A cyanopyrazolone heterobifunctional reagent and its application in constructing sugar chips

Abstract

The invention discloses a cyanopyrazolone heterodifunctional reagent and its application in constructing a sugar chip, wherein the reagent is 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone, the reagent has a stable structure and strong chemical selectivity, and the active methylene can be used to mark the reducing end of the sugar chain, and the sugar is efficiently derivatized by Knoevenagel condensation and Michael addition reaction, and under this condition, another functional group cyano is tolerated; subsequently, a sugar chain derivative with a single molecule active primary amino group is obtained by one-step reduction, and a sugar chain derivative monomer is further obtained by two-dimensional high performance liquid chromatography separation, and the monomer reacts with a solid phase carrier functionalized with N-succinimide to realize rapid and efficient construction of a sugar chip. The cyanopyrazolone heterodifunctional reagent of the present invention can be used to construct a variety of natural sugar chips, which has a good application prospect in the construction of sugar chips, and the obtained sugar chips can be used for the screening of sugar-binding proteins.

Description

Technical Field

[0001] The invention belongs to the technical field of sugar biology, and specifically relates to a cyanopyrazolone heterobifunctional reagent and an application thereof in the construction of a sugar chip. Background Art

[0002] Sugars, also known as carbohydrates, are the most widely distributed and abundant class of compounds in nature. They are an important component of life systems and are widely present in organisms. They play a vital role in biological recognition and biological information transmission, such as the regulatory effect of sugar-binding proteins on cell recognition, adhesion and signal transduction, mediating the infection of pathogens, causing corresponding immune responses, and the key role of sugars in pathological processes such as tumor metastasis and inflammatory attacks.

[0003] Glycosylation is one of the most important post-translational modifications of proteins and plays a very important role in the precise regulation of various biological processes in cells. Studies have reported that glycoconjugates such as glycoproteins, glycolipids, and proteoglycans participate in various molecular interactions in the body. As a key information molecule on glycoconjugates, sugar chains are of great significance for the analysis of sugar chain structure and function. Understanding glycoprotein interactions at the molecular level has always been the main interest of glycobiologists and researchers in different fields including immunology, infection, and oncology, and is the technical core of developing glycosylation methods based on glycoprotein treatment and diagnosis. Sugar chip (also called carbohydrate microarray) is a type of biochip. Its technical principle is to fix sugars or sugar complexes with a certain structure on a specific carrier, and analyze the sugar-mediated specific binding effect and the structural characteristics of the sample based on the signal after the sample to be tested hybridizes with it. In 2002, Wang et al. (Nat. Biotechnol., 2002, 20 (3), 275-281) first reported the preparation of sugar chips by fixing sugar probes on nitrocellulose-coated carriers. Subsequently, with the development of sugar science, sugar chip technology has grown rapidly and is gradually becoming an important technology applied in glycobiology and glycomics.

[0004] Sugar chip technology has the advantages of low sample usage, high sensitivity, simple operation and high throughput in analyzing the interaction between sugars and sugar-binding proteins. It is currently one of the most powerful tools for studying the specific recognition and interaction between sugar-binding proteins and sugar chains. The application of sugar chips mainly includes the detection and quantitative analysis of anti-sugar antibodies related to disease identification, analysis of the binding specificity of sugars and sugar-binding proteins (Chemical Science, 2016, 7(3), 2084-2093), detection of pathogens and screening of drug targets (J.Immunol.Res., 2015, doi:10.1155 / 2015 / 510810) and other research fields.

[0005] The construction process of natural sugar microarray can generally be divided into four steps: (1) releasing sugar chains from natural biological samples or sugar complexes; (2) derivatizing sugar chains to give them chromophores and free active groups for further separation, purification and subsequent immobilization; (3) purifying sugar chain derivatives using a variety of separation methods to obtain components with a single structure; and (4) immobilizing various sugar chain monomers on a carrier to prepare a sugar microarray for studying the specific interactions between sugars and sugar-binding proteins. One of the key factors in constructing a sugar chip is to construct a sugar library. Due to the complex structure of sugars and the difficulty in synthesis, the types and quantities of sugars that can be used to construct a sugar chip are limited (Acc. Chem. Res., 2015, 48 (5), 1450-1463). Sugar sources are mainly obtained through chemical synthesis, chemoenzymatic synthesis and separation of natural sugar molecules. Due to the limitations of chemical synthesis and chemoenzymatic synthesis of sugars, such as high cost, complex steps, harsh conditions, and limited types of glycosyltransferases, they mainly rely on the separation of sugar molecules from natural sources, mainly including free sugars, bound N / O-sugar chains, glycolipids, glycosaminoglycans, GPI anchor proteins, hyaluronic acid, etc. in various biological samples such as cells, tissues, pathogens, milk, and urine. Connectors or fluorescent tags are derived from sugar-containing mixtures for subsequent purification, separation, and immobilization (Glycoconjugate J., 2015, 32 (8), 559-574; ACS Chem. Biol., 2009, 4 (9), 741-750).

[0006] Effectively fixing sugar chains on the surface of the carrier is a necessary condition for the successful preparation of sugar microarrays. In recent years, the development of heterobifunctional reagents for the derivatization of natural sugars has greatly promoted the development of natural sugar microarrays. This type of reagent has two active functional groups, one of which can be used to label the reducing end of the sugar chain, and the other functional group can provide a reaction site for the subsequent construction of sugar microarrays for the study of sugar chain functions. At present, the developed reagents mainly include 2-aminobenzamide (2-AB) (Anal.Chem., 2007, 79, 8107-8113), 2-aminobenzoic acid (2-AA) (Int.J.Parasitol., 2015, 45, 465-475), 2,6-diaminopyridine (2-DAP) (Nat.Methods, 2005, 2, 845-850; Glycoconjugate J., 2008, 25, 15-25), N-aminoethyl-2-aminobenzamide (AEAB) (Chem. Biol., 2009, 16, 36-47; J. Biol. Chem., 2012, 287, 44784-44799) and N-alkyl oxime reagents, such as fluorenylmethyloxycarbonyl chloride (Fmoc) (Anal. Biochem., 2009, 395, 151-160), 2-aminomethyl-N, O-hydroxyethyl (AMNO) (Glycobiology, 2006, 16, 21-27), N-Fmoc-3-(methoxyamino)propylamine (F-MAPA) (Bioconj. Chem., 2019, 30, 2897-2908) and O-benzylhydroxylamine (BHA) (SLAS Technol., 2020, 25, 388-396). Although this type of reagent has been successfully used to construct a sugar library containing labels, a sugar chip has been successfully constructed and applied to the screening of sugar-binding proteins GBPs. However, the low reactivity of aromatic amines in 2-AB, 2-AA and 2-DAP molecules (directly covalently coupled to epoxy-activated carriers by secondary amine chemistry) and the weak fluorescence of benzene rings / pyridine rings limit their application in sugar chip construction. At the same time, 2-DAP and AEAB, both of which carry two amino groups, have less than ideal chemical selectivity when reacting with reducing sugar chains. The above-mentioned heterodifunctional reagents can only be used for the derivatization of reducing sugar chains. To construct a sugar chip, reducing sugar chains must be obtained first and then derivatized, which requires a two-step process to complete. The purification process of sugar chain derivatives is also relatively complicated. In addition, the reaction system of this type of reagent is an acidic system, and sulfated or sialic acid-modified acidic sugar chains have poor tolerance to them.In view of the shortcomings and deficiencies of these current reagents, the inventor's research team previously developed an aminopyrazolone-type heterobifunctional reagent: 3-amino-1-phenyl-pyrazolone (PAP for short), which successfully achieved the release and simultaneous labeling of glycoprotein O-sugar chains by PAP under alkaline conditions, and obtained O-sugar chain derivatives with primary amino groups (Analytical Biochemistry, 2018, 549, 1-11; Patent: ZL201710954115.8). However, since the active amino linker carried by PAP is too short, the sugar chain cannot be effectively fixed on the solid phase carrier, and the construction of sugar chips and the study of sugar chain functions cannot be realized.

