩藻糖乙酰基修饰，经反应模块G，H完成氨基修饰基团组装，得到五糖41，进一步经反应模块I催化氢化制得五糖IV-a。
8)当n为1，V*-为H-时，IV-b的合成步骤包括(如图18所示)：四糖35经反应模块C脱保护得到四糖受体42，进一步与单糖砌块2糖苷化反应制得五糖43。43经反应模块D，H完成乙脒基修饰，制得五糖44，进一步经反应模块I催化氢化得到五糖IV-b。
9)当n为1，V*-为H-时，IV-c的合成步骤包括(如图19所示)：四糖28经反应模块J脱保护得到四糖受体45，进一步与单糖砌块1糖苷化反应制得五糖46。五糖46经反应模块B，H完成氨基修饰基团组装，得到五糖48，进一步经反应模块I催化氢化制得五糖IV-c。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，当糖砌块1(奎诺糖)作为糖基供体时，其2号位氨基可以由叠氮基进行保护，以利于1,2-顺式-α-糖苷键的生成。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，当糖砌块2(岩藻糖)作为糖基供体时，其2号位氨基可以由叠氮基进行保护，以利于1,2-顺式-α-糖苷键的生成。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，当糖砌块2(岩藻糖)的4号位羟基作为糖基化反应受体时，为了提高4号位羟基的亲核性，该糖砌块2(岩藻糖)的2号位氨基可以由Troc保护，3号位羟基可以由萘甲基保护。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，当糖砌块3(葡萄糖醛酸)作为糖基供体时，其结构中3号位氨基可以由叠氮基作为保护基，以避免酰胺类保护基对糖基化反应的阻碍。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，当糖砌块3(葡萄糖醛酸)作为糖基供体时，其结构中2号位氨基可以由三氯乙酰基保护，以利于β-构型糖苷键的生成，且可通过催化氢化转化为乙酰氨基。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，糖砌块1(奎诺糖)结构中的2号位乙酰氨基可以在完成糖砌块1(奎诺糖)的糖基化反应后随即引入，以避免对其后糖砌块的引入造成阻碍。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，糖砌块2(岩藻糖)的3号位萘甲基可以在完成4号位糖基化反应后随即转换为乙酰基修饰，以避免对其后糖砌块的引入造成阻碍。
本发明提供的类志贺邻单胞菌O51寡糖的化学合成方法，糖砌块3(葡萄糖醛酸)结构中的3号位丁酰胺基的组装应在葡萄糖醛酸4号位脱保护之前完成，以避免对其后糖砌块的引入造成阻碍。
本发明还提供了三种用于制备杂修饰多氨基糖的正交保护中间体，分别为通式8，通式16，通式22所示的化合物。以这三种化合物可完成不同氨基修饰基团的组装，也可进一步用于寡糖链的延长。
本发明首次完成了类志贺邻单胞菌O51血清型O-抗原糖结构的化学合成。此外通过在糖类结构还原端引入相应连接臂，为合成寡糖结构偶联载体分子或固定化于相应基质提供基础，可用于类志贺邻单胞菌O51血清型O-抗原糖结构生物作用的研究，以及制备、开发糖类疫苗等。
附图说明
图1：类志贺邻单胞菌O51血清型O-抗原三糖重复单元。
图2：通式I中U x，U x+1，U x+2所示化合物。
图3：通式II-a，II-b和II-c所示化合物。
图4：通式III-a，III-b和III-c所示化合物。
图5：通式IV-a，IV-b和IV-c所示化合物。
图6：单糖砌块1，2，3和连接臂4所示化合物。
图7：2,3-二氨基葡萄糖52的合成反应式。
图8：氨基丁酰化修饰所用丁酰类化合物。
图9：硫代乙酰亚氨酸芳(烷)基酯卤酸盐。
图10：乙酰亚氨酸烷基酯卤酸盐。
图11：类志贺邻单胞菌O51三糖II-a的合成反应式。
图12：类志贺邻单胞菌O51三糖II-b的合成反应式。
图13：类志贺邻单胞菌O51三糖II-c的合成反应式。
图14：类志贺邻单胞菌O51四糖III-a的合成反应式。
图15：类志贺邻单胞菌O51四糖III-b的合成反应式。
图16：类志贺邻单胞菌O51四糖III-c的合成反应式。
图17：类志贺邻单胞菌O51五糖IV-a的合成反应式。
图18：类志贺邻单胞菌O51五糖IV-b的合成反应式。
图19：类志贺邻单胞菌O51五糖IV-c的合成反应式。
图20：化合物11*的化学合成反应式。
图21：化合物12*和13*的化学合成反应式。
图22：化合物22*的化学合成反应式。
图23：化合物35*的化学合成反应式。
具体实施方式654ew
实验中所用商品化试剂均未经处理直接使用。反应所用无水溶剂由MBraun MB-SPS 800型溶剂干燥系统制备。硅胶柱层析所用溶剂均为分析纯且经减压蒸馏后使用。薄层层析(TLC)所用硅胶板为60-F254硅胶制备的玻璃基或铝箔基硅胶板。薄层层析显色试剂为糖显色剂(0.1％(v/v)3-甲氧基苯酚，2.5％(v/v)硫酸乙醇溶液)，或CAM显色剂(5％(w/v)钼酸铵，1％(w/v)硫酸铈(II)和10％(v/v)硫酸水溶液)，或茚三酮显色剂(1.5％(w/v)茚三酮和3％(v/v)醋酸正丁醇溶液)。正相硅胶柱层析所用硅胶为200-300目硅胶。分子排阻色谱所用填料为葡聚糖凝胶
Figure PCTCN2018082660-appb-000008
LH-20(GE Healthcare)。
分别对各反应步骤产率进行计算，产率计算方式为：(目标产物物质的量/原料物质的量)×100％。利用核磁图谱，红外图谱，旋光度，高分辨质谱对于产品进行结构鉴定，利用核磁图谱和高效液相色谱对于产品进行纯度分析。氢谱、碳谱以及二维核磁谱由Bruker Ascend 400兆核磁共振仪，或Bruker Ultrashield Plus 400兆核磁共振仪，或Bruker AVIII 700兆核磁共振仪在25℃下测得。高分辨质谱由Agilent 6220电喷雾离子源-飞行时间质谱仪测得。旋光度由Schmidt & Haensch UniPol L 1000全自动旋光仪在589nm下测得，测定浓度(c)单位为 g/100mL。红外图谱由Thermo Fisher Scientific Nicolet iS5红外仪测得。分析型高效液相色谱由Agilent 1200series液相联四级杆电喷雾质谱6130进行，所用分析柱为Thermo Scientific Hypercarb柱子(150x 4.6mm)。制备型高效液相色谱由Agilent 1200 series液相联四级杆电喷雾质谱6130进行，所用半制备柱为Thermo Scientific Hypercarb柱子(150x 10mm)。
实施例1
烯丙基3,4,6-三-O-乙酰基-2-脱氧-2-三氯乙酰氨基-α-D-吡喃葡萄糖(1*)的合成
反应方程式如图20所示；
在氩气保护下，1,3,4,6-四-O-乙酰基-2-脱氧-2-三氯乙酰氨基-β-D-吡喃葡萄糖(M.Virlouvet et al.,Adv.Synth.Catal.2010,352,2657-2662)(27g,0.055mol)溶解于无水二氯甲烷(130mL)，并加入活化的
Figure PCTCN2018082660-appb-000009
分子筛和烯丙醇(18.7mL,0.274mol)。冷却至-5℃后，三氟化硼乙醚(70mL,0.548mol)逐滴加入。反应液于0℃下搅拌30min，然后升至室温反应71小时。反应结束后，将反应液倾倒于200g碎冰上，硅藻土过滤后得到的有机相分别用水、饱和碳酸氢钠溶液、饱和食盐水进行萃取，得到的有机相经硫酸钠除水后浓缩。得到粗品经硅胶柱层析(石油醚/乙酸乙酯，5:1，v/v)纯化得到黄色糖浆状的纯品1*(19.9g,0.041mol,74％)。[α] D 20＝+87.8°(c＝1.20,CHCl 3)；IR ν max(film)3429,2960,1749,1722,1517,1368,1229,1048,822,682cm -1； 1H NMR(400MHz,CDCl 3)δ＝6.93(d,J＝9.1Hz,1H,NH),5.88(dddd,J＝17.0,10.3,6.5,5.4Hz,1H,CH＝C),5.41-5.23(m,3H,4-H,C＝CH 2),5.16(dd,J＝9.8,9.8Hz,1H,3-H),5.01(d,J＝3.7Hz,1H,1-H),4.34-4.19(m,3H,6-CH 2,OCH a),4.11(dd,J＝12.4,2.4Hz,1H,OCH b),4.09-3.98(m,2H,2-H,5-H),2.11(s,3H,CH 3CO),2.05(s,3H,CH 3CO),2.02(s,3H,CH 3CO)； 13C NMR(100MHz,CDCl 3)δ＝171.1,170.6,169.3,161.9,132.6,119.1,95.5,92.1,77.3,70.7,69.0,68.1,67.8,61.8,53.9,20.7,20.63,20.59；HR-ESI-MS(m/z):calcd for C 17H 22Cl 3NO 9Na +(M+Na +):512.0258,found:512.0254。
实施例2
烯丙基4,6-O-苄叉基-2-脱氧-2-三氯乙酰氨基-α-D-吡喃葡萄糖(2*)的合成
反应方程式如图20所示；
化合物1*(50g,0.102mol)溶解于甲醇(800mL)，加入甲醇钠(2.8g,0.051mol)后在室温下搅拌5小时。反应结束后用Amberlite IR 120阳离子交换树脂中和反应液，经过滤、浓缩得到白色固体为目标3,4,6-三羟基糖(37.1g,0.102mol,quant.)。
三羟基糖(71g,0.195mol)进一步溶解于300mL无水DMF，加入苯甲醛二甲缩醛(35mL,0.234mol)和对甲苯磺酸(4.45g,0.023mol)。反应液在60℃反应24小时后，利用旋 转蒸发仪减压蒸馏除去反应中生成的甲醇，继续置于60℃反应，如此操作反复进行，待反应结束可减压蒸馏除去DMF。得到的粗品溶解于乙酸乙酯，经饱和碳酸氢钠溶液、水和饱和食盐水萃取，无水硫酸钠除水、浓缩后得到粗产物。将该粗产物用硅胶柱层析(石油醚：乙酸乙酯，10:1，v/v)纯化得到白色固体状产品2*(73.6g，0.163mol，83％)。[α] D 20＝+68.6°(c＝1.00,CHCl 3)；IR ν max(film)3339,2919,1695,1530,1451,1372,1085,1027,989,748,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.55-7.34(m,5H,Ph),6.96(d,J＝8.8Hz,1H,NH),5.86(dddd,J＝16.9,10.3,6.4,5.3Hz,1H,CH＝C),5.53(s,1H,PhCH),5.35-5.20(m,2H,C＝CH 2),4.97(d,J＝3.8Hz,1H,1-H),4.27(dd,J＝10.2,4.8Hz,1H,4-H),4.24-4.11(m,2H,2-H,OCH a),4.06-3.94(m,2H,OCH b,5-H),3.86(ddd,J＝9.9,9.8,4.7Hz,1H,6-CH a),3.74(t,J＝10.3Hz,1H,6-CH b),3.56(t,J＝9.3Hz,1H,3-H),2.78(br,1H,3-OH)； 13C NMR(100MHz,CDCl 3)δ＝162.3,137.0,132.9,129.3,128.3,126.3,118.7,101.9,96.3,92.4,81.5,69.7,68.8,68.6,62.8,55.4；HR-ESI-MS(m/z):calcd for C 18H 20Cl 3NO 6Na +(M+Na +):474.0254,found:474.0245。
实施例3
烯丙基4,6-O-苄叉基-3-O-三氟甲磺酰基-2-三氯乙酰氨基-2-脱氧-α-D-吡喃葡萄糖(3*)的合成
反应方程式如图20所示；
