A chemical synthesis method of EPS-G7

Abstract

The present invention provides a chemical synthesis method of EPS-G7, wherein β-cyclodextrin A is acetylated to obtain β-acetylcyclodextrin B, B is ring-opened in a mixed solution of acetic anhydride / concentrated sulfuric acid to obtain a chain acetylated heptose C, p-nitrophenol is condensed with intermediate C using boron trifluoride acetonitrile to obtain a key intermediate p-nitrophenyl (G1)-O-acetyl-D-maltoseptaglycoside D with α as the dominant configuration, D is removed from the acetyl group under the action of sodium methoxide to obtain pNP-G7 (p-nitrophenyl (G1)-D-maltoseptaglycoside), pNP-G7 is in the presence of p-toluenesulfonic acid and dimethoxyethane, and acetal protection is formed between the hydroxyl groups on C4 and C6 of the terminal monosaccharide to obtain EPS-G7. The reaction raw materials used in the method of the present invention are cheap and readily available, and the chemical synthesis of α-amylase detection reagent EPS-G7 is realized, which is a simple, economical and efficient synthesis method.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, relates to a synthesis method of an alpha-amylase detection reagent, and specifically relates to a chemical synthesis method of EPS-G7. Background Art

[0002] α-Amylase plays a key role in the human physiological cycle and is used as an important indicator of some related diseases, such as acute pancreatitis, acute organophosphorus pesticide poisoning, anxiety or depression, etc.

[0003] EPS-G7, as a substrate of α-amylase, is a commercial detection reagent for in vitro detection of α-amylase (AMS) activity in human serum or urine, with the advantages of high sensitivity and strong anti-interference ability. The chemical name of EPS-G7 is 4,6-dimethoxyethane-(G7)-p-nitrobenzene (G1)-α-D-maltoheptaglycoside, with a molecular weight of 1299.41. It is a white or nearly white solid powder with the following structural formula.

[0004]

[0005] EPS-G7 is obtained by one-step chemical modification of p-nitrobenzene (G1)-α-D-maltoheptaglycoside (pNP-α-G7), so the preparation of pNP-α-G7 is the key to the synthesis of EPS-G7. So far, pNP-α-G7 in the literature has been prepared by enzymatic methods, and there are two most common methods. The first is to open the α-cyclodextrin ring to obtain maltohexaose, and then synthesize pNP-α-G7 with p-nitrobenzene-α-D-glucoside (pNP-α-G1) under enzyme catalysis. The second is the transglycosylation method, in which pNP-α-G7 is prepared from pNP-α-G1 and maltoheptaglycoside under transferase catalysis.

[0006]

[0007] The key to the chemical synthesis of EPS-G7 lies in the α-selective synthesis of pNP-α-G7. However, most of the current glycosylation reaction methods generate β-glycosides, and the chemical synthesis of α-glycosides, especially 1,2-cis-α-glycosides, is difficult to achieve stereoselectivity. In addition, the existing methods have shortcomings such as long substrate activation steps, limited substrates, and the need for metal catalysts. Therefore, it is of great application value to develop a new synthetic method to achieve the construction of 1,2-cis-α-glycosidic bonds and apply it to the chemical synthesis of EPS-G7. Summary of the invention

[0008] The purpose of the present invention is to provide a chemical synthesis method of EPS-G7, and the synthesis route is as follows:

[0009]

[0010] This is achieved through the following steps:

[0011] (1) β-cyclodextrin A reacts with acetic anhydride at 20-30°C for 12 hours to obtain β-acetylcyclodextrin B;

[0012] (2) Dissolving the intermediate B in a mixture of acetic anhydride / concentrated sulfuric acid (V / V=49:1) and reacting at 40-60°C for 24 hours to obtain a chain acetylated heptasaccharide C;

[0013] (3) In the presence of a condensation reagent, the intermediate C reacts with p-nitrophenol at 0-40° C. for 1-9 h to obtain the key intermediate p-nitrophenyl (G1)-O-acetyl-D-maltoheptaglycoside D with α-dominant configuration;

[0014] (4) The key intermediate D is reacted with sodium methoxide at 20-30°C for 24-48h to obtain pNP-α-G7 (p-nitrophenyl (G1)-D-maltoheptaglycoside);

[0015] (5) pNP-α-G7 was reacted at 55 °C for 24 h in the presence of p-toluenesulfonic acid and dimethoxyethane to obtain EPS-G7 (4,6-ethylene-(G7)-p-nitrophenyl (G1)-α-D-maltoheptaglycoside).

[0016] The condensation reagent in step (3) can be boron trifluoride acetonitrile, boron trifluoride ether or tin tetrachloride, preferably boron trifluoride acetonitrile.