[0007] The technical problems in the construction of heterodifunctional reagents and oligosaccharide chips reported so far include: ① The amount of derivatization reagents for reducing sugar chains is large and the derivatization efficiency is low, and the derivatization reagents are limited to mass spectrometry separation and analysis of sugar chains; ② The heterodifunctional reagents reported for sugar chip construction can only react with reducing sugar chains, not O-sugar chains, and require a two-step process to obtain pre-column derivatized sugar chains, which increases the difficulty of sugar chain separation and purification; ③ The reaction of heterodifunctional reagents reported for sugar chip construction with reducing sugar chains is carried out in an acidic system, and the acidic sugar chains modified by sulfate or sialic acid have poor tolerance. Therefore, the development of pyrazolone heterodifunctional reagents with novel structures, bifunctional functional groups and moderate linker arms is of great significance for the construction of sugar chips and the study of the relationship between sugar chain structure and function. Summary of the invention

[0008] The purpose of the present invention is to provide a cyanopyrazolone heterobifunctional reagent in view of the shortcomings of existing heterobifunctional reagents and sugar chip construction. The reagent has two functional groups with different functions: an active methylene group and a cyano group. Under alkaline conditions, the active methylene group reacts with a reducing sugar chain or a sugar chain reducing end of a glycoprotein to obtain a sugar chain derivative with strong hydrophobicity and two molecules of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone. The reaction conditions are mild and the product purification is simple. Then, the two molecules of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone are purified by a one-step reaction. The sugar chain derivatives containing 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone were converted into sugar chain derivatives with single molecule 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone. The sugar chain derivative monomers were obtained by two-dimensional high performance liquid chromatography separation. The sugar chains can be efficiently fixed by amidation and reaction with N-succinimide (NHS) functionalized solid phase carriers, and then a variety of natural sugar chips can be constructed for the interaction between sugar probes and sugar binding proteins, providing a powerful tool for the study of the structure and function relationship of sugar chains.

[0009] To achieve the above object, the present invention provides a cyanopyrazolone heterobifunctional reagent with the following structural formula, and its chemical name is 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone (ANPMP).

[0010]

[0011] The synthesis steps of the above ANPMP are as follows:

[0012] Step 1: dissolving 4-aminobenzeneacetonitrile in an inorganic acid, stirring at -15 to -10°C for 0.5 to 1 hour, then slowly dropping a sodium nitrite aqueous solution, maintaining the temperature at -15 to -10°C, stirring and reacting for 1 to 1.5 hours to obtain a 4-acetonitrile benzene diazonium inorganic acid salt reaction solution;

[0013]

[0014] Step 2: The 4-acetonitrile benzene diazonium inorganic acid salt reaction solution is quickly filtered to remove insoluble matter to obtain a clear and transparent filtrate, which is added to a stannous chloride hydrochloric acid solution at one time, and stirred at -5 to 0°C for 1 to 2 hours. A large amount of milky white precipitate is produced by the reaction to obtain a reduction product, 4-acetonitrile phenylhydrazine hydrochloride;

[0015]

[0016] Step 3: 4-acetonitrile phenylhydrazine hydrochloride and an inorganic base are stirred in an organic solvent at room temperature for 12 to 20 hours to obtain 4-acetonitrile phenylhydrazine;

[0017]

[0018] Step 4: 4-acetonitrile phenylhydrazine and ethyl acetoacetate were refluxed at 100°C for 1 hour in the absence of solvents and with glacial acetic acid as a catalyst, then cooled to room temperature, and after adding diethyl ether and continuing to stir vigorously for 2 hours, recrystallized to obtain white solid ANPMP.

[0019]

[0020] In the above step 1, the inorganic acid is any one of concentrated hydrochloric acid, concentrated sulfuric acid, and tetrafluoroboric acid; the sodium nitrite aqueous solution is prepared on-site by dissolving solid sodium nitrite in water, and the concentration of the sodium nitrite aqueous solution is 1.4 to 2.5 mol / L; the molar ratio of 4-aminobenzyl cyanide to sodium nitrite is 1:1.08 to 1.15; and the ratio of 4-aminobenzyl cyanide to the inorganic acid is 1 mmol:1 to 1.5 mL.

[0021] In the above step 2, the molar ratio of the stannous chloride to the 4-aminobenzeneacetonitrile in step 1 is 3 to 5:1; the stannous chloride hydrochloric acid solution is prepared by stannous chloride and concentrated hydrochloric acid at a ratio of 1 mmol:0.17 to 0.2 mL, the stannous chloride hydrochloric acid solution needs to be prepared and used immediately, and is oxidized by oxygen in the air to generate tin chloride after being left for a long time, thereby losing its reducing property; the 4-acetonitrile benzene diazonium hydrochloride solution obtained in step 1 needs to be quickly filtered to remove insoluble matter, and the filtrate is added to the stannous chloride hydrochloride solution at one time to improve the yield of 4-acetonitrile phenylhydrazine hydrochloride generated in step 2.

[0022] In the above step three, the inorganic base is selected from an aqueous solution of any one of sodium hydroxide, potassium hydroxide, and sodium carbonate, wherein the mass concentration of sodium hydroxide or potassium hydroxide is 5% to 25%, and the mass concentration of sodium bicarbonate is 10% to 20%; the organic solvent is selected from any one of dichloromethane, chloroform, and 1,2-dichloroethane; and the pH of the solution after the reaction is completed is controlled to be 8 to 9.

[0023] In the above step 4, the ratio of the amount of 4-acetonitrile phenylhydrazine, ethyl acetoacetate and glacial acetic acid is 1:1-1.15:0.003-0.005; the solvent used for recrystallization is any one or more of methanol, ethanol, toluene and ethyl acetate.

[0024] The ANPMP of the present invention can be used to construct a sugar chip, and the specific method comprises the following steps:

[0025] Step (1): subjecting reducing sugar chains or glycoproteins to Knoevenagel condensation and Michael addition reaction with ANPMP under alkaline conditions to generate sugar chain derivatives with two molecules of ANPMP; the reducing sugar chains are free reducing sugar chains or sugar chains released from glycoproteins, wherein the free reducing sugar chains are reducing sugar chains extracted from plants or animals or synthesized reducing sugar chains.