化合物2*(53.1g,0.117mol)溶解于无水二氯甲烷/吡啶混合液(660mL，7:1，v/v)中，降温至-20℃后，在搅拌条件下逐滴加入三氟甲磺酸酐(40mL,0.234mol)的二氯甲烷溶液(150mL)，其后2小时中逐渐升温至10℃。反应结束后，反应液经二氯甲烷稀释，依次以1M HCl溶液、饱和碳酸氢钠、水和饱和食盐水进行萃取。有机相经无水硫酸钠除水、低温下减压蒸馏除去溶剂，所得粗产品经硅胶柱层析(石油醚：乙酸乙酯，5:1至2:1，v/v)纯化得到黄色糖浆状产品3*(66.5g,0.114mol,98％)。[α] D 20＝+35.3°(c＝1.00,CHCl 3)；IR ν max(film)3348,1698,1529,1414,1373,1203,1146,1122,959,836,753,628cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.52-7.33(m,5H,Ph),7.06(d,J＝9.6Hz,1H,NH),5.87(dddd,J＝17.0,10.2,6.8,5.4Hz,1H,CH＝C),5.62(s,1H,PhCH),5.37-5.27(m,2H,C＝CH 2),5.13(dd,J＝10.3,9.1Hz,1H,3-H),5.03(d,J＝3.7Hz,1H,1-H),4.50(ddd,J＝10.0,10.0,3.8Hz,1H,2-H),4.36(dd,J＝10.4,4.5Hz,1H,6-CH a),4.25(ddt,J＝12.7,5.5,1.3Hz,1H,OCH a),4.06(ddt,J＝12.7,6.8,1.2Hz,1H,OCH b),3.98(td,J＝9.6,4.6Hz,1H,5-H),3.92(dd,J＝9.2Hz,1H,6-CH b),3.84(dd,J＝10.1,10.1Hz,1H,4-H)； 13C NMR(100MHz,CDCl 3)δ＝162.2,136.2,132.2,129.2,128.2,125.9,119.9,116.8,101.6,96.4,91.8,83.1,78.2,69.3,68.4,63.3,53.8；HR-ESI-MS(m/z):calcd for  C 19H 19Cl 3F 3NO 8SNa +(M+Na +):605.9747,found:605.9760。
实施例4
烯丙基4,6-O-苄叉基-2-三氯乙酰氨基-2-脱氧-α-D-吡喃阿洛糖(4*)的合成
反应方程式如图20所示；
将化合物3*(0.5g,0.855mmol)溶解于无水DMF(10mL)中，加入亚硝酸钾(364mg,4.275mmol)，反应液在50℃下搅拌6小时。待反应结束，加入二氯甲烷将反应液稀释，并经饱和食盐水萃取，所得有机相经无水硫酸钠除水后减压浓缩。粗品经硅胶柱层析(石油醚：乙酸乙酯，8:1，v/v)得到白色固体状的3号位转位产物4*(278mg,0.614mmol,72％)。[α] D 20＝+74.0°(c＝1.00,CHCl 3)；IR ν max(film)3419,1711,1507,1378,1218,1102,1065,1023,820,756,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.57-7.33(m,6H,NH,Ph),5.88(dddd,J＝16.9,10.3,6.4,5.2Hz,1H,CH＝C),5.63(s,1H,PhCH),5.37-5.20(m,2H,C＝CH 2),5.00(d,J＝4.2Hz,1H,1-H),4.38(dd,J＝10.3,5.1Hz,1H,6-CH b),4.27(m,2H,3-H,OCH a),4.24-4.16(m,2H,2-H,5-H),4.05(ddt,J＝13.0,6.5,1.3Hz,1H,OCH b),3.80(t,J＝10.3Hz,1H,6-CH a),3.67(dd,J＝9.7,2.8Hz,1H,4-H),2.66(br,1H,3-OH)； 13C NMR(100MHz,CDCl 3)δ＝161.7,136.9,132.9,129.3,128.4,126.2,118.8,101.9,96.0,92.3,78.1,77.2,69.5,69.0,67.3,57.6,51.2；HR-ESI-MS(m/z):calcd for C 18H 20Cl 3NO 6Na +(M+Na +):474.0254,found:474.0245。
实施例5
烯丙基4,6-O-苄叉基-3-O-三氟甲磺酰基-2-三氯乙酰氨基-2-脱氧-α-D-吡喃阿洛糖(5*)的合成
反应方程式如图20所示；
在-20℃下，向化合物4*(178mg,0.393mmol)的无水二氯甲烷(3.93mL)溶液中加入吡啶(273μL,3.380mmol)，进一步滴加三氟甲磺酸酐(133μL,0.786mmol)，在2小时的搅拌过程中，逐渐提高反应温度至10℃。待反应结束后，以二氯甲烷稀释反应液，所得有机相分别经1M HCl溶液、饱和碳酸氢钠溶液、水和饱和食盐水萃取。所得有机相经无水硫酸钠除水后低温下减压蒸馏除去溶剂，所得粗品以硅胶柱(石油醚：乙酸乙酯，10:1-5:1-2:1，v/v)进行纯化得黄色糖浆状产物5*(160mg,0.275mmol,70％)。[α] D 20＝+40.4°(c＝1.00,CHCl 3)；IR ν max(film)2923,2853,1750,1497,1416,1211,1028,821,617cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.53-7.35(m,5H,Ph),7.33(d,J＝8.5Hz,1H,NH),5.89(dddd,J＝16.8,10.6,6.0,4.7Hz,1H,CH＝C),5.61(s,1H,PhCH),5.44(t,J＝2.9Hz,1H,3-H),5.37(dq,J＝17.3,1.6Hz,1H,C＝CH a),5.28(dq,J＝10.5,1.4Hz,1H,C＝CH b),4.96(d,J＝4.3Hz,1H,1-H),4.44(ddd,J＝8.0,4.3,3.2Hz,1H,2-H),4.41-4.31(m,2H,6-H a,OCH a),4.26(td,J＝9.9,5.2Hz,1H,5-H), 4.05(ddt,J＝13.3,6.0,1.4Hz,1H,OCH b),3.87(dd,J＝9.7,2.5Hz,1H,4-H),3.78(t,J＝10.4Hz,1H,6-H b)； 13C NMR(100MHz,CDCl 3)δ＝162.0,136.3,132.4,129.4,128.3,126.2,118.1,102.4,94.9,91.5,80.7,74.9,69.0,68.8,58.1,50.2；HR-ESI-MS(m/z):calcd for C 19H 19Cl 3F 3NO 8SNa +(M+Na +):605.9747,found:605.9753。
实施例6
烯丙基4,6-O-苄叉基-3-叠氮-2-三氯乙酰氨基-2,3-二脱氧-α-D-吡喃葡萄糖(6*)的合成
反应方程式如图20所示；
化合物5*(65mg,0.111mmol)溶解于无水DMF中，加入叠氮化钠(36mg,0.555mmol)，在室温下搅拌过夜。待反应结束后，以乙酸乙酯稀释反应液，分别经水、饱和食盐水萃取，所得有机相经无水硫酸钠除水后浓缩。所得粗品经硅胶柱层析纯化(石油醚：乙酸乙酯，20:1-8:1，v/v)得到白色固体状产物6*(44.2mg,0.093mmol,83％)。[α] D 20＝+39.7°(c＝1.00,CHCl 3)；IR ν max(film)3120,2917,2849,2110,1692,1528,1372,1258,1124,1080,1055,1016,836,750,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.55-7.33(m,5H,Ph),6.88(d,J＝9.5Hz,1H,NH),5.94-5.80(m,1H,CH＝C),5.63(s,1H,PhCH),5.36-5.23(m,2H,C＝CH 2),4.92(d,J＝3.6Hz,1H,1-H),4.32(dd,J＝10.4,4.9Hz,1H,6-CH a),4.24(ddt,J＝12.7,5.5,1.4Hz,1H,OCH a),4.13(ddd,J＝11.2,9.7,3.7Hz,1H,2-H),4.05(ddt,J＝12.8,6.6,1.4Hz,1H,OCH b),3.99-3.88(m,2H,3-H,5-H),3.79(t,J＝10.3Hz,1H,6-CH b),3.70(t,J＝9.6Hz,1H,4-H)； 13C NMR(100MHz,CDCl 3)δ＝161.9,136.7,132.6,129.1,128.3,125.9,119.2,101.5,95.9,92.3,80.3,77.2,68.9,68.7,63.3,61.2,53.5；HR-ESI-MS(m/z):calcd for C 18H 19Cl 3N 4O 5Na +(M+Na +):499.0319,found:499.0311。
实施例7
烯丙基3-叠氮-2-三氯乙酰氨基-2,3-二脱氧-α-D-吡喃葡萄糖(7*)的合成
反应方程式如图20所示；
将化合物6*(20mg,0.042mmol)加入80％醋酸水溶液(0.9mL)中，加热到55℃搅拌至反应结束。经减压蒸馏除去溶剂后，以硅胶柱层析纯化(二氯甲烷：甲醇，60:1-40:1-30:1，v/v)得到白色固体状的4,6-二羟基糖7*(16.3mg,0.042mmol,quant.)。[α] D 20＝+16.9°(c＝0.35,CHCl 3)；IR ν max(film)3411,2924,2108,1712,1517,1262,1049,822,680cm -1； 1H NMR(400MHz,CD 3OD)δ＝5.98(dddd,J＝17.0,10.4,6.4,5.1Hz,1H,CH＝C),5.38(dq,J＝17.3,1.7Hz,1H,C＝CH a),5.24(dq,J＝10.4,1.4Hz,1H,C＝CH b),4.94(d,J＝3.6Hz,1H,1-H),4.29(ddt,J＝13.2,5.2,1.5Hz,1H,OCH a),4.10(ddt,J＝13.1,6.4,1.4Hz,1H,OCH b),3.99(dd,J＝11.5,9.2Hz,1H,3-H),3.93-3.81(m,2H,2-H,6-H a),3.81-3.68(m,2H,5-H,6-H b),3.52(t,J＝9.3Hz,1H, 4-H)； 13C NMR(100MHz,CD 3OD)δ＝166.7,137.6,120.8,98.9,76.5,73.7,72.0,67.5,64.8,58.1；HR-ESI-MS(m/z):calcd for C 11H 15Cl 3N 4O 5Na +(M+Na +):411.0006,found:411.0001。
实施例8
烯丙基3-叠氮-2-三氯乙酰氨基-2,3-二脱氧-α-D-吡喃葡萄糖醛酸苄酯(8*)的合成
反应方程式如图20所示；
在0℃下，向化合物7*(22g,0.056mol)的二氯甲烷(2.8L)溶液中加入水(1.4L)，2,2,6,6-四甲基哌啶氧化物(2,2,6,6-tetramethylpiperidine 1-oxyl，TEMPO)(4.4g,0.028mol)和二乙酰氧基碘苯(diacetoxyliodo benzene，BAIB)(45.5g,0.141mol)。反应液在室温下4小时搅拌后，经硅胶柱层析、浓缩后，所得粗品直接投入下一步反应。
粗品羧酸化合物溶解于无水DMF中(2.8L)，依次加入碳酸氢钠(21.4g,0.255mol)和溴苄(50.4mL,0.424mol)，所得反应液在室温下搅拌。待反应结束后，减压蒸馏除去溶剂，所得粗品经硅胶柱层析(石油醚：乙酸乙酯，10:1，v/v)得到黄色糖浆状的糖醛酸苄酯8*(20.4g,0.041mol,两步反应产率73％)。[α] D 20＝+54.5°(c＝1.00,CHCl 3)；IR ν max(film)3412,2930,2110,1717,1514,1264,1057,909,820,733,698cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.43-7.33(m,5H,Ph),6.79(d,J＝9.4Hz,1H,NH),5.86(dddd,J＝17.0,10.3,6.6,5.4Hz,1H,CH＝C),5.37-5.20(m,4H,PhCH 2,C＝CH 2),4.97(d,J＝3.6Hz,1H,1-H),4.31-4.20(m,2H,5-H,OCH a),4.13-4.00(m,2H,2-H,OCH b),3.91(t,J＝9.5Hz,1H,4-H),3.75(dd,J＝11.1,9.3Hz,1H,3-H),3.31(br,1H,4-OH)； 13C NMR(100MHz,CDCl 3)δ＝169.5,161.9,134.8,132.4,128.77,128.75,128.3,119.4,95.6,92.2,77.2,71.2,70.5,69.3,67.8,63.6,52.7；HR-ESI-MS(m/z):calcd for C 18H 19Cl 3N 4O 6Na +(M+Na +):515.0268,found:515.0264。
实施例9
烯丙基4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-二脱氧-α-D-吡喃葡萄糖醛酸苄酯(9*)的合成
反应方程式如图20所示；
