Application of Helicobacter pylori O2 serotype O antigen sugar fragment in preparation of Helicobacter pylori infection detection device

Abstract

The application discloses application of a Helicobacter pylori O2 serum type O antigen sugar fragment in preparation of a Helicobacter pylori infection detection device and belongs to the technical field of Helicobacter pylori infection detection.The Helicobacter pylori O2 serum type O antigen sugar fragment is dissolved in a phosphate solution, then is printed on a chip, is incubated, and is made to be covalently combined with the chip to obtain a sugar chip; the obtained sugar chip is placed in a diluted human serum solution, is incubated, and is made to combine a human serum antibody recognizing a synthetic sugar fragment with an antibody on the sugar chip; subsequently, a fluorescently labeled anti-human IgG antibody is used as a secondary antibody, is combined with the human serum antibody on the sugar chip, and a Helicobacter pylori detection chip is obtained.The application provides a new method for judging whether Helicobacter pylori is infected by detecting IgG antibodies in human serum based on the Helicobacter pylori detection chip, and the method is efficient and accurate.

Description

Technical Field

[0001] This invention specifically relates to the application of the Helicobacter pylori O2 serotype O antigen sugar fragment in the preparation of a Helicobacter pylori infection detection device, and belongs to the field of Helicobacter pylori infection detection technology. Background Technology

[0002] Helicobacter pylori infection: Approximately 50% of the world's population is infected with Helicobacter pylori (Hooi et al., Gastroenterology, 2017, 153(2):420–29). Infection with Helicobacter pylori can cause a variety of diseases, such as chronic gastritis, peptic ulcers, and gastric cancer (Sun et al., Crit. Rev. Food Sci. Nutr., 2022, 62(7):1713–24). Helicobacter pylori has been identified as a Group 1 carcinogen by the World Health Organization (Huang et al., Front. Cell. Infect. Microbiol., 2016, 6). Currently, Helicobacter pylori infection is mainly detected through the urea breath test, which may produce false positives or false negatives due to medications or the presence of other bacteria in the gastrointestinal tract. Therefore, developing a more convenient and effective detection method to identify Helicobacter pylori infection is of great significance to human health. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for detecting whether a patient is infected with Helicobacter pylori, enabling rapid detection of Helicobacter pylori infection in patients.

[0004] This invention provides the application of a Helicobacter pylori O2 serotype O antigen glycoside fragment in the preparation of a Helicobacter pylori infection detection device, wherein the Helicobacter pylori O2 serotype O antigen glycoside fragment is selected from any one or more of the following:

[0005]

[0006] In one embodiment of the present invention, the sugar fragment of Helicobacter pylori O2 serotype O antigen is preferably disaccharide 2, or a combination of disaccharide 2 and other sugar fragments.

[0007] The present invention also provides a Helicobacter pylori infection detection chip, comprising the following components: a chip, a Helicobacter pylori O2 serotype O antigen sugar fragment bound to the chip, a human serum antibody bound to the Helicobacter pylori O2 serotype O antigen sugar fragment, and a fluorescently labeled anti-human IgG antibody bound to the human serum antibody.

[0008] This invention also provides a method for preparing a Helicobacter pylori infection detection chip, comprising:

[0009] (1) The above-mentioned Helicobacter pylori O2 serotype O antigen sugar fragment was dissolved in phosphate solution, then printed on a chip, and incubated to allow the sugar fragment to covalently bind to the chip, thus obtaining a sugar chip;

[0010] (2) The obtained sugar chip was placed in a diluted human serum solution and incubated to allow the human serum antibody that recognizes the synthetic sugar fragment to bind to the antibody on the sugar chip; then, fluorescently labeled anti-human IgG antibody was used as a secondary antibody to bind to the human serum antibody on the sugar chip to obtain a Helicobacter pylori infection detection chip.

[0011] In one embodiment of the present invention, the concentration of the Helicobacter pylori O2 serotype O antigen sugar fragment relative to the phosphate solution in step (1) is 25mM-75mM. Specifically, 50mM can be selected.

[0012] In one embodiment of the present invention, the pH of the phosphate solution in step (1) is 8-9. Specifically, 8.5 is optional.

[0013] In one embodiment of the present invention, the incubation temperature in step (1) is room temperature (20-30°C); the humidity is 50%-70%. Specifically, the humidity can be 65%.

[0014] In one embodiment of the present invention, the human serum is diluted to a ratio of 1:10 to 1:100 in step (2). Specifically, a dilution of 1:20 may be selected.

[0015] In one embodiment of the present invention, the fluorescently labeled anti-human IgG antibody in step (2) refers to: cy3-labeled goat anti-human IgG antibody.

[0016] In one embodiment of the present invention, the ratio of fluorescently labeled anti-human IgG antibody to human serum in step (2) is 1:30 to 1:1000. Specifically, 1:400 may be selected.

[0017] In one embodiment of the present invention, the above-mentioned Helicobacter pylori infection detection chip is specifically prepared by the following method:

[0018] (1) The sugar fragment of Helicobacter pylori O2 serotype O antigen was dissolved in 50 mM phosphate solution (pH 8.5) and printed onto the chip using a chip spotting instrument. It was incubated overnight at room temperature and 65% humidity to allow the sugar fragment to covalently bind to the chip. After incubation, it was treated with a mixed solution of 100 nM ethanolamine and 50 nM sodium phosphate (pH 9) at 50°C for 1 hour to remove unbound chip surface sites and obtain the sugar chip.