[0017] The amount of the condensation reagent in step (3) can be 1-4 equivalents, preferably 3 equivalents.

[0018] The reaction solvent in step (3) can be dichloromethane, acetonitrile, toluene or 1,2-dichloroethane, etc., preferably dichloromethane.

[0019] The reaction temperature in step (3) can be 0-40°C, preferably 25-30°C.

[0020] The reaction time in step (3) can be 1-9 h, preferably 4-5 h.

[0021] The synthesis method provided by the present invention uses β-cyclodextrin as the starting material, and sequentially undergoes five steps of acetylation, hydrolysis ring opening, condensation, ester hydrolysis and acetal protection. The reaction conditions are mild, and the reaction raw materials used are cheap and readily available, providing a simple and easy method for the chemical synthesis of EPS-G7. The chemical synthesis method described in the present invention has not been reported in the literature, and compared with enzymatic synthesis, this method is more economical and efficient.

[0022] The key to the synthetic route provided by the present invention is to design a condensation reaction of acetylated heptasaccharide and p-nitrophenol based on boron trifluoride acetonitrile coordination, and the α / β ratio can reach 80:20. This reaction avoids the cumbersome glycosyl donor or glycosyl acceptor pre-activation process, can be carried out at room temperature, has low reagent price, and simple post-processing operation, thus providing a simple method for the synthesis of oligosaccharide derivatives and glycosylation reactions. DETAILED DESCRIPTION

[0023] The invention is further explained and illustrated below in conjunction with specific embodiments, but they do not constitute limitation or restriction of the scope of the invention.

[0024] Example 1 Acetylation reaction

[0025]

[0026] Dissolve 28g of β-cyclodextrin A in 50mL of pyridine, cool to 0℃, add 60mL of acetic anhydride and 2.8g of DMAP, and stir at room temperature (20-30℃) for 12h. After the reaction of the raw materials, pour the reaction solution into 1000mL of water, extract with ethyl acetate (200mL×3), combine the organic phases, wash with hydrochloric acid (500mL×2, 1M), saturated sodium bicarbonate (500mL×2) and saturated sodium chloride (500mL) in sequence, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 50g of β-acetylcyclodextrin B. White powder; yield 99%; Mp235.1-236.4℃; 1 H NMR (500MHz, CDCl3) δ5.31(dd,J=9.7,8.1Hz,7H),5.09(d,J=3.8Hz,7H),4.83-4.77(m,7H),4.56(dd,J=12.6,2.0Hz,7H),4.28(d d,J=12.6,4.2Hz,7H),4.15(ddd,J=9.8,4.2,1.9Hz,7H),3.71(dd,J=9.6,8.0Hz,7H),2.13(s,21H),2.10(s,21H),2.06(s,21H); 13 C NMR (125MHz, CDCl3) δ170.8(7C), 170.5(7C), 169.5(7C), 96.8(7C), 77.2(7C), 70.8(7C), 70.4(7C), 69.6(7C), 62.5(7C), 20.8(7C), 20.8(14C).

[0027] Example 2 Glycosidic bond hydrolysis

[0028]

[0029] Dissolve 50g of β-acetylcyclodextrin B in a mixture of 100mL of acetic anhydride / concentrated sulfuric acid (V / V=49:1), stir at 50℃ for 24h, pour the resulting mixture into 1000mL of water, extract with ethyl acetate (200mL×3), combine the organic layers, and wash with saturated sodium bicarbonate (500mL×2) and saturated sodium chloride (500mL×2) in sequence. After drying over anhydrous sodium sulfate, filter and concentrate, the obtained crude product is recrystallized three times with ethanol to obtain 28.2g of intermediate C. White powder; yield: 54%; Mp141.8-143.2℃; 1 H NMR (500MHz, CDCl3) δ6.23 (d, J = 3.7Hz, 1H), 5.50 (t, J = 9.5Hz, 1H), 5.42-5.27 (m, 12H), 5.06 (t, J = 9.9 Hz,1H),4.94(dd,J=10.0,3.8Hz,1H),4.84(dd,J=10.5,4.1Hz,1H),4.72(dt,J=9.9,4.5Hz,5H),4.58 -4.44(m,6H),4.36-4.09(m,8H),4.04(dd,J=10.4,6.1Hz,2H),4.00-3.84(m,11H),2.22(s,3H),2.20 (s,9H),2.18(s,6H),2.14(s,3H),2.09(s,3H),2.06-2.00(m,21H),1.99(s,12H),1.98-1.95(m,12H); 13 C NMR (125MHz, CDCl3) δ170.8(3C),170.7(3C),170.6,170.5,170.5(5C),169.9(2C),169.8,169. 8,169.6(2C),169.5(3C),169.0,95.9,95.7,95.7(2C),95.6(2C),88.9,73.2(4C),72.3,72.2,7 1.7(5C),70.5(5C),70.2,70.0,69.8(2C),69.3,69.1,68.9(3C),68.4,67.9(2C),62.3(4C),62 .1(2C),61.3,21.0,20.9(3C),20.9(5C),20.8(4C),20.8(2C),20.7,20.6(3C),20.5(3C),20.4.

