Preparation method of sialic acid galactose disaccharide

Abstract

The invention relates to the technical field of synthesis of sugar derivatives, in particular to a preparation method of sialic acid galactose disaccharide. The arylboronic acid is used for temporarily protecting hydroxyl groups at positions 4 and 6 of galactose, and after a sialic acid donor and a catalyst are added, sialic acid can be selectively coupled to hydroxyl groups at positions 3. The method can realize 3-site hydroxyl selective reaction under the condition that four hydroxyl groups at 2, 3, 4 and 6 sites of galactose are exposed at the same time, and has good reaction regioselectivity. The steps of selectively feeding and removing protective groups on 2, 4 and 6 sites are reduced, the synthetic route of sialic acid galactose disaccharide is simplified, and the synthetic yield is further improved. In addition, the sialylation reaction of the method provided by the invention has complete alpha selectivity, alpha and beta isomers do not need to be separated, and separation and purification are further simplified.

Description

Technical Field

[0001] The present invention relates to the technical field of sugar derivative synthesis, in particular to a method for preparing sialic acid galactose disaccharide. Background Art

[0002] Sialic acid (Neu5Ac, N-acetylceramidic acid) is widely present in many cell-surface glycoconjugates and secreted glycoproteins. It participates in a variety of cellular events, such as viral infection, inflammation, and intercellular adhesion. Sialic acid, located at the terminus of the glycan chain, is a key determinant of glycan-protein interactions. Therefore, the synthesis of uniform sialic acid-containing glycans is crucial for uncovering their cellular functions. In nature, the glycosidic bonds between sialic acid and other sugars are α-oriented. From a synthetic perspective, sialic acid glycosylation is one of the most challenging practices in glycochemistry, typically resulting in low yields and poor stereoselectivity. The electron-withdrawing nature of the carboxylic acid group hinders the stability of the oxonium ion intermediate produced during glycosylation, thereby inhibiting the glycosylation reaction. Furthermore, glycoene byproducts, generated through the elimination of hydrogen at the 2- and 3-positions, often constitute a significant fraction. Furthermore, the lack of a directing group at the adjacent position (the 3-position) makes the stereoselectivity of the product difficult to control, often resulting in a mixture of α and β forms, posing significant purification challenges.

[0003]

[0004] In the human body, the most common structure of sialic acid is linked to galactose (Gal) via a 2,6- or 2,3-linkage. Compared to the former, because the 3-hydroxyl group of the galactose is a secondary hydroxyl group, the latter often has lower yields and stereoselectivity. When synthesizing complex structures, mixtures of α and β are difficult to separate, and products with a single configuration cannot be obtained. To address this challenge, scientists have proposed pre-establishing a sialic acid galactose disaccharide module with an α-2,3 linkage at an early stage and using this disaccharide module to construct larger and more complex polysaccharides.

[0005] However, even the synthesis of α-2,3-linked sialic acid galactosyl disaccharide modules usually requires a long synthetic route. To achieve regioselectivity at the 3-position linker, it is usually necessary to first protect the 2-, 4-, and 6-position hydroxyl groups. After the sialic acid coupling is completed, the protecting groups at these three positions are removed and further replaced with acetyl groups. To reduce the use of protecting groups, sometimes only the 6-position hydroxyl group is protected, but the above-mentioned steps of adding and removing protecting groups are still required. In addition, commonly used methods are usually unable to achieve complete α selectivity, which in many cases poses a great challenge to purification. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for preparing sialylgalactosyl disaccharide, which adopts the following technical scheme: using a galactose derivative I as an acceptor, adding an arylboronic acid and a desiccant to react, and then adding a sialic acid derivative donor II and a catalyst to react to generate sialylgalactosyl disaccharide III.

[0007]

[0008] In the preparation method provided by the present invention, compound I first reacts with arylphenylboronic acid to form intermediate IV: Ar is an aryl group. After adding Compound II and a catalyst, Compound II selectively couples with the 3-hydroxyl group because the 4- and 6-hydroxyl groups are shielded, while the 2-hydroxyl group is relatively inactive. After the reaction is complete, the arylboronic acid can be removed by direct extraction with water or saturated NaHCO3 solution.

[0009] Product III can be further converted into a sialic acid galactosyl disaccharide donor for subsequent attachment to other sugar structures.

[0010] Preferably, the aryl boronic acid is selected from phenylboronic acid, 2-ethylphenylboronic acid, 3-bromophenylboronic acid, 3-acetamidophenylboronic acid, 3-methanesulfonylaminophenylboronic acid, 4-methoxyphenylboronic acid, 4-chlorophenylboronic acid, 4-cyanophenylboronic acid, 4-isopropylphenylboronic acid, 4-isobutylphenylboronic acid, 4-carboxyphenylboronic acid, 2,6-dimethylphenylboronic acid, 3,5-difluorophenylboronic acid, 3,4-dimethoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 3,4,5-trimethoxyphenylboronic acid, 2-naphthaleneboronic acid or 9-anthraceneboronic acid.