[0019] (2) Human serum was diluted and incubated with the sugar fragments on the sugar chip to allow the human serum antibody that recognizes the synthesized sugar fragments to bind to the antibody on the sugar chip, and the unbound serum antibody was washed away; then, fluorescently labeled anti-human IgG antibody was used as a secondary antibody to bind to the human IgG antibody bound to the chip, and the unbound secondary antibody was washed away.

[0020] In one embodiment of the present invention, the obtained Helicobacter pylori detection chip is scanned on a microarray scanner, and the results of the sugar chip are used to determine whether the patient is infected with Helicobacter pylori.

[0021] The present invention also provides a Helicobacter pylori infection detection device containing the above-mentioned Helicobacter pylori infection detection chip.

[0022] Beneficial effects:

[0023] This invention provides a method for determining whether a person is infected with Helicobacter pylori by detecting IgG antibodies in human serum. Using the Helicobacter pylori detection chip of this invention, serum from 153 patients who tested positive in the carbon-13 breath test was screened. The results showed that the antibodies in the serum of 135 of these patients could bind to the synthesized disaccharide fragment. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the Helicobacter pylori infection detection chip of the present invention.

[0025] Figure 2 This is a roadmap for synthesizing the target product 3* in Example 2.

[0026] Figure 3 The roadmap for synthesizing the target products 5*, 8*, 10*, 12*, and 14*.

[0027] Figure 4 A method for synthesizing oligosaccharides of Helicobacter pylori O2 serotype O antigen.

[0028] Figure 5 A schematic diagram of spotting sugar chips.

[0029] Figure 6 This image shows the results of serum analysis of the first 90 patients who tested positive in a carbon-13 breath test out of 153 patients using a sugar chip.

[0030] Figure 7 This image shows the results of serum analysis in the last 63 patients who tested positive in a carbon-13 breath test out of 153 patients using a sugar chip. Detailed Implementation

[0031] Examples are provided for the content included in the claims.

[0032] The monosaccharide building blocks and their bi, tetra, and hexasaccharide synthesis methods were prepared according to the following reference: Tian G, Qin C, Liu Z, et al. Total synthesis of the: Helicobacter pylori serotype O2 O-antigenα-(1→2)-Andα-(1→3)-linked oligoglucosides. Chem Commun. 2020; 56(3):344-347.

[0033] The structures of the tetrasaccharide and the hexasaccharide are as follows:

[0034] Synthesize fully α-linked, protected mono-, tri-, and pentasaccharides, as well as di- and tri-saccharides with different linkage sequences. Figure 2 , Figure 3 Finally, the protection was removed to obtain the Helicobacter pylori O2 serotype O antigen oligosaccharide containing amino-linked arms. Figure 4 ).

[0035] Example 1:

[0036] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-2,4-di-O-benzyl-3-O-acetylpropionyl-α-D-glucopyranoside (2*)

[0037] The reaction equation is as follows Figure 2 As shown:

[0038] N-phenyltrifluoroacetylimine ester 6-O-acetyl-2,4-di-O-benzyl-3-O-acetylpropionyl-α-D-glucopyranoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (134 mg, 0.2 mmol) and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol (131 mg, 0.4 mmol) were dissolved in toluene, azeotropically removed three times by rotary evaporation, and pre-activated [product name missing] was added to the oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (10 mL) was added under argon protection, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was cooled to 0°C, and trimethylsilyl trifluoromethanesulfonate (3.6 μL, 0.02 mmol) was added dropwise. The mixture was stirred at 0°C for 4 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 3:1, v / v) to obtain α and β products (83%, α:β > 20:1), with the α configuration being the target product 2* (135 mg, 0.166 mmol). [α] 25 D = +45.97 (c 1.0, CHCl3). 1 H NMR(400MHz,Chloroform-d)δ7.42–7.12(m,20H,Ar-H),5.53(t,J=9.6Hz,1H,3-H),5.16(d,J=11.5Hz,2H,Ar-CH) ,4.69(s,1H,1-H),4.65–4.40(m,6H,Ar-CH),4.23(t,J=2.7Hz,2H,6-H),3.85(s,1H,5-H),3.56(s,1H,Linker-OCH a ),3.51(d,J=9.5Hz,1H,4-H),3.44(dd,J=10.0,3.5Hz,1H,2-H),3.21(d,J=28.8Hz,3H,Linker-OCH b ,Linker-NCH2),2.75–2.40(m,4H,Lev-CH2),2.15(s,3H,CH3CO),2.03(s,3H,CH3CO),1.56(d,J=31.7Hz,4H,Linker-CH2),1.28(d,J=23.4Hz,2H,Linker-CH2). 13C NMR (101MHz, CDCl3) δ206.37,171.79,170.64,138.09,137.93,137.50,128.53,128.47,128.43,1 28.15,127.97,127.90,127.85,127.82,127.25,96.63(C-1),77.35,77.24,77.04,76.72,76.06, 74.38,74.06,72.62,68.32,67.14,62.92,50.56,50.26,47.12,46.22,37.84,29.92,29.09,28.1 2,28.00,27.52,23.46,20.87.IR(film):ν=1743,1697,1417,1362,1231,1073,699.HRMS(ESI)m / z calcd for C 47 H 55 NO 11 Na[M+Na] + 832.3667, found 832.3666.