[0030] Example 3 Condensation reaction

[0031]

[0032] Mix 3.18g of intermediate C and 2.8g of p-nitrophenol in 6mL of dichloromethane, add 0.486g of solid boron trifluoride acetonitrile after dissolving, and stir at 25-30℃ for 4h. After the reaction is completed, add 20mL of water to the reaction solution to quench the reaction, stir, separate, and wash the organic layer with saturated sodium bicarbonate and saturated sodium chloride in turn. After drying over anhydrous sodium sulfate, filter and concentrate. The crude product is separated by silica gel column chromatography (petroleum ether: ethyl acetate = 2:3) to obtain 1.43g of a mixture of intermediates D-α and D-β (α / β = 80:20). White powder; yield: 43%; Mp135.9-137.6℃; 1 H NMR (500MHz, CDCl3) δ8.23(d,J=9.3Hz,2H),7.24(d,J=9.2Hz,2H),5.76-5.66(m,1H),5.42-5.24(m,13H),5.05(t,J=9.9Hz ,1H),4.93(dd,J=10.2,3.6Hz,1H),4.84(dd,J=10.5,4.0Hz,1H),4.75-4.66(m,5H),4.54-4.43(m,6H),4.26(dddd,J=16.4, 10.0,6.9,3.5Hz,5H),4.20(dd,J=12.3,2.5Hz,1H),4.15(dd,J=12.3,2.6Hz,1H),4.08-4.00(m,3H),3.95-3.86(m,11H),2 .20-2.16(m,12H),2.13(s,6H),2.08(s,3H),2.05-2.03(m,12H),2.02-2.00(m,9H),1.99-1.98(m,15H),1.97-1.93(m,9H); 13CNMR(125MHz, CDCl3)δ170.8,170.7(2C),170.7(2C),170.6,170.5,170.5,170.5,170.4(2C),170.3(2C),170.2,169.8(2 C),169.6(2C),169.6(2C),169.5,169.5,160.7,143.2,125.9(2C),116.7(2C),95.9,95.7,95.7(2C),95.6(2C),94.1,73 .2(4C),72.3,72.1(2C),71.7(3C),71.6(2C),70.5(3C),70.5(3C),70.1(2C),69.4(2C),69.2(2C),68.9(4C),68.4,67.9 ,62.3(3C),62.1,61.4,20.9,20.9(4C),20.9(3C),20.8(2C),20.8,20.7(2C),20.6(2C),20.6(3C),20.5(2C),20.5(2C).

[0033] Example 4 Condensation reaction

[0034]

[0035] Mix 3g of intermediate C and 2.7g of p-nitrophenol in 6mL of dichloromethane, add 0.47mL of boron trifluoride ether after dissolving, and stir at 25-30℃ for 60-72h. Add 20mL of water to the reaction solution to quench the reaction, stir, separate, and wash the organic layer with saturated sodium bicarbonate and saturated sodium chloride in sequence. After drying over anhydrous sodium sulfate, filter and concentrate. The obtained crude product is separated by silica gel column chromatography (petroleum ether: ethyl acetate = 2:3) to obtain 1.2g of a mixture of intermediates D-α and D-β (α / β = 75:25), with a yield of 38%.

[0036] Example 5 Condensation reaction

[0037]

[0038] Mix 3g of intermediate C and 2.7g of p-nitrophenol in 6mL of dichloromethane, add 0.5mL of tin tetrachloride after dissolving, and stir at 25-30℃ for 12-20h. Add 20mL of water to the reaction solution to quench the reaction, stir, separate, and wash the organic layer with saturated sodium bicarbonate and saturated sodium chloride in sequence. After drying over anhydrous sodium sulfate, filter and concentrate. The obtained crude product is separated by silica gel column chromatography (petroleum ether: ethyl acetate = 2:3) to obtain 1.26g of a mixture of intermediates D-α and D-β (α / β = 60:40), with a yield of 40%.