[0011] Preferably, the catalyst is selected from trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate or tert-butyldimethylsilyl trifluoromethanesulfonate.

[0012] Preferably, the desiccant is selected from molecular sieves, anhydrous calcium chloride or anhydrous calcium sulfate.

[0013] Preferably, the molar ratio of the arylboronic acid to the galactose derivative I is 1.2 to 1:1.

[0014] Preferably, the molar ratio of the sialic acid derivative II to the galactose derivative I is 2 to 0.5:1.

[0015] Preferably, the solvent used is selected from acetonitrile, propionitrile, a mixture of dichloromethane and acetonitrile, a mixture of dichloromethane and propionitrile, a mixture of 1,2-dichloroethane and acetonitrile, a mixture of 1,2-dichloroethane and propionitrile, a mixture of ethyl acetate and acetonitrile, or a mixture of ethyl acetate and propionitrile.

[0016] Preferably, the reaction temperature of the second step is -100°C to -20°C.

[0017] The beneficial effects of the present invention are:

[0018] The method provided by the present invention can achieve selective reaction of the hydroxyl group at position 3 when all four hydroxyl groups at positions 2, 3, 4, and 6 in Compound I are simultaneously exposed, demonstrating excellent regioselectivity. This reduces the steps for selectively adding and removing protecting groups at positions 2, 4, and 6, streamlining the synthesis pathway for sialylated galactosyl disaccharide and further improving synthesis yield. Furthermore, the sialylation reaction method provided by the present invention exhibits complete α selectivity, eliminating the need for separation of α and β isomers and further simplifying separation and purification. DETAILED DESCRIPTION

[0019] Compound II used in this example can be synthesized according to existing literature reports (Org. Lett. 5, 3827-3830 (2003)) or purchased from the market. Other raw materials used in this example can be purchased from the market.

[0020] The arylboronic acid and catalyst used in Examples 1 to 4 are summarized as follows:

[0021] Example Arylboronic Acid catalyst 1 4-Methoxyphenylboronic acid TBSOTf 2 Phenylboronic acid TMSOT 3 3,5-Dimethoxyphenylboronic acid TfOH 4 4-Chlorophenylboronic acid TESOTf

[0022] Example 1. Product III was synthesized using 4-methoxyphenylboronic acid and tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) as a catalyst.

[0023] Compound I (413 mg, 1.47 mmol), 4-methylphenylboronic acid (235 mg, 1.55 mmol) and Molecular sieves (4 g) were mixed in anhydrous DCM / MeCN (1 / 1, v / v, 34 mL) and stirred at room temperature under nitrogen for 16 hours. After the reaction system was cooled to -65°C, compound II (1.47 g, 2.21 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 5 mL) was added, followed by TBSOTf (102 μL, 0.444 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 1 mL). The reaction was stirred at -65°C for 1.5 hours, then Et3N (4 eq.) was added to stop the reaction, and the temperature was raised to room temperature. The reaction mixture was then diluted with DCM (40 mL) and filtered through celite. The organic phase was separated and washed three times with saturated NaHCO3 solution (80 mL), twice with distilled water (80 mL), and once with saturated brine (80 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was further separated and purified by silica gel column chromatography (eluent: DCM / MeOH 50 / 1→45 / 1→40 / 1) to give product III (630 mg, 0.835 mmol) in a yield of 57%.

[0024] The high-resolution mass spectrometry and nuclear magnetic resonance data of product III are as follows:

[0025] HRMS m / z:Calcd.for C 31 H 51 NO 18 Si[M+H] + :1807.5991;found:1807.5981.

[0026] 1H NMR (400MHz, CDCl3) δ5.43(ddd,J=8.6,5.8,2.7Hz,1H),5.30(dd,J=8.8,1.9Hz,1H),5.24(d,J=9.6Hz,1H),4.95(ddd,J =12.1,9.9,4.6Hz,1H),4.42(d,J=7.7Hz,1H),4.30(dd,J=12.5,2.7Hz,1H),4.10–3.96(m,5H),3.93–3.84(m,2H),3.82 (s,3H),3.74(dd,J=3.4,1.2Hz,1H),3.71–3.61(m,2H),3.54(t,J=5.5,1.1Hz,1H),2.69(dd,J=13.0,4.7Hz,1H),2.13( s,3H),2.12(s,3H),2.08(dd,J=3.8,2.8Hz,1H),2.03(s,3H),2.03(s,3H),1.89(s,3H),1.15–0.93(m,2H),0.01(s,9H).

