CN111233949A - Ganglioside GM3 and/or its analogue, synthetic method and application - Google Patents

Ganglioside GM3 and/or its analogue, synthetic method and application Download PDF

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CN111233949A
CN111233949A CN202010083701.1A CN202010083701A CN111233949A CN 111233949 A CN111233949 A CN 111233949A CN 202010083701 A CN202010083701 A CN 202010083701A CN 111233949 A CN111233949 A CN 111233949A
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acetyl
hydrogen atom
fluorine atom
sphingosine
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孟欣
李晓
李婷申
张慧明
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Tianjin University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract

The invention relates to a method for synthesizing ganglioside GM3 and/or its analogues, which comprises ⑴ selecting lactose donor and/or its analogues shown in general formula I and sphingosine derivatives shown in general formula II, ⑵ synthesizing lactose sphingosine and/or its derivatives from compounds shown in general formula I and general formula II by glycosylation, then removing protective groups, ⑶ synthesizing sialylated lactose sphingosine and/or its derivatives by one-pot three-enzyme method, ⑷ synthesizing ganglioside GM3 and/or its derivatives by condensation reaction of fatty acids with general formula IV.

Description

Ganglioside GM3 and/or its analogue, synthetic method and application
Technical Field
The invention belongs to the technical field of organic compound synthesis, and particularly relates to ganglioside GM3 and/or analogues thereof, a synthesis method and application.
Background
Ganglioside GM3 was the first ganglioside found to be the simplest in structure, and was first isolated from equine erythrocytes in 1952, Yamakawa et al (Yamakawa, T.; Suzuki, S.J.biochem.1952,39,393), whose chemical structure was NeuNAc α (2-3) Gal β (1-4) Glc β (1-3) Cer, which was formed by coupling a molecule of sialic acid with GM3 synthase, the sugar chain of GM3 further increased to form ganglioside compounds, ganglioside GM3 has important biological functions, such as maintaining structural stability, cell surface differentiation and antigens, cell-cell interaction and recognition, as markers, cell growth regulation and information transfer, as receptors for certain bioactive factors, and influences cell membrane functions, cell adhesion, and the like.
Although GM3 is present in some tissues and cells of mammals, its content is extremely low, and although the isolation and extraction techniques are greatly improved, it is still very difficult to obtain large amounts of GM3, which is extremely expensive, reaching 2000 yuan or more per 0.1 mg (Sigma,2020, Product No.860074), and does not meet the needs for large scale bioactivity experiments. Some documents report the preparation of GM3 and its analogues by total chemical synthesis and chemical enzymatic methods (Shumichi Hashimoto et al, Tetrahedron Lett.2000,41,7691; Tomoya Ogawa, Carbohydr. Res.,1988,174, 73-85; Uri Zehavi, Glycoconjugate J.1998,15, 657. eye 662; Yukeishi Ito; James C.Paulson, J.Am.chem.Soc.1993,115, 1603-1605; Auubin, Y.; Yukeishi Ito; James C.Paulson; Prestegard, J.H.Biochemistry,1993,32, 1342012; Laura Mauri, Riccardo Caselll, Guner Kirschnson & santron 0668, Glycoshi J.biochronshi, 1993,32, 1342012; Laura Mauri, Yokoshii, Yokohyo, Yodhi et J.201, Yodhi et 2, Yodhuk J.103, Yodhisan J.103, Yodhi et J.201, Yodhi et 2, Yodhi et 2, Yodhi, J.Sho Kodhi, J.Sho Kodhi, Kodhuk et 2, Kohyo koohne et 2, Ko.
The synthesis of ganglioside compounds mainly comprises three key steps, 1) the construction of thermodynamically unstable N-acetylneuraminic acid α -type glycosidic bonds, 2) the preparation of sphingosine acceptors, 3) the coupling of trisaccharide glycosyl donors with sphingosine, wherein the construction of N-acetylneuraminic acid α -type glycosidic bonds is the important one, wherein N-acetylneuraminic acid is one of sialic acids, and sialic acid is a nine-carbon-containing natural saccharide widely present in various biological tissues, in the reaction for chemically synthesizing glycosides, the construction of sialyl glycosidic bonds is the key step for synthesizing sialic acid conjugates, but the research is far less intensive than other building blocks, the reaction of general glycosyl donors can obtain higher yield and stereoselectivity, while the reaction of sialic acid donors is relatively poor, mainly due to the structural characteristics of the glycosyl groups themselves: 1) 2-position isobaric ion with electron head carbon, the formation of sialic acid ion leads to a more efficient steric hindrance than the conventional steric hindrance, no efficient steric hindrance of sialic acid donor, no significant steric hindrance to the formation of sialic acid ion, no significant steric hindrance of the sialic acid ion, no significant steric hindrance of the C2. A.2. A. a sialic acid donor, B. A. a sialic acid donor, B. a number of sialic acid donor, B. A. a number of sialic acid donor, B. a number of sialic acid acceptor, a variety of sialic acid, C. A. a variety of sialic acid.
It is worth mentioning that some progress has been made in the catalytic synthesis of the glycoside of the ganglioside GM3 by glycosyltransferases. But also face several difficulties: firstly, expensive active sugar is often needed for reaction as an intermediate, secondly, a multi-enzyme system is often needed for catalysis of the reaction, the corresponding enzymes are difficult to obtain, particularly, sialyltransferase from mammals is II type transmembrane protein, and the prior technical means is difficult to realize large-scale expression of soluble protein; thirdly, the series of enzymes often have stronger substrate specificity, narrow substrate applicability and are difficult to tolerate non-natural modification. Therefore, the search for a rapid, efficient and large-scale synthesis method for preparing ganglioside GM3 and its derivatives is a problem to be solved.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides ganglioside GM3 and/or analogues thereof, a synthesis method and application, wherein the method can be used for efficiently, quickly and simply synthesizing ganglioside GM3 and analogues thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
ganglioside GM3 and/or its analogues, having the following structural formula:
Figure BDA0002381239030000021
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
A method for the synthesis of ganglioside GM3 and/or analogues thereof as described above, comprising the steps of:
⑴ lactose donor represented by formula I and/or its analogues are selected;
Figure BDA0002381239030000031
wherein:
R1selected from p-tolylthio, fluorine atom, bromine atom, trichloroacetimidate;
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
and selecting a sphingosine derivative of formula II:
Figure BDA0002381239030000032
wherein:
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑵ the compounds shown in the general formula I and the general formula II are synthesized into lactose sphingosine and/or derivatives thereof by glycosylation reaction, and then the protecting group is removed;
the general formula III of the lactose sphingosine and/or the derivative thereof is as follows:
Figure BDA0002381239030000033
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑶ Synthesis of sialylated lactosphingosine and/or derivatives thereof by a one pot three enzyme process, the sialylation employing sialic acid and/or analogues thereof, wherein the analogue of sialic acid is R7Or R8Substituted sialic acid, the three enzymes used in the one-pot three-enzyme sialylation being aldolase, sialic acid CMP-synthetase and α 2,3 sialyltransferase, respectively;
the sialylated lactose sphingosine and/or derivatives thereof have the general formula IV:
Figure BDA0002381239030000041
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
⑷ condensing general formula IV and fatty acid to synthesize ganglioside GM3 and/or its derivative;
Figure BDA0002381239030000042
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
Furthermore, the lactose donor and/or the analogue I thereof in said step ⑴ is synthesized by the following method:
reacting lactose and/or its analogs with acetic anhydride and sodium acetate solution under reflux, i.e. protecting hydroxyl group with acetyl group; then, adding ammonium acetate into a tetrahydrofuran and/or methanol system to remove an acetyl group at the C1-position at the reducing end of the acylation product to obtain an intermediate; then under the ice bath condition, adding trichloroacetonitrile and DBU, and reacting at room temperature to obtain trichloroacetimidate, namely lactose donor and/or the analogue thereof.