[0039] Example 2:

[0040] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside (3*)

[0041] The reaction equation is as follows Figure 2 As shown:

[0042] Under argon protection, 2* (63 mg, 0.078 mmol) was dissolved in a 20:1 (v / v) dichloromethane / methanol mixture (0.84 mL), and hydrazine acetate (11 mg, 0.119 mmol) was added. The mixture was stirred at room temperature for 3.5 hours. After complete reaction of the starting material by TLC, the reaction solution was diluted with dichloromethane and washed successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1, v / v) to obtain the target product 3* (50 mg, 0.07 mmol, 90%). [α] 25 D = +68.10(c 1.0, CHCl3). 11H NMR (400 MHz, Chloroform-d) δ 7.60–6.87 (m, 20H, Ar-H), 5.16 (d, J = 10.0 Hz, 2H, Ar-CH), 4.90 (d, J = 11.2 Hz, 1H, Ar-CH), 4.70 (s, 1H, 1-H), 4.64 (m, 3H, Ar-CH), 4.48 (d, J = 7.9 Hz, 2H, Ar-CH), 4.25 (qd, J = 11.9, 3.4 Hz, 2H, 6-H), 4.09 (t, J = 9.1 Hz, 1H, 3-H), 3.78 (d, J = 9.7 Hz, 1H, 5-H), 3.54 (d, J = 8.9 Hz, 1H, Linker-OCH a ), 3.44–3.39 (m, 1H, 4-H), 3.35 (dd, J = 9.6, 3.5 Hz, 1H, 2-H), 3.32–3.11 (m, 3H, Linker-OCH b , Linker-NCH2), 2.00 (s, 3H, CH3CO), 1.66–1.45 (m, 4H, Linker-CH2), 1.28 (d, J = 22.5 Hz, 2H, Linker-CH2). 13 13C NMR (101 MHz, CDCl3) δ 170.75, 138.06, 137.91, 128.59, 128.55, 128.49, 128.45, 128.26, 128.14, 128.02, 127.92, 127.84, 96.23 (C-1), 79.67, 77.36, 77.04, 76.89, 76.72, 74.56, 73.61, 72.84, 68.32, 68.11, 67.17, 63.21, 50.54, 50.25, 47.12, 46.18, 29.15, 28.00, 27.53, 23.51, 20.86. IR (film): ν = 2935, 1697, 1454, 1235, 1072, 699. HRMS (ESI) m / z calcd for C 42 H 49 NO9Na [M+Na] + 734.3300, found 734.3300.

[0043] Example 3:

[0044] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside (5*)

[0045] The reaction equation is as follows Figure 3 As shown:

[0046] N-phenyltrifluoroacetylimine ester 6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (126.6 mg, 0.174 mmol) and 3* (41.3 mg, 0.058 mmol) were dissolved in toluene, azeotropically removed three times on rotary evaporator, and pre-activated [agent / concentrate] was added to an oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (2.9 mL) was added under argon protection, and the mixture was stirred at 0°C for 15 minutes. Trimethylsilyl trifluoromethanesulfonate (3.15 μL, 0.0174 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 4.5 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction mixture was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 4:1, v / v) to obtain α and β products (69%, α:β = 20:1), where the α configuration was the target product 5* (50 mg, 0.04 mmol). [α] 25 D = +62.48 (c 1.0, CHCl3). 1H NMR(400MHz,Chloroform-d)δ7.98–7.88(m,2H,Ar-H),7.69–7.62(m,1H,Ar-H),7.58–7.52(m,1H,Ar-H),7.49–7.45(m,1H,Ar-H),7.43–7.13(m,29H,Ar-H),7.08–7.03(m,2H,Ar-H),7.01(dd,J=8.4,1.7Hz,1H,Ar-H),5.96(dd,J=10.2,9.1Hz,1H,3-H),5.63(d,J=3.5Hz,1H,1-H),5.17(d,J=11.6Hz,2H,NCbz-CH2),5.03(d,J=11.6Hz,1H,Ar-CH),4.76(s,1H,1-H'),4.63(dd,J=11.6,6.1Hz,2H,Ar-CH),4.57–4.40(m,7H,5-H,Ar-CH),4.36(d,J=10.9Hz,1H,Ar-CH),4.29–4.22(m,2H,3-H',6-H'-1H),4.20–4.07(m,2H,6-H-1H,6-H'-1H),3.90(d,J=12.4Hz,1H,6-H-1H),3.82(s,1H,5-H'),3.72–3.52(m,5H,2-H,2-H',4-H,4-H',Linker-OCH),3.35–3.14(m,3H,Linker-NCH2,Linker-OCH),2.02(d,J=10.5Hz,6H,CH3CO),1.50(s,4H,Linker-CH2),1.40–1.21(m,2H,Linker-CH2). 13C NMR (101MHz, CDCl3) δ170.71,170.66,165.55,138.06,137.93,137.60,137.44,134.62,133.04,132.90,132.85,130.11,129.71,128.54 ,128.46,128.39,128.35,128.23,128.16,128.04,127.97,127.85,127.80,127.76,127.58,127.47,127.25,126.92,126.80,125.91,12 5.89,125.82,97.00(C-1),96.20(C-1'),78.59,78.55,77.35,77.24,77.03,76.72,75.88,74.34,74.21,73.74,73.28,72.83,68.43,68 .21,68.12,67.17,62.82,50.61,46.21,29.16,27.54,23.45,20.96,20.91.IR(film):ν=2935,1739,1454,1235,1072,699.HRMS(ESI)m / z calcd for C 75 H 79 NO 16 Na[M+Na] + 1272.5291, found1272.5291.