化合物8*(20.3g,0.041mol)溶解于无水二氯甲烷(400mL)，在0℃下，依次加入溴苄(49mL,0.411mol)和氧化银(28.6g,0.123mol)，所得反应液在0℃下搅拌5小时后升至室温搅拌28小时。待确认反应结束后，经硅藻土过滤和浓缩，所得粗品经硅胶柱层析(石油醚：乙酸乙酯，40:1-10:1，v/v)得到白色固体状产物9*(14.7g,0.025mol,62％)。[α] D 20＝+66.3°(c＝1.00,CHCl 3)；IR ν max(film)3357,2935,2108,1743,1713,1514,1253,1185,1111,1063,1028,820,750,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.43-7.10(m,10H,2Ph),6.81(d,J＝9.6Hz,1H,NH),5.84(dddd,J＝17.0,10.3,6.6,5.4Hz,1H,CH＝C),5.32-5.22(m,2H, C＝CH 2),5.21(s,2H,COOCH 2Ph),4.95(d,J＝3.5Hz,1H,1-H),4.69(d,J＝10.5Hz,1H,PhCH 2),4.49(d,J＝10.5Hz,1H,PhCH 2),4.35-4.27(m,1H,5-H),4.24(ddt,J＝12.8,5.4,1.4Hz,1H,OCH a),4.17-4.08(m,1H,2-H),4.04(ddt,J＝12.8,6.6,1.2Hz,1H,OCH b),3.83-3.74(m,2H,3-H,4-H)； 13C NMR(100MHz,CDCl 3)δ＝168.4,161.8,136.9,134.9,132.4,128.7,128.59,128.56,128.5,128.4,128.24,128.21,128.14,128.12,119.44,119.39,95.5,92.3,78.2,75.1,71.0,69.1,67.6,64.3,53.0；HR-ESI-MS(m/z):calcd for C 25H 25Cl 3N 4O 6Na +(M+Na +):605.0737,found:605.0731。
实施例10
4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-二脱氧-D-吡喃葡萄糖醛酸苄酯(10*)的合成
反应方程式如图20所示；
将化合物9*(95.8mg,0.164mmol)和醋酸(28μL,0.492mmol)溶解于1,4-二氧六环(2mL)，加入二氧化硒(91mg,0.820mmol)后加热至回流温度反应。待反应结束后，降至室温，滴加三乙胺淬灭反应。浓缩得到的粗品经硅胶柱层析纯化(石油醚：乙酸乙酯，8:1-4:1，v/v)得到淡黄色固体状的半缩醛产物10*(58.0mg,0.107mmol,65％)。[α] D 20＝+33.2°(c＝1.00,CHCl 3)；IR ν max(film)3410,2944,2113,1712,1518,1457,1357,1264,1187,1113,1068,822,752,698cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.47-7.06(m,10H,2Ph),6.92(d,J＝9.5Hz,1H,NH),5.29(t,J＝3.5Hz,1H,1-H),5.21(d,J＝12.1Hz,1H,PhCH a1),5.17(d,J＝12.1Hz,1H,PhCH a2),4.70(d,J＝10.5Hz,1H,PhCH b1),4.56-4.44(m,2H,PhCH b2,5-H),4.11(td,J＝9.7,3.4Hz,1H,2-H),3.81(m,2H,3-H,4-H),3.57(d,J＝3.7Hz,1H,1-OH).； 13C NMR(100MHz,CDCl 3)δ＝168.6,162.1,136.8,134.7,128.8,128.7,128.5,128.3,128.2,92.2,90.9,78.0,75.0,70.8,67.8,63.7,53.1；HR-ESI-MS(m/z):calcd for C 22H 21Cl 3N 4O 6Na +(M+Na +):567.0395,found:567.0414。
实施例11
(4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-脱氧-1-(N-苯基)-2,2,2-三氟乙酰亚胺酯-D-吡喃葡萄糖)醛酸苄酯(11*)的合成
反应方程式如图20所示；
向化合物10*(22.7mg,0.042mmol)的无水二氯甲烷(0.4mL)溶液中加入碳酸钾(12mg,0.084mmol)和N-苯基三氟乙酰亚胺氯(13μL,0.084mmol)，所得反应液在室温下搅拌过夜。待反应结束后过滤得到有机相，经浓缩后得到的粗品以硅胶柱层析纯化(正己烷：乙酸乙酯，18:1，v/v)得到无色浆状产品11*(27.4mg,0.038mmol,91％)。IR ν max(film)3346,2112,1723,1524,1317,1213,1166,1090,823,752,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.44(s,1H),7.35-7.11(m,20H),7.03(q,J＝7.5Hz,2H),6.80(d,J＝8.8Hz,1H),6.73(d,J＝7.8 Hz,2H),6.68(d,J＝7.8Hz,2H),6.38(s,1H),6.12(s,1H),5.30-5.02(m,3H),4.61(d,J＝10.5Hz,1H),4.56(d,J＝10.6Hz,1H),4.52-4.44(m,2H),4.37(d,J＝7.6Hz,1H),4.24(s,1H),4.07(dt,J＝14.0,7.2Hz,1H),3.92(s,1H),3.85(dd,J＝13.4,7.8Hz,2H)； 13C NMR(100MHz,CDCl 3)δ＝167.4,167.2,162.0,142.5,136.2,136.1,134.7,134.5,128.83,128.77,128.69,128.67,128.64,128.58,128.56,128.5,128.41,128.37,124.8,124.5,119.3,119.1,75.0,74.3,73.8,72.7,67.9,67.8,62.5,51.7；HR-ESI-MS(m/z):calcd for C 30H 25Cl 3F 3N 5O 6Na +(M+Na +):738.0691,found:738.0722。
实施例12
乙基1-硫-3,4-二-O-乙酰基-2-叠氮基-2-脱氧-L-吡喃岩藻糖(12*)的合成
反应方程式如图21所示；
将3,4-二-O-乙酰-2-叠氮-2-脱氧-L-吡喃岩藻糖(C.L.Pereira et al.,Angew.Chem.Int.Ed.2015,54,10016-10019)(44.4mg,0.16mmol)溶解于无水二氯甲烷/吡啶混合液(1.6mL，4:1，v/v)中，降至0℃后，滴加醋酸酐(150μL,1.6mmol)，待加入催化量二甲基氨基吡啶后，于室温下搅拌反应液。待确认反应结束后，反应液经饱和碳酸氢钠溶液萃取，所得有机相经无水硫酸钠除水后浓缩。所得粗品经硅胶柱层析纯化(石油醚：乙酸乙酯，10:1，v/v)得到1-氧乙酰糖(50.5mg,0.16mmol,quant.)。
1-氧乙酰岩藻糖(50.5mg，0.16mmol)与乙硫醇(18μL，0.242mmol)溶解于无水二氯甲烷(1.6mL)，加入活化的
Figure PCTCN2018082660-appb-000010
分子筛搅拌30分钟。待降温至-10℃后加入三氟甲烷磺酸三甲基硅酯(35μL,0.193mmol)，所得反应液在0℃下搅拌。待确认反应结束后，加入三乙胺将反应淬灭，减压蒸馏除去溶剂后所得粗品经硅胶柱层析纯化(石油醚：乙酸乙酯，10:1，v/v)得到产物12*(44.0mg,0.14mmol,88％,α:β＝1:0.7)。 1HNMR(400MHz,CDCl 3)δ＝5.45(d,J＝5.6Hz,1H,1α-H),5.28(dd,J＝3.4,1.2Hz,1H,4α-H),5.26-5.21(m,1H,4β-H),5.13(dd,J＝11.0,3.3Hz,1H,3α-H),4.88(dd,J＝10.2,3.3Hz,1H,3β-H),4.49(qd,J＝6.5,1.3Hz,1H,5α-H),4.37(d,J＝10.2Hz,1H,1β-H),4.22(dd,J＝11.0,5.5Hz,1H,2α-H),3.82-3.72(m,1H,5β-H),3.67(t,J＝10.2Hz,1H,2β-H),2.87-2.71(m,2H,β-SCH 2CH 3),2.61(dddd,J＝20.3,12.9,7.4,5.4Hz,2H,α-SCH 2CH 3),2.18(s,5H,2CH 3CO),2.05(s,6H,2CH 3CO),1.34(t,J＝7.4Hz,2H,β-SCH 2CH 3),1.31(t,J＝6.4Hz,3H,α-SCH 2CH 3),1.21(d,J＝6.5Hz,2H,6β-CH 3),1.16(d,J＝6.5Hz,3H,6α-CH 3)。
实施例13
3,4-二-O-乙酰-2-叠氮-2-脱氧-L-吡喃岩藻糖三氟乙酰亚胺酯(13*)的合成
反应方程式如图21所示；
3,4-二-O-乙酰-2-叠氮-2-脱氧-L-吡喃岩藻糖(C.L.Pereira et al.,Angew.Chem.Int.Ed.2015,54,10016-10019)(70mg,0.256mmol)溶解于无水二氯甲烷(2.6mL)，加入碳酸钾(71mg,0.512mmol)和N-苯基三氟乙酰亚胺氯(77μL,0.512mmol)后，在室温下搅拌过夜。待反应结束后，过滤除去固形物，并以二氯甲烷冲洗，经浓缩得到的粗品由硅胶柱层析纯化(正己烷：乙酸乙酯，10:1，v/v)得到产物13*(96.7mg,0.218mmol,85％)。 1HNMR(400MHz,CDCl 3)δ＝7.40-6.75(m,5H,Ph),5.58(m,1H,1-H),5.20(m,1H,4-H),4.84(m,1H,3-H),3.90(t,J＝9.3Hz,1H,2-H),3.74(m,1H,5-H),2.20(s,3H,CH 3CO),2.07(s,3H,CH 3CO),1.21(d,J＝6.4Hz,3H,6-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝170.3,169.7,143.0,128.8,124.6,119.2,95.7,71.5,70.4,69.1,59.9,20.6,15.9。
实施例14
苯基4-O-苄基-3-O-乙酰基-2-叠氮-2-脱氧-1-硒-α-D-吡喃葡萄糖(14*)的合成
反应方程式如图22所示；
苯基 4,6-O-苄叉基-3-O-乙酰基-2-叠氮基-2-脱氧-1-硒-α-D-吡喃葡萄糖(F.Santoyo-González et al.,Synlett,1994,6,454-456)(1g,2.1mmol)与甲苯共沸三次后，在氩气保护下，溶解于18mL无水二氯甲烷中。向反应瓶中加入1M的硼烷四氢呋喃溶液(12.4mL,12.4mmol)，在降温至0℃后，加入三氟甲烷磺酸三甲基硅酯(190μL,1.05mmol)。所得反应液在室温下搅拌，经TLC确认反应结束后，以二氯甲烷稀释反应液，并以饱和碳酸氢钠溶液萃取。有机相经无水硫酸钠除水后减压蒸馏浓缩，得到粗品经硅胶柱层析纯化(正己烷：乙酸乙酯，4:1，v/v)得到黄色糖浆状的化合物14*(148.9mg,0.313mmol)。[α] D 20＝+159.5°(c＝1.00,CHCl 3)；IR ν max(film)3459,2924,2106,1748,1364,1228,1088,737,694cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.70-7.14(m,10H,2Ph),5.87(d,J＝5.4Hz,1H,1-H),5.38(t,J＝9.8Hz,1H,3-H),4.74-4.54(m,2H,PhCH 2),4.19(dt,J＝9.9,2.9Hz,1H,6-CH a),3.90(dd,J＝10.3,5.4Hz,1H,2-H),3.72(m,3H,4-H,5-H,6-CH b),2.04(s,3H,CH 3CO),1.64(dd,J＝7.7,5.2Hz,1H,6-OH)； 13C NMR(100MHz,CDCl 3)δ＝169.7,137.4,134.9,129.2,128.6,128.2,128.1,128.0,127.9,84.0,75.4,74.7,74.2,73.8,63.0,61.1,20.9；HR-ESI-MS(m/z):calcd for C 21H 23N 3O 5SeNa +(M+Na +):500.0701,found:500.0682。
实施例15
苯基4-O-苄基-3-O-乙酰基-2-叠氮基-2-脱氧-6-O-(对甲苯磺酰基)-1-硒-α-D-吡喃葡萄糖(15*)的合成
反应方程式如图22所示；