Moreover, the step of synthesizing the sphingosine derivative in step ⑴ is as follows:
in 1H-imidazole-1-sulfonyl azide hydrochloride and/or CuSO4And/or K2CO3Under the condition (1), firstly, converting the C2 site amino group of the sphingosine into an azido group; thereafter, the hydroxyl group at C1 position of sphingosine was protected with TBDPS and then subjected to SOCl2/Et3N and RuCl3·3H2O and NaIO4Protecting 3, 4-position simultaneously in one pot, refluxing with tetrabutyl ammonium iodide and/or DBU at 110 deg.C and THF/H2O/H2SO4Under the condition, unsaturated double bonds at C4, 5-positions are generated in two steps, and then benzoyl protection is carried out on hydroxyl at C3 position and TBDPS protecting group at C1 position is removed, thus obtaining the sphingosine derivative.
In addition, the synthesis of the lactose sphingosine and/or the derivatives thereof in the step ⑵ comprises the following specific steps:
adding 1.2-10.0 equivalent of compound I and 1.0 equivalent of compound II into eggplant shapeAdding anhydrous dichloromethane and the same mass of reactants into a bottle
Figure BDA0002381239030000051
Molecular sieve, the reaction system is protected by argon, the mixture is stirred for 1 hour at room temperature, the reaction solution is placed at-18 ℃, and 0.1-2.0 equivalent weight of BF is dripped3OEt2Stirring for 0.5-3 h at the temperature of-18 ℃, and adding 2-5 drops of triethylamine to stop reaction after the detection reaction of the thin layer chromatography is finished; filtering, collecting filtrate, evaporating to dryness, and separating and purifying with flash column to obtain lactose sphingosine and/or its derivatives;
wherein, petroleum ether is used for the rapid column separation and purification: and (3) ethyl acetate.
Further, the sialylated lactosphingosine and/or its derivative synthesis in the step ⑶ comprises adding 1.0 equivalent of lactosphingosine and/or its derivative, 1.0-20.0 equivalent of N-acetyl-mannose and/or its analogue, 1.0-10.0 equivalent of sodium pyruvate, 1.0-20.0 equivalent of CTP, 5.0-100mM MgCl2Preparing aqueous solution with 10-500mM Tris-HCl buffer solution with pH5.0-10.5, and adding aldolase, sialic acid CMP-synthetase and sialyltransferase to realize one-kettle three-enzyme sialylation; and in the product purification stage, a C18 solid phase extraction column is used firstly, a crude product is obtained by separation and purification, and then an LH20 gel column is used for separation, so that a pure product of sialyllactose sphingosine and/or an analogue compound thereof is obtained.
In addition, the ganglioside GM3 and/or its derivative in the step ⑷ is synthesized by dissolving sialyllactose sphingosine and/or its derivative in DMF, adding 1-1.5 equivalents of fatty acid, 1-1.5 equivalents of HOBT, 1-1.5 equivalents of EDC, and 1.2-2.0 equivalents of Et under the protection of argon gas3N, stirring for 12h at room temperature, detecting by thin-layer chromatography, performing rotary evaporation to concentrate the reaction solution, and performing rapid silica gel column to obtain ganglioside GM3 and/or derivatives thereof;
wherein, the volume ratio of the rapid silica gel column is 1: 4 ethyl acetate/methanol.
Further, the enzymes used in the one-pot three-enzyme sialylation in the step ⑶ were aldolase PmAldolase, Neisseria meningitidis CMP-sialic acid synthase and Passteuerllurgica sialytransferase 1 of bacterial origin, and the reaction time was 5 minutes to 2 hours.
In addition, in the step ⑶, the reaction temperature in the enzymatic synthesis is 0-37 ℃, the rotation speed is 0-240 rpm, and the enzyme reaction stopping method is to add equal volume of 4 ℃ anhydrous methanol into the reaction and incubate the mixture at 4 ℃ for 0-30 minutes.
Use of the synthetic method as described above for the preparation of the ganglioside GM3 and/or derivatives thereof.
The invention has the advantages and positive effects that:
1. the method can efficiently, quickly and simply synthesize the ganglioside GM3 and analogues thereof, adopts a chemoenzymatic method, can be applied to the preparation of the ganglioside GM3 and derivatives thereof, and is used for drug development.
2. The method combines the flexibility of a chemical synthesis method and the high regioselectivity, the high stereoselectivity and the high efficiency of enzymatic synthesis together, realizes the high-efficiency chemical enzymatic synthesis of the ganglioside GM3 and the analogues thereof, and solves the defects of low substrate reaction activity, multiple synthesis steps, low yield and the like in the existing full-chemical synthesis of the ganglioside GM3 and the analogues thereof.
3. The method has wide application prospect in developing and synthesizing other types of gangliosides for drug development.