[0047] Example 4:

[0048] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-2,4-di-O-benzyl-3-acetylpropionyl-α-D-glucopyranosyl-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranosyl glycoside (8*)

[0049] The reaction equation is as follows Figure 3 As shown:

[0050] N-phenyltrifluoroacetylimine ester 6-O-acetyl-2,4-di-O-benzyl-3-acetylpropionyl-α-D-glucopyranosyl glycoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (140 mg, 0.176 mmol) and 6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranosyl glycoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (165.4 mg, 0.246 mmol) were dissolved in toluene, azeotropically removed three times by rotary evaporation, and pre-activated [product name missing] was added to the oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (8.8 mL) was added under argon protection, and the mixture was stirred at 0°C for 15 minutes. Trimethylsilyl trifluoromethanesulfonate (4.45 μL, 0.0246 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 3.5 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction mixture was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 2:1, v / v) to obtain the α-configuration target product 8* (157.4 mg, 0.13 mmol, 74%).

[0051] Example 5:

[0052] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside (10*)

[0053] The reaction equation is as follows Figure 3 As shown:

[0054] N-phenyltrifluoroacetylimine ester 6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (91.7 mg, 0.126 mmol) and N-benzyl-N-benzyloxycarbonyl-5-aminopentyl 6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside (46.7 mg, 0.042 mmol) (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (91.7 mg, 0.126 mmol) Commun. 2020; 56(3):344-347.) Dissolve in toluene, azeotropically remove water three times on rotary evaporator, and add pre-activated [product / material] to the oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (2.1 mL) was added under argon protection, and the mixture was stirred at 0°C for 15 minutes. Trimethylsilyl trifluoromethanesulfonate (2.3 μL, 0.0126 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 4 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction mixture was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 3:2, v / v) to obtain the α-configuration target product 10* (36.5 mg, 0.022 mmol, 53%). [α] 25 D = +94.41(c 1.0, CHCl3). 1H NMR(400MHz,Chloroform-d)δ8.12–6.87(m,47H,Ar-H),5.99–5.80(m,2H,3-H,3-H”),5.42(d,J=3.4Hz,1H,1'-H),5.15(d,J=11.5Hz,2H,Ar-CH),5.01(d,J=3.4Hz,1H,1”-H),4.96(s,1H,1-H),4.76(d,J=11.9Hz,1H,Ar-CH),4.66–4.42(m,8H,5”-H,Ar-CH),4.38(d,J=10.9Hz,1H,Ar-CH),4.33–4.23(m,5H,6”-H,Ar-CH),4.11–3.99(m,3H,6-H-1H,3'-H,6'-H-1H),3.96(s,1H,5-H),3.89(d,J=12.2Hz,1H,6-H-1H),3.83(dd,J=10.1,3.4Hz,1H,2-H),3.78–3.64(m,3H,6'-H-1H,4-H,5'-H),3.60(d,J=9.3Hz,1H,4'-H),3.56(d,J=6.6Hz,1H,Linker-OCH),3.54–3.46(m,3H,2'-H,2”-H,4”-H),3.29(d,J=11.9Hz,1H,Linker-OCH),3.15(d,J=31.6Hz,2H,Linker-NCH),2.13–1.95(m,9H,CH3CO),1.45(s,4H,Linker-CH2),1.24–1.08(m,2H,Linker-CH2). 13C NMR (101MHz, CDCl3) δ170.65,170.51,165.56,165.18,137.99,137.47,137.17,137.0 0,134.56,133.15,132.99,132.87,132.83,130.10,129.84,129.68,129.64,128.52, 128.50,128.47,128.42,128.35,128.17,128.15,128.12,128.05,128.01,127.98,12 7.85,127.79,127.68,127.58,127.24,126.81,126.53,125.89,125.81,96.58(C-1'), 95.87(C-1),94.78(C-1”),77.95,77.68,77.36,77.04,76.72,76.12,75.82,75.56,7 4.83,74.62,74.21,74.13,73.48,73.05,72.84,72.48,68.67,68.50,68.21,67.13,6 2.87,62.63,62.16,50.49,50.17,47.06,29.72,29.24,27.92,27.45,23.41,20.98,2 0.91,20.86.IR(film):ν=2929,2359,1735,1453,1365,1237,1071,700.HRMS(ESI)m / z calcd for C 97 H 101 NO 23 Na[M+Na] + 1671.6690, found 1671.6692.