[0026] Step (2): The sugar chain derivative with two molecules of ANPMP is reduced to a sugar chain derivative with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone (AEPMP) by ammonium formate under the action of palladium-carbon catalyst.

[0027] Step (3): The sugar chain derivative with a single molecule of AEPMP is separated and purified by two-dimensional high performance liquid chromatography to obtain a sugar chain derivative monomer with a single molecule of AEPMP.

[0028] Step (4): The sugar chain derivative monomer carrying a single molecule of AEPMP is fixed on a solid phase carrier functionalized with N-succinimide (NHS) through an amidation reaction to obtain a sugar chip.

[0029] In the above step (1), the reducing sugar chain is added to the methanol or ethanol solution of ANPMP, so that the concentration of ANPMP in the obtained reaction system is 0.1-0.8 mol / L, and the molar ratio of ANPMP to the reducing sugar chain is 5-15:1; then, sodium hydroxide aqueous solution or ammonia aqueous solution is added to the reaction system until the pH of the reaction system is 8-9, and then heated to 40-70°C, and stirred at constant temperature for 1.5-4 hours; after the reaction is completed, it is naturally cooled to room temperature, the pH of the reaction solution is adjusted to 7.0, and purified with a C18 solid phase extraction column to obtain a sugar chain derivative with two molecules of ANPMP. Excess labeling reagents can be directly extracted and removed with an organic solvent, which can achieve rapid separation of sugar chain derivatives.

[0030] In the above step (1), glycoprotein is added to a methanol or ethanol aqueous solution of ANPMP, so that the concentration of ANPMP in the obtained reaction system is 0.8-1.5 mol / L and the mass ratio of ANPMP to glycoprotein is 15-50:1; then, sodium hydroxide aqueous solution or ammonia aqueous solution is added to the reaction system until the pH of the reaction system is 9-11, and then heated to 60-90° C., and stirred at a constant temperature for 12-48 hours; after the reaction is completed, it is naturally cooled to room temperature, the pH of the reaction solution is adjusted to 6.0, extracted with chloroform or dichloromethane, concentrated and purified by a C18 solid phase extraction column to obtain a sugar chain derivative with two molecules of ANPMP.

[0031] In the above step (2), a sugar chain derivative with two molecules of ANPMP, a palladium carbon catalyst, and ammonium formate are added to a mixture of water and methanol or ethanol or isopropanol, and stirred at 25 to 45° C. for 12 to 48 hours; after the reaction is completed, the mixture is cooled to room temperature, centrifuged to remove the catalyst, and the supernatant is concentrated and the residue is purified by a graphite carbon column to obtain a sugar chain derivative with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone.

[0032] In the above step (3), the method for separation and purification by two-dimensional high performance liquid chromatography is to first separate the sugar chain derivative with single molecule AEPMP by one-dimensional normal phase column high performance liquid chromatography, and then separate by two-dimensional reverse phase column high performance liquid chromatography; the chromatographic conditions of the one-dimensional normal phase column high performance liquid chromatography separation are: Inertsil Amide column, column temperature 25°C, detection wavelength 254nm, flow rate 0.8-1.0mL / min, mobile phase A is acetonitrile, mobile phase B is 100mmol / L acetic acid-ammonium acetate buffer with pH=6, gradient elution, and the eluate of each component is collected separately, concentrated under reduced pressure and then freeze-dried; the chromatographic conditions of the two-dimensional reverse phase column high performance liquid chromatography separation are: Cosmosil C18 column, column temperature 25°C, detection wavelength 254nm, flow rate 0.8-1.0mL / min, mobile phase A is acetonitrile, mobile phase B is 10mmol / L acetic acid-ammonium acetate buffer with pH=5.5, gradient elution, and the eluate of each component is collected separately, and concentrated under reduced pressure.

[0033] In the above step (4), the sugar chain derivative monomer with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone is dissolved in ultrapure water and diluted with 100-600 mmol / L phosphate buffer with a pH of 8.0-9.5 to a final concentration of 10-500 μmol / L, and then spotted on the NHS-functionalized solid phase carrier using a non-contact spotter, the spotting table temperature is controlled at 15-20° C. and the humidity is controlled at 50%-70%. After spotting, the solid phase carrier is placed in a constant temperature and humidity incubator at a temperature of 25-30° C. and a humidity of 50%-70% for incubation for 8-12 hours, and then dried in an oven at 50-60° C. for 3-4 hours.

[0034] Taking glycoprotein O-sugar chains as an example, the reaction principle of the sugar chip constructed in the present invention is:

[0035]

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention uses cheap 4-aminobenzeneacetonitrile as a starting material, and synthesizes 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone (ANPMP) in high yield through key steps such as diazotization and reduction reaction, condensation ring-closing reaction, etc. The compound can be used as a derivatization reagent for carbohydrate substances. Under alkaline conditions, the reagent has a stable structure and strong chemical selectivity. The active methylene group undergoes Knoevenagel condensation and Michael addition reaction with the reducing end of the sugar chain, while the active cyano group does not undergo hydrolysis reaction under this condition, and there is no by-product. The sugar chain derivatives labeled with the cyanopyrazolone heterodifunctional reagent carry two molecules of cyano groups, have strong hydrophobicity, and are easy to purify. At the same time, the derivatization method has high derivatization efficiency, short reaction time, is suitable for various reducing sugar chains, has strong versatility, and has good repeatability and reliability. The reagent can be applied to the derivatization labeling of sugar chains in a variety of standard glycoproteins and complex biological samples, successfully realizes the large-scale preparation of natural sugar chain derivatives, and provides a rich source of sugar libraries for the construction of sugar chips. The sugar chain derivatized with this reagent can convert the cyano group into an active amino group through a one-step reduction reaction, so that each sugar chain molecule carries one active amino group. The prepared sugar chain derivatives with single-molecule AEPMP are separated by a high-performance liquid chromatography separation method combining normal phase and reverse phase, thereby obtaining high-purity sugar chain derivative monomers. As a linking reagent for the construction of sugar chips, AEPMP has strong chemical selectivity and moderate linker arm length compared with existing heterodimer reagents. It can effectively react with N-succinimide functionalized solid phase carriers to successfully construct sugar chips for the interaction between sugar probes and sugar-binding proteins, providing a powerful tool for the study of the structural and functional relationship of sugar chains. On this basis, each sugar chain derivative monomer can be subjected to electrospray ionization tandem mass spectrometry (ESI-MS) in positive ion mode after chemical derivatization (N-acetylation and permethylation). n ) analysis to resolve the fine structure of sugar chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the ESI-MS spectrum of the maltodextrin derivative with two molecules of ANPMP prepared under alkaline conditions in Example 2.

[0039] Figure 2 This is the ESI-MS spectrum of the maltodextrin derivative with single molecule AEPMP prepared in Example 2.

[0040] Figure 3 These are the one-dimensional (A) and two-dimensional (B) HPLC separation profiles of the maltodextrin derivative with single-molecule AEPMP in Example 2.