将化合物14*(7.9g,16.6mmol)溶解于无水吡啶(110mL)，加入对甲苯磺酰氯(8.0g,42mmol)后，将反应液在室温下搅拌过夜。当TLC显示原料已完全转化后，对反应液进行减压蒸馏浓缩，以二氯甲烷稀释后，分别经饱和碳酸氢钠溶液、水萃取。经过无水硫酸钠除水后，减压蒸馏除去溶剂，所得粗品以硅胶柱层析纯化(石油醚：乙酸乙酯，5:1，v/v)得到黄色糖浆状的产品15*(9.06g,14.37mmol,87％)。[α] D 20＝+123.2°(c＝1.00,CHCl 3)；IR ν max(film)2107,1749,1363,1214,1176,1094,977,939,815,738,681cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.83-7.14(m,15H,3Ph),5.76(d,J＝5.4Hz,1H,1-H),5.38-5.22(m,1H,3-H),4.53(s,2H,PhCH 2),4.38-4.21(m,2H,5-H,6-CH a),4.09-3.97(m,1H,6-CH b),3.85(dd,J＝10.3,5.4Hz,1H,2-H),3.65(t,J＝9.1Hz,1H,4-H),2.42(s,3H,PhCH 3),2.05(s,3H,CH 3CO)； 13C NMR(100MHz,CDCl 3)δ＝169.5,145.0,137.0,134.5,132.6,129.8,129.2,128.6,128.2,128.12,128.05,128.0,127.9,84.1,75.3,74.9,74.1,71.3,67.8,62.7,21.7,20.8；HR-ESI-MS(m/z):calcd for C 28H 29N 3O 7SSeNa +(M+Na +):654.0789,found:654.0795。
实施例16
苯基4-O-苄基-3-O-乙酰基-2-叠氮基-2,6-二脱氧-6-碘-1-硒-α-D-吡喃葡萄糖(16*)的合成
反应方程式如图22所示；
化合物15*(400mg,0.64mmol)与碘化钠(480mg,3.2mmol)加入正丁酮(8mL)中，在80℃下回流6小时。冷却至室温后，加入乙酸乙酯，经1M硫代硫酸钠溶液和水萃取，所得有机相以无水硫酸钠处理除水。减压蒸馏除去溶剂所得粗品经硅胶柱层析纯化(正己烷：乙酸乙酯，10:1，v/v)得到黄色浆状的产品16*(340mg,0.58mmol,91％)。[α] D 20＝+170.3°(c＝1.20,CHCl 3)；IR ν max(film)2928,2108,1746,1367,1226,1091,1044,739,694cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.40(m,10H,2Ph),5.89(d,J＝5.3Hz,1H,1-H),5.41(t,J＝9.7Hz,1H,3-H),4.71(q,J＝11.0Hz,2H,PhCH 2),3.94(dd,J＝10.3,5.4Hz,1H,2-H),3.87(dt,J＝9.1,3.2Hz,1H,5-H),3.59(t,J＝9.2Hz,1H,4-H),3.48(dd,J＝11.1,3.9Hz,1H,6-CH a),3.32(dd,J＝11.1,2.8Hz,1H,6-CH b),2.06(s,3H,CH 3CO)； 13C NMR(100MHz,CDCl 3)δ＝169.6,137.2,134.6,129.3,128.7,128.2,128.1,128.0,127.9,84.0,79.9,75.1,73.8,71.3,62.9,20.9,7.3；HR-ESI-MS(m/z):calcd for C 21H 22IN 3O 4SeNa +(M+Na +):609.9718,found:609.9698。
实施例17
苯基4-O-苄基-3-O-乙酰基-2-叠氮基-2-脱氧-1-硒-α-D-吡喃奎诺糖(17*)的合成
反应方程式如图22所示；
化合物16*(150mg,0.26mmol)溶解于无水DMF(3.5mL)中，加入氰基硼氢化钠(82mg,1.30mmol)后将反应液加热至95℃搅拌过夜。待反应结束后，将反应液降至室温，倾倒入水中，以乙酸乙酯萃取两次。有机相经水洗后，以无水硫酸钠处理除水，浓缩得到粗品经硅胶柱层析纯化(甲苯)得到白色固体状的产品17*(84.4mg,0.18mmol,71％)。[α] D 20＝+201.3°(c＝1.00,CHCl 3)；IR ν max(film)2925,2108,1752,1367,1223,1083,735,692cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.63-7.22(m,10H,2Ph),5.81(d,J＝5.4Hz,1H,1-H),5.34(dd,J＝10.3,9.1Hz,1H,3-H),4.71-4.51(m,2H,PhCH 2),4.26(dq,J＝9.6,6.2Hz,1H,5-H),3.91(dd,J＝10.3,5.4Hz,1H,2-H),3.26(t,J＝9.4Hz,1H,4-H),2.04(s,3H,CH 3CO),1.26(d,J＝6.2Hz,3H,6-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝169.6,137.5,134.7,129.1,128.5,128.4,128.03,127.97,127.9,84.1,81.8,74.8,74.1,69.9,63.3,20.9,17.6；HR-ESI-MS(m/z):calcd for C 21H 23N 3O 4SeNa +(M+Na +):484.0751,found:484.0755。
实施例18
4-O-苄基-3-O-乙酰基-2-叠氮基-2-脱氧-D-吡喃奎诺糖(18*)的合成
反应方程式如图22所示；
将化合物17*(4.81g,10.5mmol)溶解于THF/水混合液(25mL，1:1，v/v)中，加入溴代丁二酰亚胺(4.45g,25.0mmol)后，反应液在室温下搅拌6小时。待反应结束后，以二氯甲烷稀释反应液，经10％Na 2S 2O 3/1M NaHCO 3混合液(1:1，v/v)萃取有机相。减压蒸馏除去溶剂后，粗品经硅胶柱层析纯化(正己烷：乙酸乙酯，5:1，v/v)得到无色浆状的产物18*(3.31g,10.0mmol,99％)。[α] D 20＝+33.9°(c＝1.00,CHCl 3)；IR ν max(film)3402,2934,2111,1751,1363,1227,1077,752,700cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.40-7.18(m,10H,Ph),5.57(dd,J＝10.3,9.4Hz,1H,3-H),5.30(d,J＝3.3Hz,1H,1-H),5.06(dd,J＝10.2,9.4Hz,1H,3-H),4.69(d,J＝8.0Hz,1H,1-H),4.66-4.54(m,4H,PhCH 2),4.13(dq,J＝12.5,6.2Hz,1H,5′-H),3.76(s,1H,1-OH),3.52(dq,J＝12.4,6.2Hz,1H,5-H),3.35(dd,J＝10.3,8.0Hz,1H,2-H),3.30-3.05(m,4H,2-H,4-H,4′-H,1-OH),2.05(s,6H,CH 3CO),1.34(d,J＝6.2Hz,3H,6-CH 3),1.29(d,J＝6.3Hz,3H,6′-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝170.01,169.95,137.6,137.4,128.52,128.49,128.0,127.93,127.88,127.8,95.8,92.2,82.1,81.4,75.0,74.8,73.9,71.8,71.6,66.9,65.6,62.3,20.9,17.8；HR-ESI-MS(m/z):calcd for C 15H 19N 3O 5Na +(M+Na +):344.1222,found:344.1224。
实施例19
4-O-苄基-3-O-乙酰基-2-叠氮-2-脱氧-D-吡喃奎诺糖三氯乙酰亚氨酯(19*)的合成
反应方程式如图22所示；
在氮气保护下，向化合物18*(11mg,0.034mmol)的无水二氯甲烷(0.4mL)溶液中，加入三氯乙腈(34μL,0.34mmol)和1,8-二氮杂二环十一碳-7-烯(DBU)(0.6μL,0.004mmol)。所得反应液在室温下搅拌5小时，当反应结束后，低温下减压蒸馏除去溶剂，所得粗品经硅胶柱层析纯化(正己烷：乙酸乙酯，7:3，v/v，含0.5％三乙胺)得到产品19*(13.6mg,0.029mmol,86％)。 1H NMR(400MHz,CDCl 3)δ＝9.49(s,1H,NH),7.46-7.23(m,5H,Ph),6.48(d,J＝3.5Hz,1H,1-H),5.62-5.42(m,1H,3-H),4.72(s,2H,PhCH2),4.04(dq,J＝12.5,6.2Hz,1H,5-H),3.93(dd,J＝10.7,3.5Hz,1H,2-H),3.49(t,J＝9.5Hz,1H,4-H),2.08(s,3H,CH 3CO),1.29(d,J＝6.2Hz,3H,6-CH 3)。
实施例20
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-3-O-乙酰基-2-叠氮基-2-脱氧-D-吡喃奎诺糖(20*)的合成
反应方程式如图22所示；
在氮气保护下，三氯乙酰亚氨酯糖供体19*(2.48g,5.325mmol)和N-苄基-N-苄氧羰基-3-氨基丙-1-醇(H.Ishida et al.,Org.Biomol.Chem.2015,13,7762-7771)(1.91g,6.390mmol)溶解于无水乙醚/无水二氯甲烷混合液(130mL，3:1，v/v)，加入活化的分子筛(Aw-300型)搅拌30分钟。将反应液降温至-40℃后，缓慢滴加三氟甲烷磺酸三甲基硅酯(1.16mL,6.390mmol)，保持反应液在此温度下搅拌至反应结束。待反应结束后滴加三乙胺淬灭反应，过滤除去分子筛后，减压蒸馏除去溶剂。所得粗品经硅胶柱层析纯化(石油醚：乙酸乙酯，15:1，v/v)后得到产物20*(3.1g,5.14mmol,96％,α:β＝3:1)。IR ν max(film)2919,2107,1749,1696,1454,1421,1361,1222,1044,735,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.44-7.13(m,20H,α-3Ph,β-3Ph),5.49(dd,J＝10.6,9.0Hz,1H,α3-H),5.18(d,J＝8.7Hz,2.6H,α-PhCH 2,β-PhCH 2),4.98(s,0.3H,β3-H),4.82(s,1H,α1-H),4.61(m,2.6H,α-PhCH 2,β-PhCH 2),4.57-4.43(m,2.6H,α-PhCH 2,β-PhCH 2),4.29(d,J＝8.2Hz,0.3H,β1-H),3.98-3.52(m,2.4H,linker-OCH a,α5-H),3.50-3.26(m,4.7H,linker-NCH 2,linker-OCH b,β5-H,β2-H),3.20(t,J＝9.3Hz,1.3H,α4-H,β4-H),3.06(d,J＝10.4Hz,1H,α2-H),2.05(s,3H,α-CH 3CO),2.04(s,1H,β-CH 3CO),1.86(d,J＝27.9Hz,2.7H,linker-CH 2),1.30(d,J＝6.2Hz,1H,β6-CH 3),1.26(d,J＝6.1Hz,3H,α6-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝169.9,169.8,163.5,156.7,156.2,137.9,137.8,137.7,137.5,136.8,128.6,128.54,128.52,128.49,128.0,127.93,127.9,127.8,127.4,101.6,97.9,91.9,82.3,81.6,77.2,74.9,73.9,72.0,71.4,67.3,66.9,66.0,65.8,64.6,61.6,50.8,44.8,43.9,28.3,27.9, 20.9,17.8；HR-ESI-MS(m/z):calcd for C 33H 38N 4O 7Na +(M+Na +):625.2638,found:625.2629。
实施例21
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-3-O-乙酰基-2-乙酰氨基-2-脱氧-α-D-吡喃奎诺糖(21*)的合成
反应方程式如图22所示；
在0℃下，向化合物20*(15.0mg,0.025mmol)的无水吡啶(0.42mL)溶液中加入硫代乙酸(0.42mL)，将反应液置于室温下搅拌过夜。待反应结束后，将反应液与甲苯共沸除去溶剂，所得粗品经硅胶柱层析纯化(石油醚：丙酮，6:1，v/v)得到无色浆状产品21*(11.8mg,0.019mmol,76％)。[α] D 20＝+53.1°(c＝1.00,CHCl 3)；IR ν max(film)3343,2936,1745,1696,1455,1423,1366,1233,1120,1048,737,700cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.48-7.01(m,15H,3Ph),6.51(d,J＝8.9Hz,1H,NH),5.37-5.01(m,3H,3-H,PhCH 2),4.80-4.31(m,5H,1-H,2PhCH 2),4.24(m,1H,2-H),3.85-3.51(m,3H,5-H,linker-2H),3.43-3.13(m,3H,4-H,linker-2H),1.99(s,3H,CH 3CO),1.96(s,3H,CH 3CO),1.75(m,2H,linker-CH 2),1.27(d,J＝6.2Hz,3H,6-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.1,156.3,137.9,137.6,136.6,128.7,128.5,128.1,127.9,127.8,127.5,127.3,97.2,81.9,75.1,74.0,67.4,67.1,64.1,52.3,49.9,43.2,27.3,23.1,21.0,17.9；HR-ESI-MS(m/z):calcd for C 35H 42N 2O 8Na +(M+Na +):641.2839,found:641.2828。