[0055] Example 6:

[0056] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-2,4-di-O-benzyl-3-O-acetylpropionyl-α-D-glucopyranoside-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside (12*)

[0057] The reaction equation is as follows Figure 3 As shown:

[0058] N-phenyltrifluoroacetylimide 6-O-acetyl-2,4-di-O-benzyl-3-O-acetylpropionyl-α-D-glucopyranosyl-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (40.5 mg, 0.0379 mmol) and 3* (22.5 mg, 0.0316 mmol) were dissolved in toluene, azeotropically removed three times on rotary evaporator, and pre-activated [agent / concentrate] was added to the oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (1.7 mL) was added under argon protection, and the mixture was stirred at 0°C for 15 minutes. Trimethylsilyl trifluoromethanesulfonate (0.69 μL, 0.0038 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 4 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction mixture was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 5:1, v / v) to obtain the α-configuration target product 12* (29 mg, 0.0182 mmol, 58%). [α] 25 D = +59.82(c 1.0, CHCl3). 1H NMR(600MHz,Chloroform-d)δ8.05–7.74(m,2H,Ar-H),7.53–6.93(m,38H,Ar-H),5.89(t,J=9.4Hz,1H,3-H'),5.73(d,J=3.6Hz,1H,1-H),5.50–5.41(m,1H,3-H”),5.18(dd,J=19.6,14.3Hz,3H,Ar-CH),4.75–4.65(m,3H,1-H',1-H”,Ar-CH),4.63–4.58(m,1H,5-H'),4.56(d,J=7.8Hz,1H,Ar-CH),4.52–4.12(m,13H,3-H,6-H,6-H'-1H,Ar-CH),4.07(s,2H,6-H”),3.98(d,J=12.4Hz,1H,6-H'-1H),3.94(d,J=10.2Hz,1H,5-H”),3.80(d,J=13.1Hz,1H,5-H),3.68(dd,J=10.1,8.5Hz,1H,4-H),3.61–3.44(m,4H,2-H,2-H',4-H',Linker-OCH),3.38(t,J=9.6Hz,1H,4-H”),3.27(m,4H,2-H”,Linker-OCH,Linker-NCH2),2.67–2.51(m,2H,Lev-CH2),2.47–2.29(m,2H,Lev-CH2),2.13(s,3H,CH3CO),2.07(s,3H,CH3CO),2.03(s,3H,CH3CO),1.82(s,3H,CH3CO),1.58(s,4H,Linker-CH2),1.26(t,J=18.6Hz,2H,Linker-CH2). 13C NMR (151MHz, CDCl3) δ206.22,171.23,171.05,170.63,170.34,165.35,1 38.89,138.22,137.93,137.77,137.50,137.33,132.72,130.38,129.52 ,128.52,128.43,128.38,128.34,128.31,128.22,128.18,128.13,128. 04,127.88,127.84,127.82,127.78,127.72,127.39,127.19,127.10,98. 61(C-1”),96.30(C-1’),95.88(C-1),79.12,78.02,77.23,77.01,76.80 ,76.12,75.30,74.84,74.07,73.84,73.60,72.88,72.69,68.95,68.09, 68.01,67.90,62.11,37.68,29.89,29.12,27.89,23.40,20.97,20.81,2 0.70.IR(film):ν=2980,1740,1454,1365,1239,1072,700.HRMS(ESI)m / z calcd for C 91 H 101 NO 24 Na[M+Na] + 1614.6606, found 1614.6608.

[0059] Example 7:

[0060] Synthesis of N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranoside-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside (14*)

[0061] The reaction equation is as follows Figure 3 As shown:

[0062] N-phenyltrifluoroacetylimide 6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside (Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347. (25.8 mg, 0.0355 mmol) and 6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranosyl-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranosyl-(1→3)-6-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranosyl-(1→2)-6-O-acetyl-4-O-benzyl-3-O-benzoyl-α-D-glucopyranoside (13.5 mg, 0.0071 mmol) (Tian G, Qin C, Liu Z, et al. Chem. Commun. 2020; 56(3):344-347. (25.8 mg, 0.0355 mmol) was dissolved in toluene, azeotropically removed three times by rotary evaporation, and pre-activated [agent / concentrate] was added to the oil pump. Molecular sieves were used and evacuated overnight on an oil pump. Anhydrous dichloromethane (0.25 mL) was added under argon protection, and the mixture was stirred at 0°C for 15 minutes. Trimethylsilyl trifluoromethanesulfonate (0.65 μL, 0.0036 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 3.5 hours. After complete reaction of the starting materials as determined by TLC, triethylamine was added to quench the reaction, and the molecular sieves were removed by diatomaceous earth filtration. The filtered reaction mixture was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration, and purified by silica gel column chromatography (petroleum ether:ethyl acetate, 4:1, v / v) to obtain the α-configuration target product 14* (6.1 mg, 0.025 mmol, 35%). [α] 25 D = +104.88 (c 1.0, CHCl3). 1H NMR(600MHz,Chloroform-d)δ8.05–6.75(m,67H,Ar-H),5.97(t,J=9.7Hz,1H,3-H””),5.83(m,2H,3-H,3-H”),5.51(d,J=3.4Hz,1H,1-H””),5.23(d,J=3.5Hz,1H,1-H”),5.15(d,J=19.4Hz,2H,NCbz-CH2),4.95–4.87(m,3H,1-H,1-H',1-H”'),4.67(m,4H,Ar-CH),4.53(d,J=10.8Hz,1H,Ar-CH),4.51–4.37(m,9H,5-H”,5-H””,NBn-CH2,Ar-CH),4.34(d,J=10.8Hz,1H,Ar-CH),4.31–4.12(m,10H,3-H',6-H””-H,6-H”-2H,6-H”'-H,Ar-CH),4.10–4.01(m,2H,6-H-H,6-H””-H),3.93(m,2H,5-H,6-H”'-H),3.90–3.84(m,2H,3-H”',6-H-H),3.80(m,3H,2-H,5-H',6-H'-H),3.72(dd,J=10.2,3.4Hz,1H,2-H””),3.64(m,3H,4-H,4-H””,5-H”'),3.57–3.40(m,6H,2-H”,4-H',4-H”,4-H”',6-H'-H,Linker-OCH),3.35(m,2H,2-H',2-H”'),3.17(m,3H,Linker-OCH,Linker-NCH2),2.14(s,3H,CH3CO),2.05(d,J=8.0Hz,6H,CH3CO),1.98(s,3H,CH3CO),1.69(s,3H,CH3CO),1.41(d,J=32.5Hz,4H,Linker-CH2),1.15(s,2H,Linker-CH2). 13C NMR (151MHz, CDCl3) δ170.72,170.57,165.47,165.05,138.03,137.52,137.37,136.97,134.65,133.31,132.89,130.15,129.96,129.62,12 8.57,128.52,128.39,128.34,128.30,128.22,128.12,128.05,127.8 5,127.75,127.62,127.56,127.22,126.61,125.85,125.80,96.58(C-1 ),95.92(C-1),94.98(C-1),93.63(C-1),92.67(C-1),77.23,77.02,7 6.81,75.11,74.54,74.13,73.99,73.38,73.00,72.77,71.11,68.74,6 8.44,68.22,67.13,66.66,62.69,62.40,62.17,46.13,29.24,23.37,21.04,20.83,20.44.IR(film):ν=1738,1238,1071,699.HRMS(ESI)m / z calcd for C 141 H 147 NO 36 Na[M+Na] + 2453.9629, found 2453.9629.