[0041] Figure 4 This is the ESI-MS spectrum of neutral free oligosaccharides in breast milk after purification by DEAE-52 weak anion exchange chromatography column in Example 3.

[0042] Figure 5 This is the ESI-MS spectrum of breast milk acidic free oligosaccharides after purification by DEAE-52 weak anion exchange chromatography column in Example 3.

[0043] Figure 6 This is the ESI-MS spectrum of the neutral free oligosaccharide derivative of breast milk with two molecules of ANPMP in Example 3.

[0044] Figure 7 These are the one-dimensional (A) and two-dimensional (B) HPLC separation profiles of the neutral free oligosaccharide derivative of breast milk with two molecules of ANPMP in Example 3.

[0045] Figure 8 This is the ESI-MS spectrum of the porcine gastric mucin O-sugar chain derivative with two molecules of ANPMP in Example 4.

[0046] Fig. 9 This is the ESI-MS spectrum of the porcine gastric mucin O-sugar chain derivative with a single molecule of AEPMP in Example 4.

[0047] Fig.10 These are the one-dimensional (A) and two-dimensional (B) HPLC separation profiles of the porcine gastric mucin O-sugar chain derivative with a single molecule of AEPMP in Example 4.

[0048] Fig.11 This is the fluorescence spectrum of the shotgun natural sugar chip containing 83 sugar chains in Example 5 combined with plant lectins AAL, HPA and SNA.

[0049] Fig.12 It is the quantitative binding map of the shotgun natural sugar chip containing 83 sugar chains in Example 5 and the plant lectins AAL, HPA and SNA. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the technical solution of the present invention, the above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0051] In the following examples, 4-aminophenylacetonitrile, sodium nitrite, stannous chloride, and ethyl acetoacetate were purchased from Beijing Bailingwei Technology Co., Ltd., China; concentrated hydrochloric acid, ether, and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium hydroxide, maltodextrin polysaccharide, porcine gastric mucin, etc. were purchased from Sigma-Aldrich; ammonium acetate was purchased from Tianjin Damao Chemical Reagent Factory; chromatographically pure acetonitrile (ACN) was purchased from Thermo Scientific; the sugar chain standard lactose-N-difucohexaose was purchased from Dextra; Shimadzu amide-based liquid chromatography column Inertsil Amide column was purchased from Shimadzu Corporation, Japan; reversed phase Cosmosil C18 (ODS) chromatography column was purchased from Nacalai Tesque, Japan; Sep-Pak C18 solid phase extraction column (100 mg / 1 mL) was purchased from Waters; porous graphite carbon solid phase extraction column (150 mg / 4 mL) was purchased from Alltech Associates; NHS functionalized glass slide (H 75×25×1) was purchased from Schott, Germany; ethanolamine was purchased from Sigma-Aldrich; PBST (10×) and PBS (10×) were purchased from Beijing Coolaibo Technology Co., Ltd.; biotinylated lectins: Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Helix pomatia agglutinin (HPA) were all purchased from Vector Laboratories, USA; Cy3-labeled streptavidin fluorescent reagent (Cy3-SA) was purchased from Beijing Solebow Technology Co., Ltd.; ultrapure water was prepared by the Milli-Q ultrapure water system of Millipore, USA; other chemical reagents were of analytical grade.

[0052] The parameters of the electrospray electrolysis linear ion trap mass spectrometry (ESI-MS) are: working voltage is 4 kV; sheath gas flow rate is 20 arb; auxiliary gas flow rate is 10 arb; capillary voltage is 37 V; capillary lens voltage is 250 V; capillary temperature is 300 ° C; scanning type is one-stage full scan; maximum injection time is 1000 ms; micro scan is 3 times; data acquisition uses LTQTune software. The ESI-MS detection in the present invention is all detected in the positive ion mode.

[0053] Microarray biochip platform: chip spotter sciFLEXARRAYER (Scienion AG, Germany), chip scanner RPAS710G (Biotech, Inc, France); desktop centrifuge (5418, Eppendorf AG, Germany); freeze dryer (SCIENTZ-18N, Ningbo Xinzhi Biotechnology Co., Ltd.).

[0054] Example 1

[0055] Synthesis of Cyanopyrazolone Heterobifunctional Reagents

[0056] Step 1: Weigh 5.58g (42.3mmol) of 4-aminobenzeneacetonitrile and dissolve it in 55mL of concentrated hydrochloric acid, stir at -10°C for about 30 minutes until 4-aminobenzeneacetonitrile is completely dissolved to prepare a 4-aminobenzeneacetonitrile hydrochloride solution; dissolve 3.15g (45.7mmol) of NaNO2 in 20mL of double distilled water, and then slowly add it dropwise to the 4-aminobenzeneacetonitrile hydrochloride solution, during which the temperature is kept at -15 to -10°C. After the addition is complete, stir and react for 60 minutes to obtain a 4-acetonitrile benzene diazonium inorganic acid salt reaction solution.

[0057] Step 2: Weigh 47.7g (210mmol) SnCl2·2H2O and dissolve it in 37mL concentrated hydrochloric acid to prepare a stannous chloride hydrochloric acid solution, and cool the solution to -10°C; quickly filter the 4-acetonitrile benzene diazonium inorganic acid salt reaction solution obtained in step 1 to remove insoluble matter, add the filtrate to the stannous chloride hydrochloric acid solution at one time, keep the temperature control range at -5 to 0°C, stir and react for 1 hour, the reaction produces a large amount of milky white precipitate, filter after the reaction is complete, collect the white solid, wash it with saturated brine, ethanol and ether respectively, and vacuum dry it to obtain the reduced product 4-acetonitrile phenylhydrazine hydrochloride.

[0058] Step 3: Weigh 36g (0.2mol) of 4-acetonitrile phenylhydrazine hydrochloride and dissolve it in 150mL of dichloromethane, add 170mL of 5% NaOH aqueous solution for neutralization, stir and react at room temperature for 12 hours, neutralize with 1mol / L hydrochloric acid to pH=8-9, extract the aqueous phase with dichloromethane until there is no obvious fluorescent spot under ultraviolet light, combine the dichloromethane phases, dry over anhydrous sodium sulfate, and remove dichloromethane by rotary evaporation to obtain 29.09g of light yellow solid 4-acetonitrile phenylhydrazine.

[0059] Step 4: Weigh 3.11g (0.021mol) of 4-acetonitrile phenylhydrazine, 3.31g (0.023mol) of ethyl acetoacetate, and 0.42mL (7.28mmol) of glacial acetic acid and add them to a reaction flask. After stirring and reacting in an oil bath at 100°C for 1 hour, cool the reaction flask to room temperature, add 10mL of anhydrous ether to dilute and vigorously stir at room temperature for 2 hours, filter the reaction solution, collect the white solid, wash with ether (3×20mL), and vacuum dry to obtain the crude target product. The crude product is recrystallized from ethanol to obtain 3.85g of white solid, i.e., cyanopyrazolone heterodifunctional reagent: 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone (ANPMP), with a yield of 86%. The spectral data are: 1 H NMR (400MHz, DMSO-d6) δ11.58(s,1H),7.75(d,J=8.4Hz,2H),7.39(d,J=8.6Hz,2H),4.04(s,2H),2.13(s,3H)ppm; 13 C NMR (100MHz, DMSO-d6) δ171.5,158.7,138.3,129.1,128.1,121.0,119.7,118.8,89.0,21.6,14.7ppm; HRMS (ESI) m / z: C 12 H 12 N3O[M+H] + Theoretical value 214.0980, measured value m / z 214.0982.