实施例22
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-2-脱氧-α-D-吡喃奎诺糖(22*)的合成
反应方程式如图22所示；
向化合物21*(7mg,0.012mmol)的甲醇(0.5mL)溶液中加入甲醇钠(0.3mg,0.006mmol)，所得反应液在室温下搅拌。待反应结束，以Amberlite IR 120阳离子交换树脂中和反应液，经过滤所得有机相在减压下蒸馏除去。所得粗品经硅胶柱层析纯化(石油醚：丙酮，4:1，v/v)得到无色浆状产物22*(6.7mg,0.0116mmol,97％)。[α] D 20＝+27.9°(c＝1.10,CHCl 3)；IR ν max(film)3325,2938,1695,1544,1424,1369,1235,1120,1070,736,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.51(d,J＝6.9Hz,1H,NH),7.44-7.12(m,15H,3Ph),5.22-5.10(m,2H,PhCH 2),5.02(d,J＝11.0Hz,1H,PhCH a1),4.78-4.63(m,2H,PhCH b1,PhCH a2),4.50(d,J＝3.6Hz,1H,1-H),4.26(d,J＝15.9Hz,1H,PhCH b2),4.21(s,1H,3-OH),4.00(m,2H,2-H,linker-1H),3.91(t,J＝9.4Hz,1H,3-H),3.67(m,2H,5-H,linker-1H),3.21-3.07(m,2H,4-H,linker-1H),3.00(dt,J＝14.2,4.5Hz,1H,linker-1H),2.12(s,3H,CH 3CO),1.70(dq,J＝9.6,5.4Hz,2H,linker-CH 2),1.25(d,J＝6.2Hz,3H,6-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝174.1,156.7,138.7, 137.3,136.5,128.7,128.5,128.4,128.3,128.2,127.7,127.6,127.3,96.9,84.7,77.3,76.0,75.1,67.5,66.5,62.9,55.3,49.4,42.3,26.9,22.6,17.9；HR-ESI-MS(m/z):calcd for C 33H 40N 2O 7Na +(M+Na +):599.2733,found:599.2750。
实施例23
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(3,4-二-O-乙酰基-2-叠氮基-2-脱氧-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(23*)的合成
反应方程式如图23所示；
在氮气保护下，三氟亚胺酯糖供体13*(159mg,0.358mmol)和受体22*(207mg,0.358mmol)溶解于无水二氯甲烷/无水乙醚混合液(12mL,1/3,v/v)中，加入噻吩(344μL,4.296mmol)和活化的分子筛(AW-300型)后，所得反应液在室温下搅拌30分钟。待降温至-40℃后，加入三氟甲磺酸三甲基硅酯(6.5μL,0.036mmol)，其后逐渐升高反应温度至室温。待TLC确认反应结束后，在0℃下滴加三乙胺淬灭反应，硅藻土过滤除去分子筛。有机相经饱和碳酸氢钠溶液萃取后，浓缩得到的粗品经硅胶柱层析纯化(石油醚：丙酮，6:1-3:1，v/v)得到目标二糖23*(262mg,0.315mmol,88％,α:β＝10:1)。[α] D 20＝-178.6°(c＝0.90,CHCl 3)；IR ν max(film)3337,2936,2112,1749,1680,1371,1233,1044,975,751,700cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.48-7.08(m,15H,3Ph),6.79(d,J＝9.3Hz,1H,NH),5.40-5.30(m,2H,1′-H,3′-H),5.14(s,2H,PhCH 2),5.01(s,1H,4′-H),4.80-4.69(m,2H,PhCH 2),4.60(m,2H,1-H,NCH aPh),4.40(m,2H,NCH bPh,2-H),4.30-4.18(m,1H,5′-H),3.96(dd,J＝19.8,9.9Hz,1H,3-H),3.80(dd,J＝9.1,6.3Hz,1H,5-H),3.70-3.62(m,2H,OCH aCCH aN),3.58(dd,J＝11.4,2.9Hz,1H,2′-H),3.29(dd,J＝10.3,4.7Hz,2H,OCH bCCH bN),3.21(t,J＝9.3Hz,1H,4-H),2.11(s,3H,CH 3CO),2.06(s,3H,CH 3CO),2.03(s,3H,CH 3CO),1.74(s,2H,OCCH 2CN),1.34(d,J＝6.2Hz,3H,6-CH 3),0.71(d,J＝6.4Hz,3H,6′-CH 3).； 13C NMR(100MHz,CDCl 3)δ＝170.4,169.8,156.4,137.7,137.5,136.5,128.7,128.6,128.5,128.1,128.0,127.8,127.7,127.5,127.2,97.8,97.5,83.6,75.7,75.5,70.7,68.4,67.6,67.4,64.9,63.9,57.8,53.4,49.8,43.0,27.3,23.1,20.8,20.7,18.2,15.5；HR-ESI-MS(m/z):calcd for C 43H 53N 5O 12Na +(M+Na +):854.3588,found:854.3582。
实施例24
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(3,4-二-O-乙酰基-2-氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(24*)的合成
反应方程式如图23所示；
化合物23*(253mg,0.304mmol)和三苯基膦(104mg,0.395mmol)溶解于5mL四氢呋喃中，反应液在40℃下搅拌，待TLC确认原料反应完全后，向反应液中加入水(66μL,3.65mmol)。加热反应至65℃回流，当反应结束后，减压蒸馏除去溶剂，所得粗品经硅胶柱层析纯化(二氯甲烷：甲醇，40:1，v/v)得到无色浆状的氨基化合物24*(224mg,0.278mmol,91％)。[α] D 20＝-55.0°(c＝1.10,CHCl 3)；IR ν max(film)3332,2937,1744,1676,1424,1369,1225,1132,1072,1029,972,750,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.49-7.12(m,15H,3Ph),7.08(d,J＝9.1Hz,1H,NH),5.25-5.07(m,3H,1′-H,PhCH 2),4.99(dd,J＝11.0,3.0Hz,1H,3′-H),4.93(s,1H,4′-H),4.83-4.66(m,2H,PhCH 2),4.60(d,J＝15.8Hz,1H,NCH aPh),4.53(d,J＝2.9Hz,1H,1-H),4.49-4.28(m,2H,NCH bPh,2-H),4.27-4.10(m,1H,5′-H),4.00(t,J＝9.7Hz,1H,3-H),3.82(dd,J＝9.0,6.3Hz,1H,5-H),3.68(ddd,J＝15.0,12.0,6.1Hz,2H,OCH aCCH aN),3.30(d,J＝7.7Hz,2H,OCH bCCH bN),3.23-3.09(m,2H,4-H,2′-H),2.07(s,3H,CH 3CO),2.03(s,6H,2CH 3CO),1.74(s,2H,OCCH 2CN),1.59(s,2H,NH 2),1.35(d,J＝6.1Hz,3H,6-CH 3),0.61(d,J＝6.3Hz,3H,6′-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝170.5,137.7,137.4,128.6,128.5,128.0,127.8,127.7,127.5,127.2,99.6,97.6,83.3,77.2,75.6,75.0,71.8,70.7,67.7,67.4,64.8,63.6,53.7,49.7,49.2,42.9,27.2,23.3,20.9,20.6,18.2,15.6；HR-ESI-MS(m/z):calcd for C 43H 55N 3O 12Na +(M+Na +):828.3683,found:828.3733。
实施例25
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(3,4-二-O-乙酰基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(25*)的合成
反应方程式如图23所示；
化合物24*(224mg,0.278mmol)溶解于无水吡啶(7mL)，在0℃下，缓慢逐滴加入2,2,2-三氯乙氧羰酰氯(96μL,0.695mmol)。将反应液置于室温搅拌，待反应结束后，加入3.5mL甲醇淬灭反应。减压蒸馏除去溶剂，所得粗品溶解于二氯甲烷中，经水萃取后，有机相以无水硫酸钠除水并浓缩。粗品经硅胶柱纯化(石油醚：丙酮，7:1，v/v)得到无色浆状产物25*(239mg,0.244mmol,88％)。[α] D 20＝-34.1°(c＝1.00,CHCl 3)；IR ν max(film)3322,2938,1744,1679,1522,1425,1369,1225,1077,1045,740,700cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.48-7.07(m,15H,3Ph),6.89(d,J＝8.9Hz,1H,NH),5.45(d,J＝9.2Hz,1H,N’H),5.21(d,J＝2.8Hz,1H,1′-H),5.13(m,3H,3′-H,PhCH 2),5.00(d,J＝11.9Hz,1H,Troc-CH a),4.81(m,2H,4′-H,PhCH a),4.59(m,3H,PhCH a,PhCH b,Troc-CH b),4.45(s,1H,1-H),4.43-4.28(m,2H,2-H,PhCH b),4.27-4.07(m,2H,2′-H,5′-H),3.96(t,J＝9.5Hz,1H,3-H),3.87-3.68(m,2H,5-H,OCCCH aN),3.69-3.54(m,1H,OCH aCCN),3.19(m,3H,OCH bCCH bN,4-H),2.09(s,3H,CH 3CO), 1.96(s,6H,2CH 3CO),1.72(m,2H,OCCH 2CN),1.35(d,J＝6.1Hz,3H,6-CH 3),0.58(d,J＝6.3Hz,3H,6′-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.0,170.5,156.4,154.9,137.5,137.4,136.4,128.7,128.53,128.46,128.1,128.0,127.7,127.4,127.2,97.7,97.2,83.3,77.2,75.8,74.7,74.5,70.5,68.9,67.8,67.4,64.9,63.5,53.3,49.7,49.4,42.8,27.1,23.0,20.73,20.65,18.2,15.4；HR-ESI-MS(m/z):calcd for C 46H 56Cl 3N 3O 14Na +(M+Na +):1002.2726,found:1002.2787。
实施例26
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(26*)的合成
反应方程式如图23所示；