4. The invention combines the flexibility of the chemical synthesis method and the high regioselectivity, stereoselectivity and high efficiency of the enzyme method together, realizes the chemical enzyme method synthesis of the ganglioside GM3 and the derivatives thereof, simplifies the whole process route, has mild reaction conditions and is easy to control the reaction, particularly, the three enzymes applied in the synthesis are all enzymes from bacteria, and has the advantages of high expression level, wide substrate adaptability, soluble expression and easy purification. In conclusion, the synthesis strategy of the one-kettle three-enzyme method is applied to the chemoenzymatic synthesis of the ganglioside GM3, so that the defects of multiple synthesis steps, low stereoselectivity, low yield, use of heavy metal salt and the like in the conventional chemoenzymatic synthesis are overcome.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of compound 14 in deuterated methanol according to the invention;
FIG. 2 is a nuclear magnetic carbon spectrum of compound 14 in deuterated methanol according to the invention;
FIG. 3 is a nuclear magnetic hydrogen spectrum of compound 15 in deuterated methanol according to the invention;
FIG. 4 is a nuclear magnetic carbon spectrum of compound 15 in deuterated methanol according to the invention;
FIG. 5 is a nuclear magnetic hydrogen spectrum of compound 16 in deuterated methanol according to the invention;
FIG. 6 is a nuclear magnetic hydrogen spectrum of compound 17 in deuterated methanol according to the invention;
FIG. 7 is a nuclear magnetic hydrogen spectrum of compound 18 in deuterated methanol according to the invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Ganglioside GM3 and/or its analogues, having the following structural formula:
Figure BDA0002381239030000071
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selectingFrom fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
A method for the synthesis of ganglioside GM3 and/or analogues thereof as described above, comprising the steps of:
⑴ lactose donor represented by formula I and/or its analogues are selected;
Figure BDA0002381239030000072
wherein:
R1selected from p-tolylthio, fluorine atom, bromine atom, trichloroacetimidate;
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
and selecting a sphingosine derivative of formula II:
Figure BDA0002381239030000081
wherein:
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑵ the compounds shown in the general formula I and the general formula II are synthesized into lactose sphingosine and/or derivatives thereof by glycosylation reaction, and then the protecting group is removed;
the general formula III of the lactose sphingosine and/or the derivative thereof is as follows:
Figure BDA0002381239030000082
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑶ Synthesis of sialylated lactosphingosine and/or derivatives thereof by a one pot three enzyme process, the sialylation employing sialic acid and/or analogues thereof, wherein the analogue of sialic acid is R7Or R8Substituted sialic acid, the three enzymes used in the one-pot three-enzyme sialylation being aldolase (PmAldolase), sialic acid CMP-synthetase (NmCSS) and α 2,3 sialyltransferase (PmST1), respectively;
the sialylated lactose sphingosine and/or derivatives thereof have the general formula IV:
Figure BDA0002381239030000083
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
R7selected from the group consisting of N-acetamido, N-propionylamino, N-trifluoroacetylamino, N-azido-acetamidoA group;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
⑷ condensing general formula IV and fatty acid to synthesize ganglioside GM3 and/or its derivative;
Figure BDA0002381239030000091
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
Preferably, the lactose donor and/or the lactose donor analogue I in step ⑴ is synthesized by the following method:
reacting lactose and/or its analogs with acetic anhydride and sodium acetate solution under reflux, i.e. protecting hydroxyl group with acetyl group; then, adding ammonium acetate into a tetrahydrofuran and/or methanol system to remove an acetyl group at the C1-position at the reducing end of the acylation product to obtain an intermediate; then under the ice bath condition, adding trichloroacetonitrile and DBU, and reacting at room temperature to obtain trichloroacetimidate, namely lactose donor and/or the analogue thereof.
Preferably, the lactose donor and/or the lactose analogue in the step (1) is synthesized by the following method: and adding lactose and/or the like into a refluxing solution of acetic anhydride (10.0-50.0 equivalent) and sodium acetate (1.0-10.0 equivalent) in batches for acylation reaction, wherein the reaction time is 2-10 h. And then dissolving the acylated product in a tetrahydrofuran/methanol (volume ratio of 1:1) system, and adding ammonium acetate (1.2-10.0 equivalent) to remove acetyl groups at the C1-position at the reducing end of the acylated product to obtain an intermediate. And then dissolving the intermediate in anhydrous dichloromethane, adding trichloroacetonitrile (2.0-30.0 equivalent) and DBU (0.1-2.0 equivalent) under an ice bath condition, and stirring at room temperature for 2-12 h to obtain trichloroacetimidate, namely the lactose donor and/or the analogue I thereof required by the subsequent glycosylation reaction.
Preferably, the step ⑴ for synthesizing the sphingosine derivative is as follows:
in 1H-imidazole-1-sulfonyl azide hydrochloride and/or CuSO4And/or K2CO3Under the condition (1), firstly, converting the C2 site amino group of the sphingosine into an azido group; thereafter, the hydroxyl group at C1 position of sphingosine was protected with TBDPS and then subjected to SOCl2/Et3N and RuCl3·3H2O and NaIO4Protecting 3, 4-position simultaneously in one pot, refluxing with tetrabutyl ammonium iodide and/or DBU at 110 deg.C and THF/H2O/H2SO4Under the condition, unsaturated double bonds at C4, 5-positions are generated in two steps, and then benzoyl protection is carried out on hydroxyl at C3 position and TBDPS protecting group at C1 position is removed, thus obtaining the sphingosine derivative.
Preferably, the sphingosine derivative in step (1) is synthesized by the following method: in 1H-imidazole-1-sulfonyl azide hydrochloride/CuSO4/K2CO3Under the condition (1), firstly, converting the C2 site amino group of the sphingosine into an azido group; in the second step, the hydroxyl group at C1 position of sphingosine was protected with TBDPS and then subjected to SOCl2(1.0-5.0 equivalent)/Et3N (1.0-5.0 equiv.) and RuCl3·3H2O (0.01-0.1 equivalent) and NaIO4(1.0-5.0 equivalent) protecting hydroxyl at C3, 4-position simultaneously in one pot, and refluxing for 2-8H and THF/H at 110 ℃ by tetrabutyl ammonium iodide/DBU2O/H2SO4Under the condition, unsaturated double bonds at C4, 5-positions are generated in two steps, and then benzoyl protection is carried out on hydroxyl at C3 position and TBDPS protecting group at C1 position is removed, thus obtaining sphingosine derivative II.
Preferably, the synthesis of the lactose sphingosine and/or the derivatives thereof in the step ⑵ comprises the following specific steps:
combining 1.2-10.0 equivalentAdding the material I and 1.0 equivalent of the compound II into an eggplant-shaped bottle, and adding anhydrous dichloromethane and the materials with the same mass as the reactants
Figure BDA0002381239030000101
Molecular sieve, the reaction system is protected by argon, the mixture is stirred for 1 hour at room temperature, the reaction solution is placed at-18 ℃, and 0.1-2.0 equivalent weight of BF is dripped3OEt2Stirring for 0.5-3 h at the temperature of-18 ℃, and adding 2-5 drops of triethylamine to stop reaction after the detection reaction of the thin layer chromatography is finished; filtering, collecting filtrate, evaporating to dryness, and separating and purifying with flash column to obtain lactose sphingosine and/or its derivatives;
wherein, petroleum ether is used for the rapid column separation and purification: and (3) ethyl acetate.