[0063] Example 8:

[0064] Synthesis of 5-aminopentyl α-D-glucopyranoside (16*)

[0065] The reaction equation is as follows Figure 4 As shown:

[0066] N-benzyl-N-benzyloxycarbonyl-5-aminopentyl-6-O-acetyl-4-O-benzyl-3-O-benzoyl-2-O-(2-naphthylmethyl)-α-D-glucopyranoside (prepared according to the reference Tian G, Qin C, Liu Z, et al. Chem Commun. 2020; 56(3):344-347.) (23.9 mg, 0.028 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 3.5 mL), and sodium methoxide (24 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 36 hours. After the reaction was complete, the reaction solution was diluted with methanol and analyzed using an Amberlite IR120 (H + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 100:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (30 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluent: water and methanol) to give compound 16* (5.53 mg, 74%). 1 H NMR(600MHz, Deuterium Oxide)δ4.92(d,J=3.8Hz,1H,1-H),3.87(dd,J=12.3,2.2Hz,1H),3.81–3.63(m,4H),3.56(ddd,J=12.0,9.6,5.0Hz, 2H), 3.42(t,J=9.5Hz,1H), 3.03(t,J=7.6Hz,2H), 1.70(ddt,J=17.3,12.2,7.2Hz,4H), 1.49(dp,J=22.1,6.8Hz,2H). 13 C NMR(151MHz,D2O)δ98.13,73.22,71.89,71.34,69.71,67.84,60.71,39.46,28.11,26.56,22.49.HRMS(ESI)m / z calcd for C 11 H 23 NO6Na[M+Na] + 288.1418, found 288.1419.

[0067] Example 9:

[0068] Synthesis of 5-aminopentyl α-D-glucopyranosyl-(1→3)-α-D-glucopyranoside (17*)

[0069] The reaction equation is as follows Figure 4 As shown:

[0070] Compound 5* (7.5 mg, 0.006 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 1 mL), and sodium methoxide (15 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 36 hours. After the starting material was completely reacted, the reaction solution was diluted with methanol and analyzed using an Amberlite IR 120 (H) spectrometer. + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 100:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (20 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluting with water and methanol) to give compound 17* (1.1 mg, 43%). 1 H NMR(600MHz, Deuterium Oxide)δ5.35(d,J=3.9Hz,1H,1-H),5.18(d,J=3.4Hz,1H,1-H),5.11(d,J=3.8Hz, 1H, 1-H), 4.03 (dt, J=10.2, 3.4Hz, 1H), 3.93 (dp, J=12.8, 3.4, 2.9Hz, 2H), 3.89–3. 73(m,10H),3.72–3.63(m,4H),3.58(ddd,J=10.0,6.7,3.7Hz,2H),3.47(ddd,J=1 0.5,9.0,1.8Hz,2H),3.01(t,J=7.6Hz,2H),1.77–1.63(m,6H),1.51–1.42(m,2H). 13C NMR(151MHz,D2O)δ99.31(1-C),95.74(1-C),94.97(1-C),79.74,74.53,73.14,72.91,71.74,71.68,71.59,71.46,69 .97,69.78,69.61,69.50,69.31,67.68,60.58,60.32,60.15,39.35,29.83,27.92,26.50,22.39.HRMS(ESI)m / zcalcd for C 23 H 43 NO 16 Na[M+Na] + 612.2474, found 612.2473.

[0071] Example 10:

[0072] Synthesis of 5-aminopentyl α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (18*)

[0073] The reaction equation is as follows Figure 4 As shown:

[0074] Compound 8* (20 mg, 0.018 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 2 mL), and sodium methoxide (15 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 10 hours. After the starting material was completely reacted, the reaction solution was diluted with methanol and analyzed using an Amberlite IR 120 (H) spectrometer. + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 20:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (20 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluent: water and methanol) to give compound 18* (5.5 mg, 78%).