向化合物25*(239mg,0.244mmol)的甲醇(8mL)溶液中加入甲醇钠(6.6mg,0.122mmol)，室温下搅拌至反应结束，以Amberlite IR 120阳离子交换树脂中和反应液，所得粗品经硅胶柱层析纯化(石油醚：丙酮，4:1，v/v)得到白色固体状产品26*(181mg,0.202mmol,83％)。[α] D 20＝+6.4°(c＝1.00,CHCl 3)；IR ν max(film)3331,2934,1679,1533,1456,1225,1042,818,737,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.40-7.14(m,15H,3Ph),7.12(d,J＝9.1Hz,1H,NH),6.19(d,J＝6.7Hz,1H,N’H),5.13(m,3H,1′-H,PhCH 2),4.77(m,3H,Troc-CH 2,PhCH a),4.60(m,2H,PhCH a,PhCH b),4.44(s,1H,1-H),4.35(m,2H,2-H,PhCH b),4.15-4.01(m,2H,5′-H,3′-OH),3.96(m,2H,3-H,2′-H),3.83(m,3H,OCCCH aN,5-H,3′-H),3.69-3.55(m,1H,OCH aCCN),3.36(s,1H,4′-H),3.33-3.05(m,3H,OCH bCCH bN,4-H),2.60(s,1H,4′-OH),2.00(s,3H,CH 3CO),1.72(m,2H,OCCH 2CN),1.32(d,J＝6.1Hz,3H,6-CH 3),0.82(d,J＝6.4Hz,3H,6′-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝172.0,157.5,156.5,137.7,137.3,136.4,128.7,128.5,128.4,128.1,127.8,127.7,127.5,127.2,127.1,97.7,96.9,95.5,83.2,77.2,75.4,74.8,74.7,71.5,71.0,67.8,67.4,65.6,63.3,53.6,51.8,49.6,42.6,27.0,23.0,18.1,15.7；HR-ESI-MS(m/z):calcd for C 42H 52Cl 3N 3O 12Na +(M+Na +):918.2514,found:918.2558。
实施例27
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(3-O-萘亚甲基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(27*)的合成
反应方程式如图23所示；
将化合物26*(200mg,0.223mmol)与甲苯共沸除水后抽真空半小时。在氮气保护下，溶解于3.2mL无水甲苯，加入二丁基氧化锡(83mg,0.335mmol)和活化的
Figure PCTCN2018082660-appb-000011
分子筛。反应液加热回流1小时后，降至室温加入萘亚甲基溴(148mg,0.669mmol)和四丁基溴化铵(108 mg,0.335mmol)，反应液加热至60℃搅拌3小时。待反应结束，将反应液过滤、浓缩，所得粗品经硅胶柱层析纯化(石油醚：丙酮，7:1，v/v)得到白色固体状的产物27*(212mg,0.204mmol,92％)。[α] D 20＝-14.2°(c＝1.00,CHCl 3)；IR ν max(film)3013,2932,1735,1670,1515,1454,1215,1091,1053cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.93-6.95(m,22H,Ar-22H),6.88(d,J＝9.5Hz,1H,2-NH),5.43(d,J＝9.7Hz,1H,2’-NH),5.27-5.09(m,3H,1'-H,CH 2),5.05(d,J＝12.1Hz,1H,CH 2-1H),4.86-4.69(m,2H,CH 2),4.65-4.51(m,3H,NCH aPh,CH 2),4.45(d,J＝3.5Hz,1H,1-H),4.34(m,3H,NCH bPh,CH 2-1H,2-H),4.20(ddd,J＝10.2,10.2,3.6Hz,1H,2'-H),4.01-3.84(m,2H,5'-H,3-H),3.83-3.70(m,2H,5-H,linker-1H),3.69-3.60(m,1H,linker-1H),3.57(dd,J＝10.7,3.0Hz,1H,3'-H),3.43(s,1H,4'-H),3.26(d,J＝9.1Hz,1H,linker-1H),3.20-3.10(m,1H,linker-1H),3.04(t,J＝9.2Hz,1H,4-H),2.38(s,1H,4'-OH),1.91(s,3H,CH 3CO),1.71(t,J＝6.2Hz,2H,linker-CH 2),1.27(d,J＝6.9Hz,3H,6-CH 3),0.85(d,J＝6.5Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.2,156.4,155.0,137.6,137.4,136.4,135.1,133.1,128.7,128.5,128.3,128.1,127.9,127.8,127.7,127.5,127.2,127.0,126.7,126.4,126.2,125.7,97.9,97.7,95.8,83.4,77.2,76.1,75.2,75.0,74.6,71.8,68.8,67.7,67.4,65.7,63.5,53.4,50.1,49.7,42.7,27.1,23.1,18.1,15.8；HR-ESI-MS(m/z):calcd for C 53H 60Cl 3N 3O 12Na +(M+Na +):1058.3140,found:1058.3133。
实施例28
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-萘亚甲基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(28*)的合成
反应方程式如图23所示；
在氮气保护下，三氟乙酰亚胺酯糖供体11*(69mg,0.096mmol)和二糖受体27*(20mg,0.019mmol)溶解于3.2mL无水二氯甲烷，加入活化的分子筛(Aw-300型)后，在室温下搅拌30分钟。在室温下加入三氟甲烷磺酸三甲基硅酯(1.8μL,0.01mmol)后，继续室温下搅拌反应液。反应结束后，在0℃下加入4滴吡啶淬灭反应，过滤所得有机相经饱和碳酸氢钠溶液萃取后减压蒸馏除去溶剂。粗品经硅胶柱层析纯化(石油醚：丙酮，6:1，v/v)得到白色固体状的目标三糖28*(24.6mg,0.016mmol,82％,β-only)。[α] D 20＝-22.9°(c＝1.00,CHCl 3)；IR ν max(film)3016,2107,1743,1676,1516,1454,1265,1216,1092,1047,752,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.91-7.07(m,33H,2”-NH,Ar-32H),6.81(d,J＝9.4Hz,1H,2-NH),5.37(d,J＝9.9Hz,1H,2’-NH),5.22-5.05(m,6H,2CH 2,1'-H,1”-H),5.01(d,J＝12.0Hz,1H,CH 2-1H),4.78(d,J＝11.2Hz,1H,CH 2-1H),4.70-4.52(m,6H,5CH 2-1H,3”-H),4.51-4.40(m, 3H,2CH 2-1H,1-H),4.40-4.26(m,2H,CH 2-1H,2-H),4.20(ddd,J＝10.9,10.6,3.5Hz,1H,2'-H),4.04(d,J＝9.7Hz,1H,5”-H),3.97(t,J＝6.2Hz,1H,5'-H),3.88(t,J＝9.7Hz,1H,3-H),3.80(s,1H,4'-H),3.78-3.67(m,2H,linker-1H,5-H),3.66-3.54(m,2H,linker-1H,3'-H),3.50(t,J＝9.4Hz,1H,4”-H),3.34-3.11(m,3H,linker-2H,2”-H),3.06(t,J＝9.3Hz,1H,4-H),1.98(s,3H,CH 3CO),1.74(s,2H,linker-CH 2),1.30-1.18(d,J＝5.3Hz,3H,6-CH 3),0.80(d,J＝6.6Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.2,167.7,162.4,156.4,154.6,137.7,137.4,137.2,136.4,134.8,134.3,133.3,133.2,128.8,128.7,128.64,128.62,128.5,128.47,128.4,128.1,128.0,127.8,127.51,127.46,127.3,127.2,126.32,126.27,126.2,100.0,97.8,97.6,97.0,95.9,91.9,83.7,78.7,77.2,75.6,75.2,74.7,74.6,73.1,72.5,67.6,67.49,67.45,66.8,63.6,61.9,57.8,53.3,50.9,49.8,42.9,27.2,23.2,18.2,16.6；HR-ESI-MS(m/z):calcd for C 75H 79Cl 6N 7O 17Na +(M+Na +):1584.3532,found:1584.3595。
实施例29
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(29*)的合成
反应方程式如图23所示；
化合物28*(1.15g,0.734mmol)溶解于二氯甲烷(6.6mL)和水(2.6mL)混合液中，加入2,3-二氯-5,6-二氰对苯醌(2,3-dichloro-5,6-dicyano-1,4-benzoquinone，DDQ)(246mg,1.101mmol)后，反应液在室温下搅拌5小时。分离得到有机相经5％硫代硫酸钠溶液萃取后，浓缩得到的粗品经硅胶柱层析(石油醚：丙酮，7:1-6:1，v/v)得到化合物29*(0.89g,0.625mmol,85％)。[α] D 20＝-6.5°(c＝1.00,CHCl 3)；IR ν max(film)3317,2934,2107,1704,1517,1216,1039,826,751,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.44-7.12(m,26H,5Ph,2”-NH),6.90(d,J＝9.4Hz,1H,2-NH),5.69(d,J＝8.6Hz,1H,2’-NH),5.23(q,J＝12.2Hz,2H,CH 2),5.18-5.03(m,4H,CH 2,1'-H,1”-H),4.80-4.65(m,3H,CH 2,CH a1),4.67-4.50(m,4H,CH 2,CH a2,CH b1),4.46(d,J＝3.5Hz,1H,1-H),4.41-4.29(m,3H,CH b2,3”-H,2-H),4.09(d,J＝9.6Hz,1H,5”-H),4.06(d,J＝6.8Hz,1H,5'-H),4.01-3.84(m,2H,2'-H,3-H),3.83-3.57(m,5H,5-H,3'-H,linker-2H,4”-H),3.47(s,1H,3'-OH),3.41(s,1H,4'-H),3.23(m,3H,linker-2H,2”-H),3.12(t,J＝9.3Hz,1H,4-H),2.00(s,3H,CH 3CO),1.73(s,2H,linker-CH 2),1.31(d,J＝6.2Hz,3H,6-CH 3),0.70(d,J＝6.4Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.5,167.1,162.0,156.4,155.8,137.7,137.4,136.9,136.4,134.8,128.71,128.67,128.6,128.53,128.47,128.4,128.2,128.1,127.9,127.8,127.5,127.2,127.1,99.6,97.7,95.7,91.8,83.4,83.0,78.5,77.3,75.4,75.1,74.9,74.7,74.6, 69.4,67.9,67.5,66.5,63.6,62.7,58.1,53.4,52.2,49.7,42.8,27.1,23.1,18.2,16.1；HR-ESI-MS(m/z):calcd for C 64H 71Cl 6N 7O 17Na +(M+Na +):1444.2906,found:1444.2916。
实施例30
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O[4-O-苄基-3-叠氮基-2-三氯乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-乙酰基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(30*)的合成