Preferably, the sialylated lactosphingosine and/or its derivatives in step ⑶ are synthesized by adding 1.0 equivalent of lactosphingosine and/or its derivatives, 1.0-20.0 equivalents of N-acetyl-mannose and/or its analogues, 1.0-10.0 equivalents of sodium pyruvate, 1.0-20.0 equivalents of CTP, 5.0-100mM MgCl2Preparing aqueous solution with 10-500mM Tris-HCl buffer solution with pH5.0-10.5, and adding aldolase, sialic acid CMP-synthetase and sialyltransferase to realize one-kettle three-enzyme sialylation; and in the product purification stage, a C18 solid phase extraction column is used firstly, a crude product is obtained by separation and purification, and then an LH20 gel column is used for separation, so that a pure product of sialyllactose sphingosine and/or an analogue compound thereof is obtained.
Preferably, the method for synthesizing sialylated lactosphingosine and/or derivatives thereof in step (3): mixing compound III (1.0 equivalent), N-acetyl mannose or its analogue (1.0-20.0 equivalents), 1.0-10.0 equivalents of sodium pyruvate, CTP (cytidine triphosphate) (1.0-20.0 equivalents), MgCl2(5.0-100mM) and Tris-HCl buffer (10-500mM, pH5.0-10.5) to prepare an aqueous solution, adding aldolase, sialic acid CMP-synthetase (H.Yu and X.Chen, Org.Lett.,2006,8,2393-ia and X.Chen, J.Am.chem.Soc.,2005,127, 17618-17619; k.lau, h.yu, v.thon, z.khedri, m.e.leon, b.k.tran and x.chen, org.biomol.chem.,2011,9, 2784-. The reaction solution was then centrifuged at 12000r/min at 4 ℃ for 30min, and the concentrated supernatant was collected. Firstly, C18 solid phase extraction column is carried out to remove most inorganic salt, crude product is obtained by separation and purification, and pure product sialyllactose sphingosine and/or analogue compound IV thereof is obtained by LH20 gel column separation.
Preferably, the ganglioside GM3 and/or its derivative in the step ⑷ is synthesized by dissolving sialyllactose sphingosine and/or its derivative in DMF, adding 1-1.5 equivalents of fatty acid, 1-1.5 equivalents of HOBT, 1-1.5 equivalents of EDC, and 1.2-2.0 equivalents of Et under the protection of argon gas3N, stirring for 12h at room temperature, detecting by thin-layer chromatography, performing rotary evaporation to concentrate the reaction solution, and performing rapid silica gel column to obtain ganglioside GM3 and/or derivatives thereof;
wherein, the volume ratio of the rapid silica gel column is 1: 4 ethyl acetate/methanol.
Preferably, the enzymes used in the one-pot three-enzyme sialylation in step ⑶ are aldolase of bacterial origin, PmAldolase, Neisseria meningitidis CMP-colloidal acid synthase (NmCSS) and Pasteurella multocida sizing transferase 1(PmST1), with a reaction time of 5 minutes to 2 hours.
Preferably, the reaction temperature in the enzymatic synthesis in the step ⑶ is 0-37 ℃, the rotation speed is 0-240 rpm, and the method for stopping the enzymatic reaction is to add equal volume of 4 ℃ anhydrous methanol into the reaction and incubate the mixture at 4 ℃ for 0-30 minutes.
Use of the synthetic method as described above for the preparation of the ganglioside GM3 and/or derivatives thereof.
More specifically, the preparation and detection are as follows:
chemical synthesis of lactose analogs and sphingosine analogs
1. The reaction equation is as follows:
Figure BDA0002381239030000111
preparation of [2,3,4, 6-tetra-O-acetyl- β -D-galactopyranose ] - [1 → 4] -1,2,3, 6-tetra-O-acetyl- β -D-glucopyranose, compound 2:
anhydrous NaOAc (0.96g, 11.60mmol) was added to acetic anhydride (5.30mL,56mmol), heated under reflux, lactose (1g,2.92mmol) was added to the reflux in equal portions four times, refluxed for 3h, and after completion of the detection reaction by thin layer chromatography, extracted with ethyl acetate/water, the organic phase was collected, concentrated by rotary evaporation, and purified by flash silica gel column to give compound 2(1.90g, 95%).
Preparation of [2,3,4, 6-tetra-O-acetyl- β -D-galactopyranose ] - [1 → 4] -2,3, 6-tri-O-acetyl- β -D-glucopyranose, compound 3:
dissolving compound 2(1.90g,2.80mmol) in tetrahydrofuran/methanol (volume ratio 1:1), adding ammonium acetate (430mg,5.60mmol), stirring overnight at room temperature, detecting reaction completion by thin layer chromatography, extracting with ethyl acetate/water, collecting organic layer, drying over anhydrous sodium sulfate, concentrating to obtain compound 3(1.68g, 94%) with purity capable of directly proceeding next reaction.
Preparation of [2,3,4, 6-tetra-O-acetyl- β -D-galactopyranose ] - [1 → 4] -2,3, 6-tri-O-acetyl- β -D-glucopyranose-trichloroacetimidate, compound 4:
dissolving the compound 3(1.68g,2.64mmol) in anhydrous dichloromethane, adding trichloroacetonitrile (1.28mL,11.7mmol) and DBU (0.098mL,0.66mmol) at 0 ℃ under the protection of argon, stirring at room temperature for 5h, detecting the progress of the reaction by thin layer chromatography, concentrating the reaction solution by rotary evaporation after the reaction is completed, and purifying by a flash silica gel column to obtain the compound 4(1.45g, 70%).
2. The reaction equation is as follows:
Figure BDA0002381239030000131
preparation of [2S,3R,4E ] -2-azido-octadecane-l, 3, 4-triol, compound 6:
dissolving phytosphingosine 5(2.30g,7.24mmol) in anhydrous methanol, adding 1H-imidazole-1-sulfonyl azide hydrochloride (1.82g,10.51mmol), CuSO4(23mg,0.14mmol)，K2CO3(1.70g, 12.31mmol), stirring at room temperature for 6h, detecting the reaction progress by thin layer chromatography, carrying out rotary evaporation concentration after the reaction is finished, adding a dilute hydrochloric acid solution and ethyl acetate for extraction, collecting an organic phase, carrying out rotary evaporation concentration to obtain a crude compound 6(2.4 g, 96%), and directly carrying out the next reaction.1H NMR(400MHz,CDCl3)δ4.00(dd,J＝6.0,7.6Hz,1H),3.90–3.86(m,2H),3.81–3.74(m,2H),3.69–3.66(q,J＝4.8Hz,1H),1.60-1.51(m,2H),1.26(s,23H),0.88(t,J＝6.8Hz,3H).