[0075] Example 11:

[0076] Synthesis of 5-aminopentyl α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (19*)

[0077] The reaction equation is as follows Figure 4 As shown:

[0078] Compound 10* (12.6 mg, 0.0076 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 1 mL), and sodium methoxide (20 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 36 hours. After the starting material was completely reacted, the reaction solution was diluted with methanol and analyzed using an Amberlite IR 120 (H) spectrometer. + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 60:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (30 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluent: water and methanol) to give compound 19* (3.0 mg, 67%). 1 H NMR(600MHz, Deuterium Oxide)δ5.35(d,J=3.9Hz,1H,1-H),5.18(d,J=3.4Hz,1H,1-H),5.11(d,J=3.8Hz, 1H, 1-H), 4.03 (dt, J=10.2, 3.4Hz, 1H), 3.93 (dp, J=12.8, 3.4, 2.9Hz, 2H), 3.89–3. 73(m,10H),3.72–3.63(m,4H),3.58(ddd,J=10.0,6.7,3.7Hz,2H),3.47(ddd,J=1 0.5,9.0,1.8Hz,2H),3.01(t,J=7.6Hz,2H),1.77–1.63(m,6H),1.51–1.42(m,2H). 13 C NMR(151MHz,D2O)δ99.31(1-C),95.74(1-C),94.97(1-C),79.74,74.53,73.14,72.91,71.74,71.68,71.59,71.46,69 .97,69.78,69.61,69.50,69.31,67.68,60.58,60.32,60.15,39.35,29.83,27.92,26.50,22.39.HRMS(ESI)m / zcalcd for C 23 H 43 NO 16 Na[M+Na] +612.2474, found 612.2473.

[0079] Example 12:

[0080] Synthesis of 5-aminopentyl α-D-glucopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranoside (20*)

[0081] The reaction equation is as follows Figure 4 As shown:

[0082] Compound 12* (11 mg, 0.0069 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 1 mL), and sodium methoxide (15 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 36 hours. After the starting material was completely reacted, the reaction solution was diluted with methanol and analyzed using an Amberlite IR 120 (H) spectrometer. + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 80:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (30 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluent: water and methanol) to give compound 20* (3.2 mg, 79%). 1 H NMR(600MHz, Deuterium Oxide)δ5.53(d,J=3.7Hz,1H,1-H),5.17(d,J=3.8Hz,1H,1-H),4.91(d,J=3.6Hz,1H,1- H),4.05(ddd,J=10.3,4.7,2.4Hz,1H),3.96(ddd,J=10.1,4.6,2.5Hz,1H),3.88–3.58(m ,17H),3.54(dt,J=9.8,6.2Hz,1H),3.47(dt,J=13.4,9.6Hz,2H),3.01(dd,J=10.3,4.9 Hz,3H),1.69(dtd,J=16.7,13.5,12.3,5.4Hz,6H),1.46(ddp,J=26.6,15.1,7.6Hz,2H). 13C NMR(151MHz,D2O)δ98.22(1-C),96.35(1-C),95.89(1-C),79.75,75.24,72.79,71.75,71.48,71.24,71 .10,69.85,69.70,69.35,69.22,67.68,60.33,60.23,39.34,29.80,28.05,26.51,22.44.HRMS(ESI)m / z calcd forC 23 H 43 NO 16 Na[M+Na] + 612.2474, found 612.2474.

[0083] Example 13:

[0084] Synthesis of 5-aminopentyl α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→2)-α-D-glucopyranoside (21*)

[0085] The reaction equation is as follows Figure 4 As shown:

[0086] Compound 14* (3.7 mg, 0.0015 mmol) was dissolved in a tetrahydrofuran / methanol mixture (1:1, v / v, 1 mL), and sodium methoxide (15 mg) was added. The mixture was stirred at room temperature for half an hour, followed by the addition of 100 μL of 1M sodium hydroxide solution. The mixture was stirred at 40 °C for 36 hours. After the starting material was completely reacted, the reaction solution was diluted with methanol and analyzed using an Amberlite IR 120 (H) spectrometer. + The reaction solution was neutralized with resin to a pH less than 7, the solution was concentrated by filtration, and purified by silica gel column chromatography (dichloromethane:methanol, 50:1, v / v) to obtain the deacylated product. This product was dissolved in a tert-butanol / water / dichloromethane mixture (5:2:1, v / v / v, 5 mL), and 10% Pd / C (20 mg) was added. The mixture was reduced with hydrogen and stirred at room temperature for 24 hours. The mixture was filtered to remove palladium on carbon, and the solvent was removed by rotary evaporation. The product was purified using a Sep-Pak cartridge C18 column (eluent: water and methanol) to give compound 21* (0.7 mg, 51%). 1H NMR(600MHz, Deuterium Oxide)δ5.54(d,J=3.7Hz,1H,1-H),5.39(d,J=4.0Hz,1H,1-H),5.22(d,J=3.9Hz,1H ,1-H),5.20(d,J=3.5Hz,1H,1-H),5.12(d,J=3.8Hz,1H,1-H),4.09(d,J=10.3Hz,1H ),4.04–3.92(m,7H),3.90–3.63(m,35H),3.62–3.51(m,5H),3.46(dt,J=14.7,9.7H z,3H),3.02(t,J=7.7Hz,3H),1.78–1.64(m,6H),1.49(dq,J=15.2,7.7,7.0Hz,2H). 13 C NMR(151MHz,H2O+D2O)δ99.19(1-C),96.58(1-C),95.85(1-C),95.68(1-C),94.91(1-C),79.76,74.95,74.39,72.78,71.90,71.69,7 1.46,71.20,69.93,69.88,69.64,69.47,69.43,69.16,67.66,60.56,60.48,60.15,60.04,39.34,27.91,26.49,22.38HRMS(ESI)m / z calcd for C 35 H 63 NO 26 Na[M+Na] + 936.3531, found936.3536.

