Heparin oligosaccharide analogue as well as synthesis method and equipment thereof

Abstract

The invention provides a heparin oligosaccharide analogue as well as a synthesis method and equipment thereof. The synthesis method of the heparin oligosaccharide analogue comprises the following steps: performing enzymolysis on African snail polysaccharide to obtain a first substrate comprising at least one of 6-12 sugars; and performing sulfonation modification on the basis of the first substrate to obtain the heparin oligosaccharide analogue. The synthesis method disclosed by the invention is improved from three aspects of substrate selection, catalytic reaction process optimization and fine regulation and control, specifically, the African snail oligosaccharide is adopted as a substrate, the processes of sugar chain extension, epimerization modification and 2-O sulfonation modification are omitted, and the time and the cost are saved; the repeated utilization of the enzyme is realized through the immobilization of the magnetic beads for modifying the enzyme; by means of the liquid drop micro-fluidic chip technology, the enzyme catalysis process is finely regulated and controlled, the reaction efficiency can be improved, and automatic synthesis of a product of a specific structure is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of synthesis, and in particular relates to a heparin oligosaccharide analogue and a synthesis method and device thereof. BACKGROUND

[0002] Synthesis of heparin oligosaccharide analogues is a research hotspot in the field. Chemical enzyme method is the most important method for synthesizing heparin oligosaccharide analogues at present. The traditional chemical enzyme synthesis method generally adopts a de novo sugar chain synthesis strategy, uses monosaccharide as a reaction raw material, and gradually extends the oligosaccharide skeleton structure from monosaccharide under the action of a sugar chain extension enzyme, and then modifies the skeleton structure. The traditional chemical enzyme method has the following disadvantages: first, since the catalytic efficiency of each step of reaction cannot reach 100%, especially the isomerization and 2-O sulfation reaction efficiency is low, the conversion rate is usually only about 60%, and the generated product has strong non-uniformity and complex structure, even if subsequent separation and purification is performed, it is difficult to obtain a single structure product for subsequent catalysis; second, the modification enzyme in the traditional catalytic process is used once, and after the catalytic reaction is completed, the enzyme needs to be inactivated by boiling the reaction system, resulting in an increase in reaction cost. In order to control the cost, the ratio of enzyme to substrate is usually reduced, which in turn reduces the catalytic efficiency; third, the traditional chemical enzyme synthesis method is usually carried out in a test tube, and once the enzyme and the substrate are mixed, they cannot be separated, which is not conducive to the fine regulation of the catalytic process and the automatic synthesis of the product. SUMMARY

[0003] In order to solve the problems existing in the prior art, the present application provides a heparin oligosaccharide analogue and a synthesis method and device thereof.

[0004] Specifically, the present application relates to the following aspects:

[0005] 1. A heparin oligosaccharide analogue comprising an oligosaccharide of the following structure:

[0006]

[0007] wherein R is H or SO3H, R' is H, Ac or SO3H, and m is an integer from 2 to 10.

[0008] 2. A synthesis method of a heparin oligosaccharide analogue, comprising:

[0009] subjecting an African giant snail polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars;

[0010] subjecting the first substrate to sulfation modification to obtain a heparin oligosaccharide analogue,

[0011] wherein the structure of the African giant snail polysaccharide is as shown below:

[0012]

[0013] n is an integer from 10 to 90.

[0014] 3. The method of item 2, wherein the sulfation modification is performed using a 6-0- sulfotransferase and / or a 3-0-sulfotransferase.

[0015] 4. The method of item 2, wherein the 6-0-sulfotransferase and the 3-0-sulfotransferase are immobilized enzymes.

[0016] 5. The method of item 4, wherein the immobilized enzymes are enzymes immobilized using polymeric magnetic microspheres.

[0017] 6. The method of any one of items 2-5, wherein the sulfation modification to obtain the heparin oligosaccharide analog from the first substrate is performed directly on the first substrate to obtain the heparin oligosaccharide analog.

[0018] 7. The method of item 6, wherein the sulfation modification is performed using a 6-0- sulfotransferase 1.