Preparation of [2S,3R,4E ] -1-oxo-tert-butyldiphenylsilane-2-azido-octadecane-l, 3, 4-triol, compound 7:
compound 6(2.40g, 6.99mmol) was dissolved in anhydrous dichloromethane at 0 deg.C and Et was added3N (2.43mL, 17.47mmol), DMAP (42.7mg,0.35mmol), TBDPSCl (2.7mL,10.48mmol), stirring at room temperature for 12h, detecting by thin layer chromatography after reaction completion, NaCl and CH2Cl2Extraction, collection of the organic phase, concentration by rotary evaporation and purification on flash silica gel (petroleum ether: ethyl acetate, 4:1) gave compound 7(3.9g, 96%).
Preparation of [2S,3R,4E ] -1-oxo-tert-butyldiphenylsilyl-2-azido-3, 4-sulfonyl-octadecane-l, 3, 4-triol, compound 8:
compound 7(3.9g, 6.7mmol) was dissolved in anhydrous dichloromethane at 0 deg.C and SOCl was added2(1.54mL，8.04mmol)，Et3N (2.79mL,20.11mmol),0 ℃, reacting for 0.5h, and detecting NaHCO after the reaction is finished by thin layer chromatography3NaCl separately from CH2Cl2Extracting, and using anhydrous Na for organic phase2SO4Drying and vacuum drying for 3 h. The dried compound was then dissolved in CCl4/CH3CN/H2O (volume ratio 1: 1:1, 12mL), RuCl was added3·3H2O(42.27mg，0.19mmol),NaIO4(4.30g,20.11mmol) at room temperature for 2h, and detecting by thin layer chromatography after completion of the reaction, ethyl acetate/saturated NaHCO3Extracted with anhydrous Na2SO4Drying and spinningConcentration by evaporation and purification on flash silica gel (petrol ether: ethyl acetate, 20:1) gave compound 8(3.7g, 87%).1HNMR(400MHz,CDCl3)δ7.61–7.59(m,4H),7.41–7.33(m,6H),4.92(dd,J＝7.2,1H),4.90–4.82(m,1H),3.96(dd,J＝11.2,2.0Hz,1H),3.82(dd,J＝11.2,4.2Hz,1H),3.64–3.59(m,1H),1.89–1.81(m,1H),1.71–1.69(m,1H),1.55–1.52(br s,1H),1.30–1.19(m,23H),1.02(s,9H),0.80(t,J＝6.8Hz,3H).
Preparation of [2S,3R,4E ] -1-oxo-tert-butyldiphenylsilyl-2-azido-4-octadecene-1, 3-diol, compound 9:
dissolving compound 8(3.7g, 5.75mmol) in toluene, adding tetrabutyl ammonium iodide (4.8g, 13mmol), DBU (1.35mL,9.02mmol), refluxing at 110 deg.C for 2H, detecting reaction completion by thin layer chromatography, cooling to room temperature, adding THF (1.5mL), H2O(0.09mL)，H2SO4(0.11mL), stirred at room temperature for 45min, and after completion of the reaction, detected by thin layer chromatography, ethyl acetate/saturated NaHCO3Extracting with NaCl, and extracting the organic phase with anhydrous Na2SO4Drying, rotary evaporation concentration and flash silica gel column purification (petroleum ether: ethyl acetate, 10:1) gave compound 9(2.7g, 83%).1HNMR(400MHz,CDCl3)δ7.71–7.68(m,4H),7.45–7.38(m,6H),5.74(dt,J＝4.0,8.0Hz,1H,H-5Cer),5.44(dd,J＝8.0,4.0Hz,1H,H-4Cer),4.23(q,J＝8.0,1H),3.82–3.79(m,2H),3.52(t,J＝8.0Hz,1H),2.11(d,J＝4.0,1H),2.01(dd,J＝4.0,8.0,1H),1.56(s,1H),1.26(s,21H),1.08(s,9H),0.88(t,J＝8.0Hz,3H).
Preparation of [2S,3R,4E ] -1-oxo-tert-butyldiphenylsilyl-2-azido-3-oxo-benzoyl-4-octadecene-1, 3-diol, compound 10:
compound 9(2.70g, 4.79mmol) was dissolved in anhydrous dichloromethane and Et was added3N (3.99mL, 28.73mmol), DMAP (58.50mg, 0.48mmol), BzCl (1.12mL, 9.67mmol) was added dropwise under ice-bath conditions, stirred at room temperature for 12h, and CH was detected after completion of the reaction by thin layer chromatography2Cl2Diluted hydrochloric acid and saturated NaHCO3Extracting with NaCl, and extracting the organic phase with anhydrous Na2SO4Drying, rotary evaporation concentration, flash silica gel column purification (petroleum ether: ethyl acetate, 9:1) gave compound 10(2.85g,89％)。1H NMR(400MHz,CDCl3)δ8.02–8.00(d,J＝8.0Hz,2H),7.69–7.64(dd,J＝20.0,8.0Hz,4H),7.57(m,d,J＝4.0Hz,1H),7.46–7.31(m,8H),5.90(dt,J＝16.0,8.0Hz,1H),5.70–5.68(m,1H),5.51(dd,J＝16.0,8.0Hz,1H),3.86–3.82(m,1H),3.75(d,J＝8.0Hz,2H),2.03(q,J＝8.0Hz,2H),1.35–1.24(m,22H),1.09(s,9H),0.89(t,J＝8.0Hz,3H).13CNMR(101MHz,CDCl3)δ165.15,138.46,135.54,135.53,134.42,134.39,133.05,132.82,132.68,130.22,130.06,129.84,129.80,129.71,128.37,127.87,127.79,127.74,123.22,74.30,65.75,63.34,32.32,31.91,29.67,29.64,29.55,29.40,29.34,29.10,28.69,26.67,25.96,22.68,19.09,14.10.