[0060] Example 2

[0061] ANPMP for the construction of maltodextrin sugar chip

[0062] Step (1): ANPMP is dissolved in methanol to prepare an ANPMP solution with a concentration of 0.5 mol / L; 5 mg (0.05 mmol) of maltodextrin polysaccharide (maltodextrin polysaccharide is a macromolecular reducing oligosaccharide) is weighed and added to 500 μL of the ANPMP solution, and then 500 μL of 0.4 mol / L sodium hydroxide aqueous solution is added to the reaction system to adjust the pH of the reaction system to 8-9, and then the reaction system is stirred at a constant temperature of 70° C. for 1.5 hours. After the reaction is completed, it is cooled to room temperature, adjusted to pH=7 with a 3 mol / L hydrochloric acid aqueous solution, and then extracted with dichloromethane once, the aqueous phase is collected and 4 drops of glacial acetic acid are added, and then the aqueous phase is extracted with 2 mL of dichloromethane, and the supernatant is collected by centrifugation, concentrated under reduced pressure, and dried to obtain a crude maltodextrin derivative with two molecules of ANPMP. The Sep-PakC18 solid phase extraction column was first activated with 3 mL of acetonitrile, and then balanced with 12 mL of deionized water. The crude maltodextrin derivative with two molecules of ANPMP was redissolved with deionized water, and then sampled three times. It was eluted with 12 mL of double distilled water for desalting, and then eluted with an acetonitrile aqueous solution with a volume concentration of 20%. The eluate was collected, concentrated and dried to obtain a maltodextrin derivative with two molecules of ANPMP (referred to as Malt-2ANMP derivative). The obtained Malt-2ANMP derivative was detected and analyzed by ESI-MS; positive ion mode detection, the scan type was a full scan, and the data acquisition used LTQ Tune software to obtain Figure 1 The mass spectrum is shown. Figure 1 It can be seen that the molecular weight of each mass spectrum peak after maltodextrin polysaccharide and ANPMP derivatization increases by 408Da, that is, two ANPMP fragments are connected, proving that maltodextrin polysaccharide has been completely derivatized. The above results show that ANPMP can be used as a derivatization reagent to directly derivatize it.

[0063] Step (2): 10 mg of Malt-2ANMP derivative and 1 mg of 10% Pd / C catalyst were weighed and placed in a 38 mL thick-walled pressure-resistant bottle equipped with a stirrer, and 20 mL of 0.5 mol / L ammonium formate in methanol water (methanol to water volume ratio is 1:1) solution was added, and stirred at 40° C. for 48 hours; after the reaction was completed, the reaction solution was cooled to room temperature, transferred to a 50 mL centrifuge tube, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The black precipitate at the bottom was washed with a 50% methanol aqueous solution by volume, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken, and then washed again with a 50% methanol aqueous solution by volume for 3 times, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The supernatant solutions were combined and concentrated to dryness under reduced pressure. Double distilled water was added to the obtained sample to fully dissolve it, and the sample was transferred to a 5 mL centrifuge tube and centrifuged at 13,000 rpm for 5 minutes. After that, a porous graphite carbon solid phase extraction column was used for desalting. The specific steps were as follows: 1 g of graphite carbon filler was weighed and loaded into a 12 mL solid phase extraction column empty tube, activated with 36 mL of acetonitrile, balanced with 72 mL of double distilled water, loaded with sample, desalted with 20 mL of double distilled water, and eluted with 20 mL of 30% acetonitrile aqueous solution. The elution process was monitored by ESI-MS mass spectrometry until all target sugar chain peaks were eluted. All eluates containing the target peaks were combined, concentrated and dried under reduced pressure to obtain maltodextrin derivatives with single molecule AEPMP (referred to as Malt-AEPMP derivatives). The detection results in the positive ion mode of ESI-MS are as follows: Figure 2 As shown, all are [M+Na] + It was found that the mass spectrum peak of each sugar chain molecule was reduced by 209Da compared with that labeled with two molecules of ANPMP, which just corresponded to the subtraction of one molecule of ANPMP (M=213). The molecular weight increased by 4Da when the cyano group was reduced to the amino group.

[0064] Step (3): The Malt-AEPMP derivative is first separated by one-dimensional normal phase column high performance liquid chromatography, and the chromatographic conditions of the one-dimensional normal phase column high performance liquid chromatography separation are: using an analytical amide-based liquid chromatography column Inertsil Amide column (column size: 4.6mm×250mm); column temperature 25°C, detection wavelength 254nm, flow rate 0.8mL / min; mobile phase A is acetonitrile, mobile phase B is 100mmol / L acetic acid-ammonium acetate buffer with a pH of 6, the sample volume is 20μL (5mg Malt-AEPMP derivative is dissolved in 100μL of 75% acetonitrile aqueous solution by volume concentration), and the sample elution gradient is: 0-120 minutes, 75%-55% mobile phase A, 25%-45% mobile phase B. Figure 3A is a separation effect spectrum of Malt-AEPMP derivatives. Each component is collected for mass spectrometry detection, and 10 components are collected, namely F1 to F10. Then, the components collected by one-dimensional separation are separated by two-dimensional reverse phase column high performance liquid chromatography. The chromatographic conditions of the two-dimensional reverse phase column high performance liquid chromatography separation are: using an analytical reverse phase Cosmosil C18 chromatographic column (column size: 4.6mm×250mm); column temperature 25°C, detection wavelength 254nm, flow rate 0.8mL / min; mobile phase A is acetonitrile; mobile phase B is 10mmol / L acetic acid-ammonium acetate buffer with pH=5.5, and the sample elution gradient is: 0 to 30 minutes 2% to 6% acetonitrile mobile phase A, 98% to 94% mobile phase B. Figure 3 B is a two-dimensional liquid chromatogram of each component of the Malt-AEPMP derivative, and a total of 10 Malt-AEPMP derivative monomers with different polymerization degrees were separated.

[0065] Step (4): The above 10 Malt-AEPMP derivative monomer samples with different polymerization degrees were dissolved in ultrapure water on a 384-well plate, and diluted with 200mmol / L pH=8.5 phosphate buffer to a final concentration of 100μmol / L of maltose derivative monomers, and then spotted on an NHS functionalized glass slide using a non-contact spotter, and the temperature of the spotting platform was controlled to be 15°C and the humidity was 60%. Each Malt-AEPMP derivative monomer sample was printed with 4 spots on each array, and each array on each glass slide was printed 16 times. After the spotting was completed, the glass slide was placed in a constant temperature and humidity incubator and incubated for 12 hours at a temperature of 26°C and a humidity of 70%, and the glass slide was kept from drying. After drying in an oven at 50°C for 3 hours, it was taken out and naturally cooled to room temperature, and then the glass slide was rinsed with a phosphate buffer containing 0.05wt% Tween-20 to remove the maltose derivative monomers not bound to the glass slide, and then centrifuged and dried to obtain a maltodextrin sugar chip. The constructed sugar chip was placed in a sealed glass slide box and refrigerated at 4°C for 48 hours, and then transferred to -20°C for storage until use.