[0087] Example 14:

[0088] (1) Sugar chip manufacturing (e.g.) Figure 5 (As shown): The synthesized monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, and hexasaccharide fragments were dissolved in 50 mM phosphate solution (pH 8.5) to prepare 0.2 mM and 1 mM sugar solutions, respectively; and correspondingly, 0.1 mg / mL and 0.5 mg / mL O2 LPS solutions were prepared; then, a chip spotting instrument was used according to... Figure 5 The grid pattern shown was printed on an area of ​​the chip (9 mm long × 9 mm wide) and incubated overnight at room temperature and 65% humidity to allow the sugar fragments to covalently bind to the chip. After incubation, the chip was treated with a mixed solution of 100 nM ethanolamine and 50 nM sodium phosphate (pH 9) at 50°C for 1 hour to remove unbound sites on the chip surface, yielding the sugar chip.

[0089] (2) Block the sugar chip using PBS solution containing 3% BSA at room temperature for 1 hour. Wash once with 0.1% Tween 20 PBS solution (PBST solution), wash twice with PBS solution, centrifuge and dry, and load the sugar chip into a 16-well incubator (ProPlate). Add 120 μL of human serum sample diluted 1:20 with PBS solution containing 1% BSA to each well, and incubate overnight in a humidified chamber at 4°C in the dark to allow the human serum IgG antibody that recognizes the synthetic sugar fragments to bind to the synthetic sugar fragments on the sugar chip. Remove the sample and wash three times with 200 μL of PBST solution to remove unbound serum IgG antibody. Subsequently, goat anti-human serum IgG antibody labeled with cy3 fluorescence was used as a secondary antibody and incubated in a humidified chamber at room temperature in the dark for 45 min to bind to the human serum IgG antibody on the chip. The secondary antibody solution was removed, and the chip was washed three times with 200 μL PBST solution to remove unbound secondary antibody. The 16-well incubator was disassembled, washed once with ultrapure water, and then washed with ultrapure water for 15 minutes to obtain the final detection chip.

[0090] The obtained detection chip was used to screen serum samples from 153 patients who tested positive in the carbon-13 breath test. The results are as follows: Figure 6-7 As shown, scanning on a microarray scanner emits fluorescent signals. Among 153 positive cases, antibodies in the serum of 135 patients were able to bind to the synthesized disaccharide fragment, achieving a detection accuracy of 88.2%. Therefore, the detection chip of this invention can accurately determine whether a patient is infected with Helicobacter pylori.

[0091] The specific detection results for each sugar fragment and the sugar antigen-O2 LPS are shown in Table 1.

[0092] Table 1 shows the results of screening serum from 153 positive patients using different sugar chips.

[0093]

[0094]

[0095] Accuracy = Number of cases with fluorescent signals / 153.

[0096] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. The application of a Helicobacter pylori O2 serotype O antigen glycoside fragment in the preparation of a Helicobacter pylori infection detection device, wherein the Helicobacter pylori O2 serotype O antigen glycoside fragment is... .

2. A Helicobacter pylori infection detection chip, characterized in that, It comprises the following components: a chip, a Helicobacter pylori O2 serotype O antigen glycoprotein fragment that binds to the chip, a human serum antibody that binds to the Helicobacter pylori O2 serotype O antigen glycoprotein fragment, and a fluorescently labeled anti-human IgG antibody that binds to the human serum antibody; wherein the Helicobacter pylori O2 serotype O antigen glycoprotein fragment is... .

3. A method for preparing a Helicobacter pylori infection detection chip, characterized in that, include: (1) Dissolve in phosphate solution, then print onto chip, incubate to allow sugar fragments to covalently bind to the chip, thus obtaining a sugar chip; (2) The obtained sugar chip was placed in a diluted human serum solution and incubated to allow the human serum antibody that recognizes the synthetic sugar fragment to bind to the antibody on the sugar chip; then, fluorescently labeled anti-human IgG antibody was used as a secondary antibody to bind to the human serum antibody on the sugar chip to obtain a Helicobacter pylori infection detection chip.

4. The method according to claim 3, characterized in that, In step (1), the concentration of the O antigen sugar fragment of Helicobacter pylori O2 serotype O in the phosphate solution is 25 mM - 75 mM.

5. The method according to claim 3, characterized in that, The pH of the phosphate solution in step (1) is 8-9.

6. The method according to claim 3, characterized in that, The incubation temperature in step (1) is room temperature 20-30℃; the humidity is 50%-70%.

7. The method according to claim 3, characterized in that, In step (2), the human serum is diluted to a ratio of 1:10 to 1:

100.

8. The method according to claim 7, characterized in that, In step (2), the human serum is diluted to a ratio of 1:

20.

9. The method according to any one of claims 3-8, characterized in that, The fluorescently labeled anti-human IgG antibody in step (2) refers to: cy3-labeled goat anti-human IgG antibody.

10. A Helicobacter pylori infection detection device, characterized in that, It contains the Helicobacter pylori infection detection chip as described in claim 2.

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