preparation of [2S,3R,4E ] -2-azido-3-oxo-benzoyl-4-octadecene-1, 3-diol, compound 11:
compound 10(2.85g,4.27mmol) is dissolved in THF and hydrogen fluoride pyridine (1.37mL) is added dropwise at 0 deg.C and stirred at room temperature for 12h, ethyl acetate/saturated NaHCO3Extracting with NaCl, and extracting the organic phase with anhydrous Na2SO4Drying, rotary evaporation concentration, flash silica gel column purification (petroleum ether: ethyl acetate, 6:1) gave compound 11(1.52g, 82%).1H NMR(400MHz,CDCl3)δ8.07–8.05(d,J＝7.6Hz,2H),7.60–7.57(m,1H),7.46(t,J＝8.0Hz,2H),5.96(dt,J＝20.8,7.2Hz,1H),5.64–5.57(m,2H),3.83–3.74(m,2H),3.65–3.60(m,1H),2.11–1.98(m,3H),1.45–1.24(m,22H),0.88(t,J＝6.8Hz,3H).13C NMR(101MHz,CDCl3)δ165.48,138.74,133.27,129.74,129.68,128.42,123.14,74.64,66.15,61.88,32.31,31.86,29.62,29.61,29.59,29.51,29.35,29.29,29.08,28.62,22.63,14.06.
Secondly, synthesizing the lactulose sphingosine by a chemical method
The reaction equation is as follows:
Figure BDA0002381239030000151
preparation of [2,3,4, 6-tetra-O-acetyl- β -D-galactopyranose ] - [1 → 4] - [2,3, 6-tri-O-acetyl- β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-azido-3-O-benzoyl-4-octadecene-1, 3-diol, compound 12:
the glycosyl donor 4(1.0g,1.28mmol) and sphingosine acceptor 11(418mg,0.97mmol) were dissolved in anhydrous dichloromethane and added
Figure BDA0002381239030000152
Molecular sieve (1.0g), stirring at room temperature for 1h under the protection of argon, placing the reaction solution at-18 ℃, and dripping BF3·OEt2(0.17mL,1.34mmol), stirring for 1h at 18 ℃, adding 2-5 drops of triethylamine to stop reaction after the detection reaction of thin layer chromatography is finished, and Et3N quenching, celite filtration, rotary evaporation of the concentrated reaction solution, and flash silica gel column purification (petroleum ether: ethyl acetate, 2:1) gave compound 12(1.12g, 83%).1H NMR(400MHz,CDCl3)δ7.97(d,J＝7.4Hz,2H),7.54–7.46(m,1H),7.38(t,J＝7.7Hz,2H),5.91–5.77(m,1H),5.57–5.41(m,2H),5.27(d,J＝2.8Hz,1H),5.12(t,J＝8.0Hz,1H),5.04(dd,J＝10.4,8.0Hz,1H),4.93–4.80(m,2H),4.50–4.37(m,3H),4.09–3.94(m,3H),3.91–3.73(m,4H),3.58–3.46(m,2H),2.08–1.89(m,21H),1.35–1.14(m,24H),0.80(t,J＝6.4Hz,3H).13C NMR(101MHz,CDCl3)δ170.19,170.13,169.99,169.89,169.66,169.39,168.96,164.93,138.87,133.08,129.79,129.60,128.32,122.54,100.99,100.18,76.01,74.50,72.72,72.56,71.36,70.83,70.57,68.97,68.17,66.52,63.34,61.76,60.71,32.23,31.77,29.50,29.43,29.24,29.20,28.99,28.58,22.53,20.64,20.57,20.54,20.48,20.46,20.35,13.98.
Preparation of [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-azido-4-octadecene-1, 3-diol, i.e. Compound 13:
dissolving compound 12(1.12g, 1.07mmol) in anhydrous methanol, adding NaOMe (96mg) to detect pH to 8, stirring at room temperature for 14h, after the detection reaction by thin layer chromatography, adding cationic resin, adjusting the reaction solution to be neutral, and performing rotary evaporation and concentration to obtain compound 13(620mg, 89%) with purity which can be directly used for the next reaction.
Preparation of [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-amino-4-octadecene-1, 3-diol, i.e. Compound 14:
compound 13(620mg, 0.95mmol)Dissolving in pyridine/water (1:1), adding 1, 3-propanedithiol (0.94mL, 9.40mmol) and triethylamine (0.99mL,7.14mmol), stirring at 50 deg.C for 12h, after completion of the reaction by thin layer chromatography, concentrating the reaction solution by rotary evaporation, and purifying with flash silica gel column (ethyl acetate: methanol, 1:1) to obtain compound 14(554mg, 93%).1H NMR(400MHz,CD3OD)δ5.77–5.63(m,1H),5.42(dd,J＝15.2,7.2Hz,1H),4.29(d,J＝7.6Hz,1H),4.24(d,J＝7.8Hz,1H),3.93(t,J＝7.2Hz,1H),3.87–3.60(m,7H),3.55–3.39(m,5H),3.39–3.32(m,1H),3.28–3.20(m,2H),2.87(td,J＝6.8,3.6Hz,1H),2.02(q,J＝6.8Hz,2H),1.35(s,2H),1.22(s,21H),0.83(t,J＝6.4Hz,3H).13C NMR(101MHz,CD3OD)δ135.49,130.41,104.87,103.96,80.37,76.85,76.27,76.03,74.58,74.44,74.13,72.30,71.11,70.06,62.27,61.61,56.00,33.22,32.84,30.57,30.53,30.41,30.24,30.16,30.14,23.51,14.25.
Synthesis of sialyllactosylsphingosine and its analogues by one-pot three-enzyme method
Figure BDA0002381239030000171
General operating method for synthesizing sialyllactosylsphingosine and analogues thereof by one-pot three-enzyme method
A50 mL centrifuge tube was charged with chemically synthesized sialylated lactosylsphingosine and its analog receptor 14(30-100mg, 1.0 eq), ManNHAc or its derivative (1.5 eq), sodium pyruvate (5.0 eq), cytidine triphosphate (CTP 1.5 eq), Tris-HCl buffer (100mM, pH 8.5) and magnesium chloride (20mM), and adjusted to a total volume of 10mL with double distilled water, after shaking, enzyme Aldolase (0.6-0.8mg), NmCSS (0.5-0.8mg) and PmST1(0.2-0.3mg) were added, followed by incubation at 37 ℃ and 140rpm for 0.5 h. Thin layer chromatography (EA: CH)3OH:H2O HOAc (4: 2:1:0.1, V/V) followed by detection, color development with anisaldehyde was performed, and after completion of the reaction, an equal volume of methanol solution was added, and the mixture was allowed to stand at 4 ℃ for half an hour. The reaction solution was then centrifuged at 12000r/min at 4 ℃ for 30min, and the concentrated supernatant was collected. Firstly, C18 solid phase extraction column is carried out to remove most inorganic salt, crude product is obtained by separation and purification,separating with LH20 gel column to obtain pure sialyl lactosphingosine and its analogues 15 and 16.