[0039] Example 6 Ester hydrolysis reaction

[0040]

[0041] 1 g of intermediate D (α / β=80:20) was dissolved in a mixed solution of 10 mL of DCM / MeOH (V / V=1:4). After cooling to 0°C, 0.2 g of sodium methoxide was added in batches and stirred at room temperature (20-30°C) for 24-48 h. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed with DCM and dried to obtain 0.52 g of light yellow solid pNP-G7 with a yield of 90%.

[0042] Example 7 Acetal Protection

[0043]

[0044] 0.52g pNP-G7 was dissolved in 10mL DMF, 0.2g p-toluenesulfonic acid and 0.28g dimethoxyethane were added to the solution, and stirred at 55°C for 24h. The reaction solution was poured into 100mL ethyl acetate to precipitate the product, which was filtered and the filter cake was washed with ethyl acetate. The filter cake was separated and purified by reverse phase column chromatography (methanol / water, 20:80) to obtain 0.313g of white solid final product EPS-G7 with a yield of 59%. Mp213.6-214.8℃; [α] D 25+126.5(c 0.55,CHCl3); IR(KBr):1594,1345,1026,870cm -1 ; 1 H NMR (500MHz, DMSO-d6) δ8.22(d,J=9.2Hz,2H),7.29(d,J=9.3Hz,2H),5.65(d,J=3.6Hz,1H),5.61-5.49(m, 7H),5.44(dt,J=8.0,3.5Hz,4H),5.41(d,J=3.3Hz,1H),5.37(d,J=6.4Hz,1H),5.24(d,J=5.3Hz,1H),5.05( ddd,J=14.2,9.8,4.1Hz,6H),4.68(q,J=5.0Hz,1H),4.63(t,J=5.6Hz,1H),4.59-4.50(m,5H),3.90(td,J= 9.3,8.8,3.7Hz,2H),3.70-3.38(m,29H),3.34-3.28(m,10H),3.10(t,J=9.4Hz,1H),1.20(d,J=5.0Hz,3H); 13C NMR(125MHz,DMSO-d6)δ162.1,141.7,125.8(2C),117.0(2C),101.1,100.3(2C),100.2(2C),98.6,97.4,80.4,79.2(3C),79.1,79.0(2C),73.2,73.1(5C),73.0,72.7,72.4,72.0(5C),71.6(5C),70.9 70.0,67.5,63.2,60.3(5C),60.0,19.9;HRMS(ESI)m / z calcd for C 50 H 77 O 38 Na[M+Na] + 1322.4021,found 1322.4029。

Claims

1. A chemical synthesis method of EPS-G7, characterized in that: This is accomplished by following these steps: (1) β -Cyclodextrin A reacts with acetic anhydride at 20-30°C for 12 h to obtain β -Acetylcyclodextrin B; (2) Dissolve the intermediate B in a mixture of acetic anhydride and concentrated sulfuric acid in a volume ratio of 49:1, and react at 40-60°C for 24 h to obtain a chain acetylated heptasaccharide C; (3) In the presence of a condensation reagent, intermediate C reacts with p-nitrophenol at 0-40°C for 1-9 h to obtain α The key intermediate p-nitrophenyl (G1)-O-acetyl-D-maltoheptaglycoside D has a dominant configuration; (4) The key intermediate D is reacted with sodium methoxide at 20-30°C for 24-48 h to obtain pNP-α-G7 (p-nitrophenyl (G1)-D-maltoheptaglycoside); (5) pNP-α-G7 was reacted at 55 °C for 24 h in the presence of p-toluenesulfonic acid and dimethoxyethane to obtain EPS-G7 (4,6-ethylene-(G7)-p-nitrophenyl (G1)- α -D-maltoheptaglycoside); The synthetic route is as follows: 。 2. The chemical synthesis method according to claim 1, characterized in that The condensation reagent in step (3) is boron trifluoride acetonitrile, boron trifluoride ether or tin tetrachloride.

3. The chemical synthesis method according to claim 2, characterized in that: The condensation reagent in step (3) is boron trifluoride acetonitrile.

4. The chemical synthesis method according to claim 1, characterized in that The amount of the condensation reagent in step (3) is selected to be 1-4 equivalents.

5. The chemical synthesis method according to claim 4, characterized in that The amount of the condensation reagent in step (3) is selected to be 3 equivalents.

6. The synthesis method according to claim 1, characterized in that The reaction solvent in step (3) is selected from dichloromethane, acetonitrile, toluene or 1,2-dichloroethane.

7. The chemical synthesis method according to claim 6, characterized in that The reaction solvent was dichloromethane.

8. The chemical synthesis method according to claim 1, characterized in that The reaction temperature in step (3) is selected to be 25-30°C.

9. The chemical synthesis method according to claim 1, characterized in that: The reaction time in step (3) is selected to be 4-5 h.

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