[0027] 13 C NMR (101MHz, CDCl3) δ170.87,170.70,170.27,170.12,170.03,168.23,102.53,97.70,76.81,73.62 ,72.66,69.29,68.58,68.55,68.17,67.16,67.03,62.51,62.24,53.21,49.94,37.41,23.15,21.19 20.80,20.76,20.73,18.18,-1.43.

[0028] Example 2. Product III was synthesized using phenylboronic acid and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a catalyst.

[0029] Compound I (413 mg, 1.47 mmol), phenylboronic acid (189 mg, 1.55 mmol) and Molecular sieves (4 g) were mixed in anhydrous 1,2-dichloroethane / MeCN (1 / 1, v / v, 34 mL) and stirred at room temperature under nitrogen for 16 hours. The reaction system was cooled to -60°C, and compound II (1.47 g, 2.21 mmol) dissolved in anhydrous 1,2-dichloroethane / MeCN (1 / 1, v / v, 5 mL) was added, followed by TMSOTf (80.4 μL, 0.444 mmol) dissolved in anhydrous 1,2-dichloroethane / MeCN (1 / 1, v / v, 1 mL). The reaction was stirred at -60°C for 1 hour, then quenched by the addition of Et3N (4 eq.), and then allowed to warm to room temperature. The reaction mixture was then diluted with DCM (40 mL) and filtered through celite. The organic phase was separated and washed three times with saturated NaHCO solution (80 mL), twice with distilled water (80 mL), and once with saturated brine (80 mL). The organic phase was dried over NaSO, filtered, and concentrated to obtain a crude product, which was further purified by silica gel column chromatography (eluent: DCM / MeOH 50 / 1→45 / 1→40 / 1) to obtain product III (610 mg, 0.809 mmol) in a yield of 55%.

[0030] Example 3. Product III was synthesized using 3,5-dimethoxyphenylboronic acid and trifluoromethanesulfonic acid (TfOH) as a catalyst.

[0031] Compound I (413 mg, 1.47 mmol), 3,5-dimethoxyphenylboronic acid (282 mg, 1.55 mmol) and Molecular sieves (4 g) were mixed in anhydrous DCM / MeCN (1 / 1, v / v, 34 mL) and stirred at room temperature under nitrogen for 16 hours. After the reaction system was cooled to -55°C, compound II (1.47 g, 2.21 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 5 mL) was added, followed by TfOH (39.3 μL, 0.444 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 1 mL). The reaction was stirred at -55°C for 1.5 hours, then Et3N (4 eq.) was added to stop the reaction, and the temperature was raised to room temperature. The reaction mixture was then diluted with DCM (40 mL) and filtered through celite. The organic phase was separated and washed with saturated NaHCO3 solution (80 mL) three times, distilled water (80 mL) twice, and saturated brine (80 mL) once. The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was further separated and purified by silica gel column chromatography (eluent: DCM / MeOH 50 / 1→45 / 1→40 / 1) to give product III (602 mg, 0.799 mmol) in a yield of 53%.

[0032] Example 4. Product III was synthesized using 4-chlorophenylboronic acid and triethylsilyl trifluoromethanesulfonate (TESOTf) as a catalyst.

[0033] Compound I (413 mg, 1.47 mmol), 4-chlorophenylboronic acid (242 mg, 1.55 mmol) and Molecular sieves (4 g) were mixed in anhydrous DCM / MeCN (1 / 1, v / v, 34 mL) and stirred at room temperature under nitrogen for 16 hours. After the reaction system was cooled to -65°C, compound II (1.47 g, 2.21 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 5 mL) was added, followed by TESOTf (100 μL, 0.444 mmol) dissolved in anhydrous DCM / MeCN (1 / 1, v / v, 1 mL). The reaction was stirred at -65°C for 1.5 hours, then Et3N (4 eq.) was added to stop the reaction, and the mixture was allowed to warm to room temperature. The reaction mixture was then diluted with DCM (40 mL) and filtered through celite. The organic phase was separated and washed three times with saturated NaHCO3 solution (80 mL), twice with distilled water (80 mL), and once with saturated brine (80 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was further separated and purified by silica gel column chromatography (eluent: DCM / MeOH 50 / 1→45 / 1→40 / 1) to give product III (610 mg, 0.809 mmol) in a yield of 55%.

[0034] Product III can be further converted to generate sialic acid galactose disaccharide donor for subsequent connection to other sugar structures. Here, the conversion to disaccharide donor IV

[0035] Take this as an example to illustrate.

[0036] Example 5. Synthesis of Disaccharide Donor IV.