The following are the structural formulas of compounds 15 and 16 obtained by the one-pot three-enzyme synthesis method:
Figure BDA0002381239030000172
wherein the yield and structural characterization information of the compounds 15 and 16 obtained by the one-pot three-enzyme method:
[ 5-N-acetylamino-3, 5-di-deoxy-D-neuraminic acid ] - (2 → 3) - [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-amino-4-octadecene-1, 3-diol (15)
The yield thereof was found to be 73%.1H NMR(400MHz,CD3OD)δ5.84–5.72(m,1H),5.40(dd,J＝15.4,6.8Hz,1H),4.33(d,J＝7.8Hz,1H),4.27(d,J＝7.8Hz,1H),4.20(t,J＝4.0Hz,1H),3.96(dd,J＝9.6,2.8Hz,1H),3.90–3.43(m,19H),3.39(d,J＝7.6Hz,2H),3.30–3.19(m,8H),2.82–2.71(m,1H),2.01(dd,J＝14.0,7.0Hz,2H),1.92(s,3H),1.69–1.59(m,1H),1.33(s,3H),1.20(s,23H),0.81(t,J＝6.8Hz,3H).13C NMR(101MHz,CD3OD)δ174.21,173.66,135.21,130.99,128.49,127.07,103.68,102.42,99.72,79.14,76.39,75.65,75.16,74.76,73.53,73.09,71.64,69.71,69.41,68.71,67.86,67.70,66.10,65.27,63.22,61.34,60.23,55.30,52.60,40.57,32.02,31.69,30.32,29.43,29.39,29.28,29.10,29.05,28.83,22.36,21.32,18.87,13.11,12.69.
[ 5-N-trifluoroacetylamino-3, 5-di-deoxy-D-neuraminic acid ] - (2 → 3) - [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-amino-4-octadecene-1, 3-diol (16)
The yield thereof was found to be 84%.1H NMR(400MHz,CD3OD)δ5.83(dt,J＝15.2,7.2Hz,1H),5.49(dd,J＝14.8,6.2Hz,1H),4.42(d,J＝7.6Hz,1H),4.34(d,J＝8.0Hz,1H),4.29(t,J＝6.6Hz,1H),4.05(dd,J＝8.0,4.2Hz,1H),3.98–3.73(m,11H),3.70–3.51(m,9H),3.46(d,J＝8.0Hz,2H),2.85(dd,J＝12.8,4.6Hz,1H),2.15–1.99(m,2H),1.81–1.67(m,1H),1.29(s,24H),0.90(t,J＝6.8Hz,6H).19F NMR(376MHz,CDCl3)δ-76.92
Fourthly, synthetic ganglioside GM3 and analogues thereof
Figure BDA0002381239030000181
Dissolving compounds 15 and 16 (10-50 mg) in DMF, adding stearic acid or other fatty acid (1-1.5 equivalent), HOBT (1-1.5 equivalent), EDC (1-1.5 equivalent), Et (Et) under the protection of argon3N (1.2-2.0 equivalent), stirring at room temperature for 12h, detecting by thin-layer chromatography, and then carrying out rotary evaporation to concentrate the reaction solution, and carrying out flash silica gel column (ethyl acetate/methanol 1: 4) to obtain compounds 17 and 18.
The following are representative compounds 17 and 18 obtained by the condensation reaction:
Figure BDA0002381239030000191
the following are the yields and structural information of compounds 17 and 18 obtained by the condensation reaction:
[ 5-N-acetylamino-3, 5-di-deoxy-D-neuraminic acid ] - (2 → 3) - [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-stearamido-4-octadecene-1, 3-diol (Neu5Ac α (2-3) Gal β (1-4) Glc β (1-3) Cer,17, 84%)
The yield thereof was found to be 84%.1H NMR(400MHz,CD3OD)δ8.53(s,1H,5”’-NH),7.73-7.59(m,1H,2NH),5.68(dt,J＝8.0,15.2Hz,1H,H-5Cer),5.44(dd,J＝8.0,7.6Hz,1H,H-4Cer),4.42(d,J＝7.9Hz,1H,H-1b),4.31(d,J＝7.9Hz,1H,H-1a),4.18(dd,J＝10.0,4.4Hz,1H,H-1Cer),4.10–4.01(m,2H),4.00–3.47(m,20H),3.42(d,J＝8.2Hz,1H),2.85(dd,J＝13.2,4.0Hz,1H,3eq”’),2.17(t,J＝7.2Hz,2H,CH2CH2CO),),2.03(s,1H),2.01(s,3H,AcH),1.73(d,J＝6.9Hz,1H),1.58(s,2H),1.29(s,50H,CH2),0.89(d,J＝7.0Hz,6H,2CH3CH2).
[ 5-N-trifluoroacetylamino-3, 5-di-deoxy-D-neuraminic acid ] - (2 → 3) - [ β -D-galactopyranose ] - [1 → 4] - [ β -D-glucopyranose ] - [1 → 1] - [2S,3R,4E ] -2-stearamido-4-octadecene-1, 3-diol (Neu5TFA α (2-3) Gal β (1-4) Glc β (1-3) Cer,18, 86%)
1H NMR(400MHz,CD3OD)δ7.60-7.15(m,1H,2NH),5.68(dt,J＝6.4,14.8Hz,1H,H-5Cer),5.44(dd,J＝8.0,7.6Hz,1H,H-4Cer),4.43(d,J＝8.0Hz,1H,H-1b),4.30(d,J＝8.0Hz,1H,H-1a),4.20(dd,J＝10.0,4.8Hz,1H,H-1Cer),4.10–4.02(m,2H),3.99–3.71(m,11H),3.69–3.52(m,8H),3.46(d,J＝10.2Hz,2H),2.84(dd,J＝12.0,4.4Hz,1H,3eq”’),2.17(t,J＝5.6Hz,2H,CH2CH2CO),2.03(d,J＝7.0Hz,2H),1.75(dd,J＝14.4,9.4Hz,2H),1.58(br s,2H),1.29(s,50H),0.90(t,J＝6.8Hz,12H).