[0066] Example 3

[0067] ANPMP for the construction of human milk oligosaccharide microarray

[0068] Take breast milk, centrifuge it at 8000 rpm at 4°C for 30 minutes, take the water layer, add 2 times the volume of anhydrous ethanol, stir for 1 hour, centrifuge it at 80000 rpm at 4°C for 15 minutes, collect the supernatant, concentrate and dry it, add water to reconstitute it, and freeze-dry it to obtain the crude breast milk free oligosaccharide. 50g of crude breast milk free oligosaccharide was dissolved in 50mL of double distilled water, and the free oligosaccharide sample was first passed through a porous graphite carbon solid phase extraction column to remove lactose, and then the free neutral and acidic oligosaccharides were eluted together with an acetonitrile aqueous solution with a volume concentration of 50%, and ESI-MS detection was performed while washing, and the sample was collected and freeze-dried. The specific process was as follows: 3g of graphite carbon filler was weighed and loaded into a 36mL solid phase extraction column empty tube, activated with 100mL of acetonitrile, balanced with 300mL of double distilled water, loaded, washed with 1000mL of water to remove lactose, eluted with 2500mL of acetonitrile aqueous solution with a volume concentration of 50%, and free oligosaccharides from which lactose was removed were obtained, and ESI-MS detection was performed, the eluate was collected and evaporated to remove the solvent, and the remaining aqueous solution was freeze-dried to obtain breast milk free oligosaccharides for use. Subsequently, a DEAE-52 weak anion exchange column was used to separate neutral and acidic sugars. The specific process was as follows: first, the column was equilibrated with 1000 mL of 0.05 mol / L NaCl aqueous solution, and then 500 mL of water was used to elute the salts not bound to the column; then, the sample was loaded and eluted with double distilled water at a flow rate of 5 mL / min for 5 hours to obtain the neutral free oligosaccharide component (NHMOs) of breast milk, which was detected by ESI-MS. Figure 4 As shown; then eluted with a 0.25 mol / L NaCl aqueous solution at a flow rate of 5 mL / min for 6 hours to obtain a breast milk acid free oligosaccharide component, and the obtained breast milk acid free oligosaccharide component was placed in a dialysis bag with a cutoff of 100 Da and dialyzed for two days to desalinate, and a desalted breast milk acid free oligosaccharide mixture (SHMO S ), using ESI-MS detection, such as Figure 5 shown.

[0069] Step (1): According to the method of step (1) in Example 2, ANPMP is used to derivatize the neutral free oligosaccharides in breast milk to prepare a neutral free oligosaccharide derivative of breast milk with two molecules of ANPMP. The mass spectrometry detection result is as follows: Figure 4 shown.

[0070] Step (2): According to the method of step (2) in Example 2, the breast milk neutral free oligosaccharide derivative with two molecules of ANPMP is reduced by ammonium formate under the action of palladium carbon catalyst, and purified by porous graphite carbon solid phase extraction column to prepare a breast milk neutral free oligosaccharide derivative with a single molecule of AEPMP (referred to as NHMOs-AEPMP derivative). The mass spectrometry detection results are as follows Figure 6 shown.

[0071] Step (3): The NHMOs-AEPMP derivative is first separated by one-dimensional normal phase column high performance liquid chromatography, and the chromatographic conditions of the one-dimensional normal phase column high performance liquid chromatography separation are: using an analytical amide-based liquid chromatography column InertsilAmide column (column size: 4.6mm×250mm); column temperature 25°C, detection wavelength 254nm, flow rate 0.8mL / min; mobile phase A is acetonitrile, mobile phase B is 100mmol / L ammonium acetate aqueous solution with a pH of 6, the sample volume is 20μL (5mg NHMOs-AEPMP derivative is dissolved in 100μL of acetonitrile aqueous solution with a volume concentration of 75%), and the sample elution gradient is: 0-90 minutes 75%-67% mobile phase A, 25%-33% mobile phase B. Figure 7 A is a separation effect spectrum of NHMOs-AEPMP derivatives. Each component was collected for mass spectrometry detection, and a total of 9 components were collected, namely F1 to F9. Then, the components collected by one-dimensional separation were separated by two-dimensional reverse phase column high performance liquid chromatography. The chromatographic conditions of the two-dimensional reverse phase column high performance liquid chromatography separation were: using an analytical reverse phase Cosmosil C18 chromatographic column (column size: 4.6mm×250mm); column temperature 25°C, detection wavelength 254nm, flow rate 0.8mL / min; mobile phase A was acetonitrile, mobile phase B was 10mmol / L ammonium acetate aqueous solution with pH=5.5, mobile phase C was ultrapure water, and the sample elution gradient was: 0 to 30 minutes 2% to 6% acetonitrile mobile phase A, 98% to 94% mobile phase B. Figure 7 B is the two-dimensional liquid chromatogram of each component of NHMOs-AEPMP derivatives, and a total of 12 NHMOs-AEPMP derivative monomers were separated.

[0072] Step (4): According to the method of step (4) in Example 2, 12 NHMOs-AEPMP derivative monomers were fixed on an NHS functionalized glass slide through an amidation reaction to obtain a human milk oligosaccharide chip.

[0073] Example 4

[0074] ANPMP for the construction of porcine gastric mucin O-glycoprotein microarray

[0075] Step (1): ANPMP was added to a methanol aqueous solution with a volume concentration of 50% to prepare a 1.2 mol / L ANPMP solution; 1.44 g of porcine gastric mucin was weighed and placed in a 150 mL thick-walled pressure-resistant bottle equipped with a stirrer, and 64 mL of ANPMP solution was added, and then a 1.1 mol / L NaOH aqueous solution was added to the reaction system until the pH of the reaction system was 10 to 11, and the mixture was stirred at 80° C. for 24 hours; after the reaction was completed, the mixture was cooled to room temperature and the reaction solution was transferred to 2 Put it in a 50mL centrifuge tube, adjust the pH to 5.0 with concentrated hydrochloric acid, add 20mL of dichloromethane to extract, centrifuge at 8000 rpm for 10 minutes, take the supernatant, add glacial acetic acid to adjust the pH to 4.0, add 20mL of dichloromethane to extract, centrifuge at 8000 rpm for 10 minutes, take the supernatant, extract it three times with dichloromethane, centrifuge at 8000 rpm for 10 minutes, take the supernatant, use a rotary evaporator at 40°C to remove the solvent to obtain a khaki-colored crude product of the O-sugar chain derivative of porcine gastric mucin with two molecules of ANPMP. Add double distilled water to fully dissolve the crude product of the O-sugar chain derivative of porcine gastric mucin with two molecules of ANPMP, then transfer it to a 5mL centrifuge tube and centrifuge at 13000 rpm for 5 minutes. Use Sep-Pak C18 solid phase extraction column to remove protein, salt and unreacted ANPMP from the supernatant according to the method of step (1) in Example 2. Figure 8 As shown in the figure, 26 kinds of porcine gastric mucin O-glycosylated derivatives with high contents were detected in the positive ion mode by ESI-MS mass spectrometry, which were: m / z 652.25, 814.25, 855.25, 960.25, 1017.25, 1105.08, 1163.25, 1179.17, 1220.33, 1308.25, 1325.42, 1366.50, 1382.33, 1423.33, 1471.42, 1528.42, 1585.33, 1674.45, 1731.42, 1747.50, 1788.25, 1836.45, 1877.42, 1909.75 and 1950.5, all of which were [M+Na] + type ion peak, in addition, m / z 1060.16 is a doubly charged peak [M+2Na] 2+ This result is consistent with that reported in the literature.