[0037] Compound III (390 mg, 0.517 mmol) was dissolved in a mixed solution of Ac2O (8 mL) and pyridine (16 mL) and stirred at room temperature overnight. Subsequently, the reaction mixture was diluted with DCM (250 mL), washed 10 times with 1M hydrochloric acid solution (30 mL), 5 times with saturated NaHCO3 solution (60 mL), 3 times with distilled water (120 mL), and once with saturated brine (250 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude product was dissolved in a mixed solution of TFA (4 mL) and DCM (8 mL) and stirred at room temperature for 2 hours. The reaction mixture was concentrated, redissolved in DCM (50 mL), washed 3 times with saturated NaHCO3 solution (80 mL), 2 times with distilled water (80 mL), and once with saturated brine (80 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. Subsequently, azeotropic distillation of toluene was performed and vacuum drying was performed to ensure complete removal of residual water. The crude product was dissolved in DCM (10 mL) and CCl3CCN (1.04 mL, 10.4 mmol) was added. The reaction system was cooled to -30°C and DBU (39 μL, 0.259 mmol) was added. The reaction mixture was stirred at -30°C for 1 hour and then warmed to room temperature. Subsequently, it was filtered through celite and evaporated to dryness under reduced pressure. The crude product was separated and purified by silica gel column chromatography (eluent: hexane / acetone 3 / 1→2 / 1) to obtain product IV (350 mg, 0.379 mmol) with a yield of 73%.

[0038] The high-resolution mass spectrometry and NMR data of product IV are as follows:

[0039] HRMS m / z:Calcd.for C 34 H 45 Cl3N2O 21 [M+Na] + :945.1473; found:945.1506.

[0040] 1H NMR(400MHz,CDCl3):δ8.64(d,J=9.8Hz,1H),6.49(d,J=3.9Hz,1H),5.52(dd,J=5.6,3.1Hz,1H),5.39-5.20(m,3H),4.96(d,J=3.5 Hz,1H),4.86(dd,J=4.5,1.6Hz,1H),4.71(dd,J=10.1,3.5 Hz,1H),4.39(dd,J=12.4,2.4 Hz,1H),4.09-3.89(m,5H),3.83(s,3H),3.64(dd,J=10.7,2.7 Hz,1H),2.57(dd,J=12.6,4.6 Hz,1H),2.15(s,3H),2.13(s,3H),2.09(s,3H),2.03(s,3H),2.01(s,3H),2.00(s,3H),1.97(s,3H),1.81(s,3H),1.69(t,J=12.4 Hz,1H).

[0041] 13 C NMR(101 MHz,CDCl3):δ170.89,170.67,170.45,170.42,170.30,169.72,169.45,168.00,161.17,96.68,94.11,90.72,72.30,71.67,71.17,69.40,68.80,68.31,67.46,67.29,62.62,61.64,53.26,49.09,37.56,23.19,21.53,20.83,20.76,20.73。

Claims

1. A method for preparing sialic acid galactosyl disaccharide, characterized in that: Using galactose derivative I as an acceptor, adding arylboronic acid and a drying agent to react, then adding sialic acid derivative donor II and a catalyst to react to generate sialic acid galactosyl disaccharide III.

2. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein The aryl boronic acid is selected from phenylboronic acid, 2-ethylphenylboronic acid, 3-bromophenylboronic acid, 3-acetamidophenylboronic acid, 3-methanesulfonylaminophenylboronic acid, 4-methoxyphenylboronic acid, 4-chlorophenylboronic acid, 4-cyanophenylboronic acid, 4-isopropylphenylboronic acid, 4-isobutylphenylboronic acid, 4-carboxyphenylboronic acid, 2,6-dimethylphenylboronic acid, 3,5-difluorophenylboronic acid, 3,4-dimethoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 3,4,5-trimethoxyphenylboronic acid, 2-naphthaleneboronic acid or 9-anthraceneboronic acid.

3. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein The catalyst is selected from trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate or tert-butyldimethylsilyl trifluoromethanesulfonate.

4. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein The desiccant is selected from molecular sieves, anhydrous calcium chloride or anhydrous calcium sulfate.

5. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein The molar ratio of the arylboronic acid to the galactose derivative I is 1.2 to 1:

1.

6. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein The molar ratio of the sialic acid derivative II to the galactose derivative I is 2-0.5:

1.

7. The method for preparing sialic acid galactosyl disaccharide according to claim 1, wherein In the method, the solvent used is selected from acetonitrile, propionitrile, a mixture of dichloromethane and acetonitrile, a mixture of dichloromethane and propionitrile, a mixture of 1,2-dichloroethane and acetonitrile, a mixture of 1,2-dichloroethane and propionitrile, a mixture of ethyl acetate and acetonitrile, or a mixture of ethyl acetate and propionitrile.

8. The method for preparing O-sugar amino acids according to claim 1, wherein In the method, the reaction temperature of the second step is -100°C to -20°C.

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