反应方程式如图23所示；
化合物29*(828mg,0.582mmol)溶解于无水二氯甲烷/吡啶混合液(30mL，4:1，v/v)，降至0℃后逐滴加入醋酸酐(550μL,5.821mmol)，待加入二甲基氨基吡啶(1.4mg,0.012mmol)后，反应液在室温下搅拌。待反应结束后，以饱和碳酸氢钠溶液萃取反应液，有机相经无水硫酸钠除水后，减压蒸馏除去溶剂。粗品经硅胶柱层析纯化(石油醚：丙酮，6:1，v/v)后得到白色固体状化合物30*(798mg,0.544mmol,94％)。[α] D 20＝-37.0°(c＝1.00,CHCl 3)；IR ν max(film)3337,2964,2108,1744,1521,1456,1367,1260,1074,1021,800,752,697cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.42-7.11(m,26H,5Ph,2”-NH),6.78(d,J＝9.5Hz,1H,2-NH),5.43(d,J＝9.9Hz,1H,2'-NH),5.23-5.07(m,5H,1'-H,PhCH 2-4H),5.01(d,J＝12.0Hz,1H,CH 2-1H),4.94(dd,J＝11.8,2.7Hz,1H,3'-H),4.89(d,J＝8.1Hz,1H,1”-H),4.78(d,J＝10.8Hz,1H,PhCH 2),4.68(d,J＝10.7Hz,1H,CH 2-1H),4.64-4.48(m,4H,CH 2-4H),4.47-4.42(m,2H,1-H,3”-H),4.37(m,2H,2-H,NCH 2Ph),4.21(ddd,J＝11.4,9.8,3.5Hz,1H,2'-H),4.04(t,J＝6.5Hz,1H,5'-H),3.92(m,2H,3-H,5”-H),3.83-3.54(m,5H,5-H,4”-H,4'-H,linker-2H),3.34-3.08(m,4H,4-H,2”-H,linker-2H),1.97(s,3H,CH 3CO),1.96(s,3H,CH 3CO),1.73(m,2H,linker-2H),1.33(d,J＝6.2Hz,3H,6-CH 3),0.61(d,J＝6.7Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝170.9,167.6,162.0,156.4,154.8,137.7,137.4,137.0,136.4,134.7,128.8,128.7,128.53,128.46,128.4,128.0,127.9,127.8,127.5,127.3,98.7,97.6,97.4,95.8,91.9,83.4,79.0,77.2,75.6,75.0,74.9,74.5,67.8,67.76,67.5,65.7,62.3,58.4,53.4,49.8,49.2,42.9,27.1,23.1,20.9,18.2,15.8；HR-ESI-MS(m/z):calcd for C 66H 74Cl 6N 7O 18 +(M+H +):1464.3192,found:1464.3281。
实施例31
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-氨基-2-二氯乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-乙酰基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(31*)的合成
反应方程式如图23所示；
化合物30*(745mg,0.509mmol)溶解于吡啶(51mL)，加入水(4.76mL,265mmol)，三乙胺(1.06mL,7.632mmol)和1,3-丙二硫醇(1mL,10.176mmol)后，反应液在室温下搅拌6小时。将反应液浓缩后所得粗品经硅胶柱层析纯化(石油醚：丙酮，5:1-3:1，v/v)得到无色浆状化合物31*(460mg,0.327mmol,64％)。[α] D 20＝-63.1°(c＝1.00,CHCl 3)；IR ν max(film)3322,2940,1746,1682,1526,1454,1363,1242,1074,738,699cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.51-7.09(m,25H,5Ph),6.81(d,J＝9.3Hz,1H,2-NH),5.90(s,1H,CHCl 2),5.47(s,1H,2’-NH),5.30-5.05(m,5H,2CH 2,1'-H),4.98(m,2H,3'-H,CH 2-1H),4.77(d,J＝10.9Hz,1H,CH 2-1H),4.71(s,1H,1”-H),4.67-4.49(m,5H,NCH aPh,CH 2,2CH 2-1H),4.45(d,J＝4.0Hz,1H,1-H),4.41-4.30(m,2H,NCH bPh,2-H),4.29-4.16(m,1H,2'-H),4.14-3.98(m,1H,5'-H),3.91(m,2H,5”-H,3-H),3.78(q,J＝7.5,7.0Hz,1H,5-H),3.83-3.56(m,4H,4'-H,4”-H,linker-2H),3.47(s,1H,2”-H),3.36-3.06(m,3H,linker-2H,4-H),1.96(s,6H,2CH 3CO),1.73(m,2H,linker-CH 2),1.33(d,J＝6.1Hz,3H,6-CH 3),0.63(d,J＝6.5Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.1,168.3,156.4,137.9,137.5,137.4,136.5,134.8,128.74,128.68,128.5,128.4,128.1,127.9,127.8,127.5,127.3,100.2,97.6,95.8,83.3,77.2,75.6,74.5,74.4,69.9,67.8,67.6,67.5,66.5,66.0,63.7,54.2,53.4,49.8,49.1,27.2,23.1,20.9,18.2,15.9；HR-ESI-MS(m/z):calcd for C 66H 77Cl 5N 5O 18 +(M+H +):1404.3677,found:1404.3643。
实施例32
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-N-(R)-3-O-苄基丁酰基-2-二氯乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-乙酰基-2-(2,2,2-三氯乙氧羰基)氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(32*)的合成
反应方程式如图23所示；
(R)-3-O-苄基丁酸(D.Seebach et al.Helv.Chim.Acta 1988,71,155-167)(234mg,1.205mmol)溶解于23mL无水二氯甲烷，在0℃下，加入草酰氯(0.8mL,9.156mmol)，室温下搅拌4小时后，减压蒸馏除去溶剂和剩余试剂，所得(R)-3-O-苄基丁酰氯抽真空3小时。氨基化合物31*(113mg,0.081mmol)溶解于4mL无水二氯甲烷，加入三乙胺(167μL,1.207mmol)和(R)-3-O-苄基丁酰氯的无水二氯甲烷溶液(4mL)后，反应液在室温下搅拌过夜。待反应结束后，在0℃下加入0.3mL甲醇淬灭反应，浓缩所得粗品经硅胶柱层析(石油醚：丙酮，5:1，v/v)得到无色浆状化合物32*(92mg,0.058mmol,72％)。[α] D 20＝-47.1°(c＝1.00,CHCl 3)；IR ν max(film)3281,2935,1744,1661,1532,1454,1246,1045,737,698cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.54(d,J＝5.1Hz,1H,2-NH),7.46-6.88(m,30H,6Ph),6.76(d,J＝9.6Hz,1H,2”-NH),6.64(d,J＝9.1Hz,1H,3”-NH),5.78(s,1H,DCA-1H),5.40(d,J＝9.9Hz,1H, 2'-NH),5.29-4.94(m,6H,2CH 2,CH 2-1H,1'-H),4.88(d,J＝11.1Hz,1H,3'-H),4.70(s,2H,CH 2),4.65-4.50(m,3H,CH 2-3H),4.50-4.45(m,1H,1”-H),4.44-4.27(m,5H,1-H,2”-H,3”-H,CH 2),4.23(m,2H,2'-H,CH 2-1H),4.02-3.85(m,4H,5”-H,5'-H,3-H,2-H),3.79(m,2H,butyryl-CH,5-H),3.66(m,2H,linker-2H),3.53(t,J＝8.6Hz,1H,4”-H),3.40(s,1H,4'-H),3.26(s,2H,linker-2H),3.14(t,J＝9.2Hz,1H,4-H),2.40-2.12(m,2H,butyryl-CH 2),1.96(s,6H,2CH 3CO),1.72(s,2H,linker-2H),1.28(d,J＝6.3Hz,3H,6-CH 3),1.19(d,J＝6.2Hz,3H,butyryl-CH 3),0.65(d,J＝6.4Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝171.9,171.2,171.0,168.0,164.4,156.4,154.5,137.9,137.44,137.35,136.5,134.9,128.8,128.71,128.65,128.6,128.5,128.42,128.38,128.1,128.0,127.9,127.8,127.7,127.6,127.4,127.3,102.0,97.6,95.9,83.3,77.3,75.6,75.3,74.5,74.0,71.5,70.4,69.8,67.7,67.5,66.3,66.0,63.7,54.6,53.4,52.5,49.8,49.1,43.6,43.0,27.2,23.2,20.9,19.4,18.2,15.9；HR-ESI-MS(m/z):calcd for C 77H 88Cl 5N 5O 20Na +(M+Na +):1602.4333,found:1602.4312。
实施例33
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-N-(R)-3-O-苄基丁酰基-2-乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-乙酰基-2-氨基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(33*)的合成
反应方程式如图23所示；
向三糖32*(48.3mg,0.031mmol)的醋酸(10mL)溶液中加入过量锌粉，反应液加热至55℃搅拌4小时。反应液经硅藻土过滤后，浓缩得到的粗品经硅胶柱层析纯化(二氯甲烷：甲醇，20:1，v/v)得到化合物33*(33.4mg,0.025mmol,81％)。[α] D 20＝-73.4°(c＝0.50,CHCl 3)；IR ν max(film)3289,2924,1748,1656,1546,1367,1238,1073,739,698cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.45-6.99(m,30H,6Ph),5.13(m,5H,2PhCH 2,1'-H),4.93(m,1H,3'-H),4.72(s,2H,PhCH 2),4.68-4.44(m,4H,NCH aPh,PhCH 2-1H,1-H,3”-H),4.42-4.28(m,4H,PhCH 2,PhCH 2-1H,NCH bPh),4.27-4.07(m,2H,2-H,1”-H),4.01(d,J＝6.6Hz,1H,5'-H),3.90(m,4H,butyryl-CH,2”-H,3-H,5”-H),3.71(m,5H,5-H,4'-H,linker-2H,4”-H),3.50-3.03(m,4H,2'-H,linker-2H,4-H),2.34(d,J＝31.3Hz,2H,butyryl-CH 2),2.16-1.86(m,6H,2CH 3CO),1.82(s,3H,CH 3CO),1.77-1.62(m,2H,linker-CH 2),1.30(d,J＝6.2Hz,3H,6-CH 3),1.19(d,J＝6.1Hz,3H,butyryl-CH 3),0.80-0.48(d,J＝5.6Hz,3H,6'-CH 3)； 13C NMR(100MHz,CDCl 3)δ＝172.3,171.0,168.2,156.4,138.3,138.0,137.5,136.5,134.8,128.7,128.6,128.5,128.4,128.3,128.2,128.1,127.8,127.7,127.5,127.2,97.4,77.2,75.7,75.3,74.1,71.8,70.4,67.5,67.4,66.0,54.2,53.2,49.8, 48.7,43.5,43.1,29.7,27.2,23.3,21.3,19.2,18.1,15.9；HR-ESI-MS(m/z):calcd for C 74H 90N 5O 18 +(M+H +):1336.6281,found:1336.6243。