[0076] Step (2): According to the method of step (2) in Example 2, the porcine gastric mucin O-sugar chain derivative with two molecules of ANPMP was reduced by ammonium formate under the action of palladium carbon catalyst, and purified by porous graphite carbon solid phase extraction column to prepare a porcine gastric mucin O-sugar chain derivative with a single molecule of AEPMP. The detection results in ESI-MS positive ion mode are as follows: Fig. 9As shown in the figure, a total of 26 ion peaks with high content were detected, including neutral and acidic oligosaccharide chains, which were m / z 443.25, 605.17, 646.25, 751.17, 808.17, 896.17, 954.25, 970.17, 1011.17, 1099.17, 1116.17, 1157.17, 1173.17, 1214.17, 1262.17, 1319.17, 1376.17, 1465.25, 1522.25, 1538.50, 1579.17, 1627.25, 1668.42, 1700.25, 1741.17 and 1888.33, all of which were [M+Na] + Type ion peak.

[0077] Step (3): The O-sugar chain derivative of porcine gastric mucin with single molecule AEPMP is first separated by one-dimensional normal phase column high performance liquid chromatography. The chromatographic conditions of the one-dimensional normal phase column high performance liquid chromatography separation are: an analytical amide-based liquid chromatography column Inertsil Amide column (column size: 4.6 mm×250 mm); a column temperature of 25° C., a detection wavelength of 254 nm, and a flow rate of 0.8 mL / min; mobile phase A is acetonitrile, mobile phase B is 100 mmol / L acetic acid-ammonium acetate buffer with a pH of 6, the sample volume is 20 μL (20 mg of the O-sugar chain derivative of porcine gastric mucin with single molecule AEPMP is dissolved in 400 μL of 75% acetonitrile aqueous solution by volume), and the sample elution gradient is: 75% to 60% mobile phase A, 25% to 44% mobile phase B from 0 to 90 minutes. Fig.10 A is a separation effect spectrum of porcine gastric mucin O-sugar chain derivatives with single molecule AEPMP. After collecting each component and detecting by mass spectrometry, it was found that the target peak of porcine gastric mucin O-sugar chain derivatives began to appear from 23 minutes, and a total of 14 components were collected, namely F1 to F14. Then, the components collected by one-dimensional separation were separated by two-dimensional reversed-phase column high performance liquid chromatography. The chromatographic conditions of the two-dimensional reversed-phase column high performance liquid chromatography separation were: using an analytical reversed-phase Cosmosil C18 column (column size: 4.6mm×250mm); column temperature 25℃, detection wavelength 254nm, flow rate 0.8mL / min; mobile phase A was acetonitrile, mobile phase B was 10mmol / L pH=5.5 acetic acid-ammonium acetate buffer, sample elution gradient: 0 to 30 minutes 2% to 6% mobile phase A, 98% to 94% mobile phase B. Fig.10 B is a two-dimensional liquid chromatogram of each component of the porcine gastric mucin O-sugar chain derivatives with a single molecule of AEPMP. A total of 29 porcine gastric mucin O-sugar chain derivative monomers with a single molecule of AEPMP were separated.

[0078] Step (4): According to the method of step (4) in Example 2, 29 kinds of porcine gastric mucin O-sugar chain derivative monomers with single molecule AEPMP were fixed on NHS functionalized glass slides through amidation reaction to obtain porcine gastric mucin O-sugar chip.

[0079] Example 5

[0080] AEPMP as a linker for the combination of a shotgun natural sugar chip containing 83 sugar chains and plant lectins AAL, HPA and SNA

[0081] Shotgun natural sugar arrays have been shown to be a reliable technique for analyzing the interaction between sugars and sugar-binding proteins. Common lectins are naturally occurring sugar-binding proteins (GBPs), and their sugar-binding properties have been well studied. Taking three well-defined biotinylated lectins as an example, AEPMP was detected as a linker shotgun natural sugar array, which includes 83 sugar chain derivatives (obtained according to the steps described in Example 2, and the specific sugar chain information is shown in Table 1. The above 83 oligosaccharides are derived from porcine gastric mucin O-sugar chains, breast milk free oligosaccharides, soybean glycoprotein N-sugar chains, maltodextrin polysaccharides, etc.), and a negative control group is PB phosphate buffer to confirm the fixation and recognition of sugar chains. As shown in Table 2, the average relative fluorescence units (RFUs) and coefficient of variation (%CV) are provided. The selected lectins include: fucose-binding lectin AAL; snail lectin HPA conjugated with terminal α-GlcNAc (N-acetylglucosamine) or α-GalNAc (N-acetylgalactosamine); sialic acid-binding lectin SNA.