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims (10)

1. Ganglioside GM3 and/or analogues thereof, characterized in that: the structural formula is as follows:
Figure FDA0002381239020000011
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
2. A method of synthesis of ganglioside GM3 and/or its analogues according to claim 1, characterized in that: the method comprises the following steps:
⑴ lactose donor represented by formula I and/or its analogues are selected;
Figure FDA0002381239020000012
wherein:
R1selected from p-tolylthio, fluorine atom, bromine atom, trichloroacetimidate;
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
and selecting a sphingosine derivative of formula II:
Figure FDA0002381239020000021
wherein:
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑵ the compounds shown in the general formula I and the general formula II are synthesized into lactose sphingosine and/or derivatives thereof by glycosylation reaction, and then the protecting group is removed;
the general formula III of the lactose sphingosine and/or the derivative thereof is as follows:
Figure FDA0002381239020000022
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, trimethylAcetyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
⑶ Synthesis of sialylated lactosphingosine and/or derivatives thereof by a one pot three enzyme process, the sialylation employing sialic acid and/or analogues thereof, wherein the analogue of sialic acid is R7Or R8Substituted sialic acid, the three enzymes used in the one-pot three-enzyme sialylation being aldolase, sialic acid CMP-synthetase and α 2,3 sialyltransferase, respectively;
the sialylated lactose sphingosine and/or derivatives thereof have the general formula IV:
Figure FDA0002381239020000023
wherein:
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R5selected from azido, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, N-9-fluorenylmethoxycarbonyl, phthalimide;
R6selected from benzoyl, acetyl, benzyl;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
⑷ condensing general formula IV and fatty acid to synthesize ganglioside GM3 and/or its derivative;
Figure FDA0002381239020000031
R2selected from fluorine atom, hydrogen atom, azide, hydroxyl, acetyl, pivaloyl;
R3selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R4selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
R7selected from the group consisting of nitrogen acetamido, nitrogen propionylamino, nitrogen trifluoroacetamido, nitrogen azidoacetamido;
R8selected from fluorine atom, hydrogen atom, acetyl group, hydroxyl group;
n is an integer of 0 to 30.
3. The method of claim 2, wherein the lactose donor and/or the lactose analog I of step ⑴ are synthesized by:
reacting lactose and/or its analogs with acetic anhydride and sodium acetate solution under reflux, i.e. protecting hydroxyl group with acetyl group; then, adding ammonium acetate into a tetrahydrofuran and/or methanol system to remove an acetyl group at the C1-position at the reducing end of the acylation product to obtain an intermediate; then under the ice bath condition, adding trichloroacetonitrile and DBU, and reacting at room temperature to obtain trichloroacetimidate, namely lactose donor and/or the analogue thereof.
4. The method according to claim 2, wherein the step of synthesizing the sphingosine derivative in step ⑴ comprises:
in 1H-imidazole-1-sulfonyl azide hydrochloride and/or CuSO4And/or K2CO3Under the condition (1), firstly, converting the C2 site amino group of the sphingosine into an azido group; thereafter, the hydroxyl group at C1 position of sphingosine was protected with TBDPS and then subjected to SOCl2/Et3N and RuCl3·3H2O and NaIO4Protecting 3, 4-position simultaneously in one pot, refluxing with tetrabutyl ammonium iodide and/or DBU at 110 deg.C and THF/H2O/H2SO4Under the condition, unsaturated double bonds at C4, 5-positions are generated in two steps, and then benzoyl protection is carried out on hydroxyl at C3 position and TBDPS protecting group at C1 position is removed, thus obtaining the sphingosine derivative.
5. The synthesis method according to claim 2, wherein the step ⑵ of synthesizing the lactose sphingosine and/or its derivatives comprises the following steps:
adding 1.2-10.0 equivalent of compound I and 1.0 equivalent of compound II into a bottle shaped like a eggplant, adding anhydrous dichloromethane and the same mass of the reactant
Figure FDA0002381239020000041
Molecular sieve, the reaction system is protected by argon, the mixture is stirred for 1 hour at room temperature, the reaction solution is placed at-18 ℃, and 0.1-2.0 equivalent weight of BF is dripped3OEt2Stirring for 0.5-3 h at the temperature of-18 ℃, and adding 2-5 drops of triethylamine to stop reaction after the detection reaction of the thin layer chromatography is finished; filtering, collecting filtrate, evaporating to dryness, and separating and purifying with flash column to obtain lactose sphingosine and/or its derivatives;
wherein, petroleum ether is used for the rapid column separation and purification: and (3) ethyl acetate.
6. The method of synthesizing sialylated lactosphingosine and/or its derivatives according to claim 2, wherein the step ⑶ comprises mixing lactosphingosine and/or its derivatives in an amount of 1.0 equivalent, N-acetylmannose and/or its analogs in an amount of 1.0 to 20.0 equivalents, sodium pyruvate in an amount of 1.0 to 10.0 equivalents, CTP in an amount of 1.0 to 20.0 equivalents, MgCl in an amount of 5.0 to 100mM2Preparing aqueous solution with 10-500mM Tris-HCl buffer solution with pH5.0-10.5, and adding aldolase, sialic acid CMP-synthetase and sialyltransferase to realize one-kettle three-enzyme sialylation; in the product purification stage, a C18 solid phase extraction column is used firstly, crude products are obtained by separation and purification, and then a LH20 gel column is used for separation to obtain pure sialyllactoseSphingoid and/or analog compounds thereof.
7. The method of claim 2, wherein the ganglioside GM3 and/or its derivative in step ⑷ is synthesized by dissolving sialyllactose sphingosine and/or its derivative in DMF, adding 1-1.5 equivalents of fatty acid, 1-1.5 equivalents of HOBT, 1-1.5 equivalents of EDC, and 1.2-2.0 equivalents of Et under protection of argon3N, stirring for 12h at room temperature, detecting by thin-layer chromatography, performing rotary evaporation to concentrate the reaction solution, and performing rapid silica gel column to obtain ganglioside GM3 and/or derivatives thereof;
wherein, the volume ratio of the rapid silica gel column is 1: 4 ethyl acetate/methanol.
8. The synthesis method according to claim 2, wherein the enzymes used in the one-pot three-enzyme sialylation in step ⑶ are Aldolase Pm Aldolase, Neisseria meningitis CMP-sialic acid synthase and Pasteurella multocida sialyltransferase 1 of bacterial origin, and the reaction time is 5 minutes to 2 hours.
9. The method according to any one of claims 2 to 8, wherein the reaction temperature in the enzymatic synthesis in step ⑶ is 0 to 37 ℃, the rotation speed is 0 to 240rpm, and the enzymatic reaction is stopped by adding an equal volume of 4 ℃ absolute methanol to the reaction and incubating at 4 ℃ for 0 to 30 minutes.
10. Use of the synthesis method according to any one of claims 2 to 9 for the preparation of the ganglioside GM3 and/or derivatives thereof.
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