实施例34
N-苄基-N-苄氧羰基-3-氨基丙基4-O-苄基-2-乙酰氨基-3-O-(4-O-[4-O-苄基-3-N-(R)-3-O-苄基丁酰基-2-乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸苄酯]-3-O-乙酰基-2-乙脒基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(34*)的合成
反应方程式如图23所示；
在氩气保护下，氨基化合物33*(12.5mg,9.35μmol)溶解于2mL无水吡啶，降温至0℃后，加入硫代乙酰亚氨酸苄酯盐酸盐(3.8mg,18.7μmol)。反应液在0℃下搅拌5小时，浓缩所得粗品经硅胶柱层析纯化(二氯甲烷：甲醇，20:1，v/v)得到无色浆状化合物34*(9.0mg,6.53μmol,70％)。[α] D 20＝-72.7°(c＝0.50,CHCl 3)；IR ν max(film)3292,1749,1657,1564,1373,1232,1074,1047,739,698cm -1； 1H NMR(400MHz,CDCl 3)δ＝7.49(d,J＝8.7Hz,2H,2-NH,3”-NH),7.44-7.15(m,30H,6Ph),6.96(s,1H,2'-NH),5.28-5.07(m,4H,2PhCH 2),4.99(m,2H,1'-H,3'-H),4.83(d,J＝10.8Hz,1H,PhCH 2-1H),4.67(d,J＝15.7Hz,1H,PhCH 2-1H),4.61-4.37(m,6H,2PhCH 2,PhCH 2-1H,1-H),4.35-4.11(m,5H,PhCH 2-1H,1”-H,2'-H,2-H,3”-H),4.06(d,J＝6.6Hz,1H,5'-H),3.92(m,6H,linker-1H,butyryl-CH,3-H,2”-H,4”-H,5”-H),3.86-3.77(m,1H,5-H),3.67(m,2H,linker-1H,4'-H),3.17(m,2H,linker-1H,4-H),3.06(d,J＝14.2Hz,1H,linker-1H),2.59(s,3H,Am-CH 3),2.39(m,2H,butyryl-CH 2),2.09(s,3H,CH 3CO),2.02(s,3H,CH 3CO),1.81(s,3H,CH 3CO),1.73(m,2H,linker-CH 2),1.36(d,J＝6.4Hz,3H,6-CH 3),1.17(d,J＝5.8Hz,3H,butyryl-CH 3),0.50(d,J＝6.3Hz,3H,6'-CH 3).； 13C NMR(100MHz,CDCl 3)δ＝172.9,170.6,168.2,166.8,156.6,138.5,137.7,137.4,136.3,134.8,128.8,128.7,128.65,128.6,128.5,128.4,128.3,128.2,128.0,127.9,127.71,127.65,127.6,127.5,127.3,102.3,97.6,96.4,83.2,77.2,76.0,75.8,75.1,74.3,71.9,70.8,70.4,68.0,67.7,67.5,66.2,63.3,54.0,53.0,50.1,49.7,43.5,42.6,27.0,23.3,21.2,19.8,19.5,18.2,15.4；HR-ESI-MS(m/z):calcd for C 76H 93N 6O 18 +(M+H +):1377.6546,found:1377.6526。
实施例35
3-氨基丙基2-乙酰氨基-3-O-(4-O-[3-N-(R)-3-羟基丁酰基-2-乙酰氨基-2,3-二脱氧-β-D-吡喃葡萄糖醛酸]-3-O-乙酰基-2-乙脒基-2-脱氧-α-L-吡喃岩藻糖)-2-脱氧-α-D-吡喃奎诺糖(35*)的合成
反应方程式如图23所示；
三糖34*(4.3mg,3.1μmol)溶解于叔丁醇/水/二氯甲烷混合液(3mL，5:2:1，v/v/v)中，氮气置换反应体系后，加入10％钯碳加氢催化剂，继续氮气置换5分钟。进一步以氢气置换反应体系5分钟后，反应液在氢气环境下搅拌24小时，硅藻土过滤后浓缩得到的粗品以C18小柱(Macherey-Nagel,Düren,德国)(洗脱液为水和甲醇)初步纯化，产品进一步以反相高效液相色谱纯化(半制备Thermo Scientific Hypercarb柱)得到白色固体状的目标产物35*(1.9mg,2.4μmol,77％)。[α] D 20＝-66.26°(c＝0.10,H 2O)； 1H NMR(700MHz,D 2O)δ＝5.18(d,J＝3.9Hz,1H,1’-H),5.10(dd,J＝11.3,2.7Hz,1H,3’-H),4.74(d,J＝3.6Hz,1H,1-H),4.61(d,J＝8.3Hz,1H,1”-H),4.53(q,J＝6.7Hz,1H,5’-H),4.26(dd,J＝11.1,3.9Hz,1H,2’-H),4.21-4.12(m,3H,butyryl-CH,4’-H,2-H),4.05-3.99(m,1H,3”-H),3.92-3.84(m,1H,2”-H),3.83-3.75(m,3H,5-H,3-H,linker-CH a),3.73(d,J＝9.7Hz,1H,5”-H),3.71-3.62(m,1H,4”-H),3.55(ddd,J＝21.7,11.5,6.1Hz,1H,linker-CH b),3.34(t,J＝9.3Hz,1H,4-H),3.13-3.03(m,2H,linker-CH 2),2.49(dd,J＝14.1,7.4Hz,1H,butyryl-CH 2),2.42-2.36(m,1H,butyryl-CH 2),2.26(s,3H,Am-CH 3),2.10(s,3H,CH 3CO),2.01(s,3H,CH 3CO),2.00-1.91(m,5H,linker-CH 2,CH 3CO),1.31(d,J＝6.2Hz,3H,6-CH 3),1.20(d,J＝6.4Hz,6H,6’-CH 3,butyryl-CH 3)； 13C NMR(176MHz,D 2O)δ＝175.1,174.8,174.4,173.6,173.1,166.1,102.7,96.9,95.8,78.9,77.4,75.9,73.5,70.4,70.2,67.8,66.7,65.0,64.9,54.3,53.9,53.4,50.3,45.1,37.2,27.1,22.2,21.8,21.6,20.3,18.8,16.7,15.0；HR-ESI-MS(m/z):calcd for C 33H 57N 6O 16 +(M+H +):793.3831,found:793.3812。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (11)

  1. 类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段，其特征在于，结构如通式I所示：
    V*-[U x+2-U x+1-U x] n-V-O-L-NH 2 式I，
    其中，
    x为1，2，3；n为1，2，3；
    -V-表示：化学键，-U x+2-，或-U x+2-U x+1-；
    V*-表示：H-，H-U x-，或H-U x+1-U x-；
    L表示连接臂；U x，U x+1，U x+2如式Ⅴ所示：
    Figure PCTCN2018082660-appb-100001
  2. 根据权利要求1所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段，其特征在于，表示为通式II：
    V*-[U x+2-U x+1-U x] n-O-L-NH 2 式II，
    其中x，n，L，U x，U x+1，U x+2和V*与通式I一致；
    通式II进一步表示为通式II-a，II-b或II-c：
    Figure PCTCN2018082660-appb-100002
  3. 根据权利要求1所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段，其特征在于，表示为通式III：
    V*-[U x+2-U x+1-U x] n-U x+2-O-L-NH 2 式III，
    其中x，n，L，U x，U x+1，U x+2和V*与通式I一致，通式III进一步表示为通式III-a，III-b或III-c：
    Figure PCTCN2018082660-appb-100003
  4. 根据权利要求1所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段，其特征在于，表示为通式IV：
    V*-[U x+2-U x+1-U x]n-U x+2-U x+1-O-L-NH 2 式IV，
    其中x，n，L，U x，U x+1，U x+2和V*与通式I一致。通式IV可以具体表示为通式IV-a，IV-b和IV-c：
    Figure PCTCN2018082660-appb-100004
  5. 一种制备权利要求1～4任一所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段的方法，其特征在于，以单糖砌块1作为寡糖链中D-奎诺糖胺的原料，单糖砌块1如式Ⅵ-1所示：
    Figure PCTCN2018082660-appb-100005
    其中PG 1为乙酰基，乙酰丙酰基，苯甲酰基，氯乙酰基，二氯乙酰基，三氯乙酰基，新戊酰基，烯丙氧羰酰基，2-萘甲基，对甲氧苄基，叔丁基二甲基硅烷基，叔丁基二苯基硅烷基，三乙基硅烷基；PG 2为苄基；LG为乙硫基，对甲苯硫基，苯硫基，溴，氟，三氯乙酰亚氨酯，N-苯基三氟乙酰亚胺酯，二丁基磷酸酯。
  6. 根据权利要求5所述的方法，其特征在于，以单糖砌块2作为寡糖链中L-岩藻糖胺的原料，单糖砌块2如式Ⅵ-2所示：
    Figure PCTCN2018082660-appb-100006
    其中PG 3，PG 4为乙酰基，乙酰丙酰基，苯甲酰基，氯乙酰基，二氯乙酰基，三氯乙酰基，新戊酰基，烯丙氧羰酰基，2-萘甲基，对甲氧苄基，叔丁基二甲基硅烷基，叔丁基二苯基硅烷基，三乙基硅烷基；LG为乙硫基，对甲苯硫基，苯硫基，溴，氟，三氯乙酰亚氨酯，N-苯基三氟乙酰亚胺酯，二丁基磷酸酯。
  7. 根据权利要求5或6所述的方法，其特征在于，以单糖砌块3作为寡糖链中2,3-二氨基-D-葡萄糖醛酸的原料，单糖砌块3如式Ⅵ-3所示：
    Figure PCTCN2018082660-appb-100007
    其中PG 5为三氯乙酰基，二氯乙酰基，氯乙酰基；PG 6为苄基，2-萘甲基，对甲氧苄基，乙酰丙酰基；PG 7为苄基；LG为乙硫基，对甲苯硫基，苯硫基，溴，氟，三氯乙酰亚氨酯，N-苯基三氟乙酰亚胺酯，二丁基磷酸酯。
  8. 根据权利要求5所述的方法，其特征在于，以Ⅵ-4所示的化合物作为连接臂原料组装于寡糖链还原末端，其中PG 9，PG 10为苄基，苄氧羰基；
    Figure PCTCN2018082660-appb-100008
  9. 根据权利要求5所述的方法，其特征在于，制备单糖砌块3的重要中间体3-叠氮基葡萄糖胺的方法，进行3号位二次转位，包括以下步骤：
    1)3-三氟甲磺酰基葡萄糖胺经Lattrell-Dax反应，制得3-羟基阿洛糖胺；
    2)3-羟基阿洛糖胺经三氟甲磺酰化反应，制得3-三氟甲磺酰基阿洛糖胺；
    3)3-三氟甲磺酰基阿洛糖胺经叠氮基亲核取代，制得3-叠氮基葡萄糖胺。
  10. 一种制备权利要求1～4任一所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段的方法，其特征在于，包括11个反应模块：A：糖苷化反应；B：叠氮基还原乙酰化；C：奎诺糖胺3号位脱保护；D：叠氮基还原修饰2,2,2-三氯乙氧羰基(Troc)；E：岩藻糖胺3,4号位选择性萘甲基保护；F：岩藻糖胺3号位乙酰化；G：叠氮基还原丁酰化；H：脱去Troc修饰乙脒基；I：催化加氢全脱保护；J：葡萄糖醛酸4号位脱保护；K：叠氮基还原修饰乙脒基。
  11. 权利要求1～4任一所述的类志贺邻单胞菌O51血清型O-抗原多糖的组装有连接臂的寡糖链片段在开发或制备类志贺邻单胞菌疫苗或者类志贺邻单胞菌感染导致的疾病的药物中的应 用。
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