[0082] In terms of the specific binding properties of lectin AAL, most fucose-containing oligosaccharides have strong binding to AAL, including those with terminal fucose residues and core fucose residues. Fig.11 The results of the fluorescence binding spots of A showed that the affinity of AAL to each oligosaccharide was significantly different. Fig.12 As shown in A, about 33 types of sugar chains have strong binding to AAL (RFU>10000) and bind to terminal α-fucosylation. In addition, 13 types of sugar chains have weaker binding strength (RFU 1000-8000) and can tolerate various forms of α-fucosylation, such as: α-1-2, α-1-3 and α-1-4 linkages. In general, the fucose-modified sugar chains recognized by AAL account for 55% of the total sugar chains isolated. This result is consistent with the results of mass spectrometry-based sugar chain composition analysis and is consistent with literature reports. Most importantly, many of the sugar chain molecules observed contain Lewis antigens, including Lewis a (Lewis antigen a [Galβ1-3(Fucα1-4)GlcNAc]) (sugar chains 33, 34, 36 and 54), Lewis x (Lex [Galβ1-4(Fucα1-3)GlcNAc]) (sugar chains 17, 32 and 37), Lewis b (Le b [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc])(sugar chain 59); blood group antigen A ([GalNAcβ1-3(Fucα1-2)Gal])(sugar chains 8, 9 and 33), blood group antigen H [Fucα1-2Galβ1-3GalNAc] (sugar chains 4, 5, 20, 21, 31, 37, 51, 61 and 65), they can mediate or regulate the interaction between cells and between cells and the extracellular environment, and therefore play an important role in many biological and pathological processes. HPA is bound to the terminal GalNAc residue, which is composed of Fig.11 The results of the fluorescence binding spots in B show that the affinity of HPA to each oligosaccharide is significantly different. The analysis results show that the interaction between HPA and the terminal α-GalNAc-modified sugar chains 8, 9, and 25 is expected, and the binding effect of HPA to the terminal α-GlcNAc is also obvious, such as Fig.12 Sugar chains 2, 15 and 23 shown in B. At an HPA concentration of 10 μg / mL, 18 sugar chains have extremely strong binding effects on HPA (RFU>30000), 11 sugar chains have moderately strong binding effects on HPA (RFU 8000-30000), and the remaining 10 sugar chains have weak binding effects on HPA (RFU 1000-8000). Compared with other types of sugar chain molecules, O-sugar chains as a whole show a higher level of HPA binding properties. This result indicates that the constructed natural sugar chip contains terminal epitopes of α-GalNAc and α-GlcNAc. As a plant lectin that recognizes terminal sialic acid-modified sugar chains, SNA is able to recognize sialic acid residues attached to the α-2,6 end. In this example, 14 sialylated sugar chains separated from breast milk acidic free oligosaccharides showed strong binding to SNA (RFU>10000) ( Fig.11 C, Fig.12 C), demonstrating the existence of the α2-6-sialylated type II (Neu5Acα2-6Galβ1-4GlcNAc, 6SLN) structure.

[0083] Table 1 Summary of sugar chain information in the natural oligosaccharide array containing 83 natural sugar probes

[0084]

[0085]

[0086]

[0087] Table 2 Summary of experimental results on the binding ability of natural oligosaccharide array containing 83 natural sugar probes with plant lectins AAL, HPA and SNA

[0088]

[0089]

[0090]

Claims

1. A cyanopyrazolone heterobifunctional reagent, wherein the reagent has the following structural formula: Its chemical name is 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone.

2. Use of the 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone according to claim 1 in constructing a sugar chip.

3. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 2, characterized in that: The following steps are involved: Step (1): subjecting a reducing sugar chain or glycoprotein to a Knoevenagel condensation and Michael addition reaction with 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone under alkaline conditions to generate a sugar chain derivative with two molecules of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone; the reducing sugar chain is a free reducing sugar chain or a sugar chain released from a glycoprotein; Step (2): reducing the sugar chain derivative with two molecules of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone with ammonium formate in the presence of palladium-carbon catalyst to form a sugar chain derivative with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone; Step (3): separating and purifying the sugar chain derivative with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone by two-dimensional high performance liquid chromatography to obtain a sugar chain derivative monomer with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone; Step (4): The sugar chain derivative monomer carrying a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone is fixed on a solid phase carrier functionalized with N-succinimide through an amidation reaction to obtain a sugar chip.

4. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 3, characterized in that: In step (1), a reducing sugar chain is added to a methanol or ethanol solution of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone, so that the concentration of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone in the obtained reaction system is 0.1 to 0.8 mol / L, and the molar ratio of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone to the reducing sugar chain is 5 to 15:1; then, an aqueous sodium hydroxide solution or an aqueous ammonia solution is added to the reaction system until the pH of the reaction system is 8 to 9, and then heated to 40 to 70° C., and stirred at a constant temperature for 1.5 to 4 hours; after the reaction is completed, the reaction solution is naturally cooled to room temperature, the pH of the reaction solution is adjusted to 7.0, and purified using a C18 solid phase extraction column to obtain a sugar chain derivative with two molecules of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone.

5. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 3, characterized in that: In step (1), the glycoprotein is added to a methanol or ethanol aqueous solution of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone, so that the concentration of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone in the obtained reaction system is 0.8-1.5 mol / L, and the mass ratio of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone to the glycoprotein is 15-50:1; then, a sodium hydroxide aqueous solution or an ammonia aqueous solution is added to the reaction system until the pH of the reaction system is 9-11, and then heated to 60-90° C., and stirred at a constant temperature for 12-48 hours; after the reaction is completed, the reaction is naturally cooled to room temperature, the pH of the reaction solution is adjusted to 6.0, extracted with chloroform or dichloromethane, concentrated, and purified by a C18 solid phase extraction column to obtain a sugar chain derivative with two molecules of 1-(4-acetonitrile phenyl)-3-methyl-5-pyrazolone.

6. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 3, characterized in that: In step (2), a sugar chain derivative with two molecules of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone, a palladium-carbon catalyst, and ammonium formate are added to a mixture of water and methanol or ethanol or isopropanol, and stirred at 25 to 45° C. for 12 to 48 hours. After the reaction is completed, the mixture is cooled to room temperature, centrifuged to remove the catalyst, and the supernatant is concentrated and the residue is purified by a graphite carbon column to obtain a sugar chain derivative with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone.

7. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 3, characterized in that: In step (3), the two-dimensional high performance liquid chromatography separation and purification method is: first use one-dimensional normal phase column high performance liquid chromatography to separate the sugar chain derivative with single molecule 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone, and then use two-dimensional reverse phase column high performance liquid chromatography to separate; the chromatographic conditions of the one-dimensional normal phase column high performance liquid chromatography separation are: TSK-GELAmide-80 column, column temperature 25°C, detection wavelength 254nm, flow rate 0.8-1.0mL / min, mobile phase A is acetonitrile, and mobile phase B is 100mmol / L The method comprises the following steps: using an acetic acid-ammonium acetate buffer solution at pH=6, performing gradient elution, collecting the eluate of each component respectively, concentrating under reduced pressure and then freeze-drying; the chromatographic conditions of the two-dimensional reverse phase column high performance liquid chromatography separation are as follows: a Cosmosil C18 column, a column temperature of 25°C, a detection wavelength of 254 nm, a flow rate of 0.8-1.0 mL / min, acetonitrile as mobile phase A, and 10 mmol / L acetic acid-ammonium acetate buffer solution at pH=5.5 as mobile phase B, performing gradient elution, collecting the eluate of each component respectively, and concentrating under reduced pressure.

8. The use of 1-(4-acetonitrilephenyl)-3-methyl-5-pyrazolone in constructing a sugar chip according to claim 3, characterized in that: In step (4), the sugar chain derivative monomer with a single molecule of 1-(4-aminoethylphenyl)-3-methyl-5-pyrazolone is dissolved in ultrapure water and diluted with 100-600 mmol / L phosphate buffer with pH=8.0-9.5 to a final concentration of 10-500 μmol / L, and then spotted on an N-succinimide functionalized solid phase carrier using a non-contact spotter, the spotting table temperature is controlled at 15-20°C and the humidity is controlled at 50%-70%. After spotting, the solid phase carrier is placed in a constant temperature and humidity incubator at a temperature of 25-30°C and a humidity of 50%-70% for incubation for 8-12 hours, and then dried in an oven at 50-60°C for 3-4 hours.

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