[0019] 8. The method of any one of items 2-5, wherein the sulfation modification to obtain the heparin oligosaccharide analog from the first substrate comprises:

[0020] hydrazinolysis of the first substrate to obtain a second substrate;

[0021] sulfation modification of the second substrate to obtain the heparin oligosaccharide analog.

[0022] 9. The method of item 8, wherein the sulfation modification is performed using a 6-0- sulfotransferase 1.

[0023] 10. The method of any one of items 2-5, wherein the sulfation modification to obtain the heparin oligosaccharide analog from the first substrate comprises:

[0024] hydrazinolysis of the first substrate to obtain a second substrate;

[0025] catalysis of the second substrate using an N- position sulfotransferase to obtain a third substrate;

[0026] sulfation modification of the third substrate to obtain the heparin oligosaccharide analog.

[0027] 11. The method of item 10, wherein the sulfation modification is performed using a 6-0- sulfotransferase 1 and / or a 3-0-sulfotransferase 5.

[0028] 12. The method of any one of items 2-11, wherein the sulfation modification of the first substrate to obtain the heparin oligosaccharide analog comprises any two or three of:

[0029] direct sulfation modification of the first substrate to obtain the heparin oligosaccharide analog;

[0030] hydrazinolysis of the first substrate to obtain a second substrate, and sulfation modification of the second substrate to obtain the heparin oligosaccharide analog;

[0031] hydrazinolysis of the first substrate to obtain a second substrate, catalysis of the second substrate using N- sulfo transferase to obtain a third substrate, and sulfation modification of the third substrate to obtain the heparin oligosaccharide analog.

[0032] 13. The method of any one of items 2-12, wherein the reaction of the sulfation modification is performed using a microfluidic system.

[0033] 14. The method of any one of items 2-13, wherein the heparin oligosaccharide analog is the heparin oligosaccharide analog of item 1.

[0034] 15. An apparatus for performing the method of any one of items 2-14, comprising a chip comprising:

[0035] an inlet channel for introducing at least one of the first substrate, the second substrate, and the third substrate;

[0036] a reaction cell immobilized with 6-O-sulfo transferase 1 and optionally 3-O-sulfo transferase 5 for sulfation modification of at least one of the first substrate, the second substrate, and the third substrate to obtain the heparin oligosaccharide analog;

[0037] an outlet channel for flowing out the synthesized heparin oligosaccharide analog to enable real-time monitoring of the product structure and reaction process control.

[0038] 16. The apparatus of item 15, wherein the apparatus further comprises:

[0039] a first substrate generating unit for hydrolyzing Lissomeris polysaccharide to generate the first substrate;

[0040] optionally a second substrate generating unit for hydrazinolysis of the first substrate to obtain the second substrate;

[0041] optionally a third substrate generating unit for catalysis of the second substrate to obtain the third substrate.

[0042] 17. The apparatus of item 15, wherein the chip is a microfluidic chip.

[0043] The synthetic method of the present application is improved from three aspects of substrate selection, catalytic reaction process optimization and fine regulation and control. Specifically, the African snail oligosaccharide is used as the substrate, the sugar chain extension, epimerization modification and 2-O sulfation modification processes are omitted, and the time and cost are saved; the magnetic bead immobilization of the modified enzyme is realized, and the reuse of the enzyme is realized; through the liquid droplet microfluidic chip technology, the fine regulation and control of the enzyme catalytic process can improve the reaction efficiency and realize the automatic synthesis of specific structure products. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The magnetic bead immobilization of the sulfotransferase is shown in the figure;

[0045] Figure 2 The sulfation reaction based on the liquid microfluidic technology is shown in the figure. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with examples. It should be understood that the examples are only used to further illustrate and explain the present application, and are not used to limit the present application.

[0047] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the materials and methods are described herein are presented for the purpose of illustrating and describing what is currently known. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The present application will be further described below in conjunction with specific examples, but not to limit the scope of the present application.

[0048] Heparin oligosaccharide analogues have important physiological functions such as anticoagulation, anti-inflammation, and anti-tumor. The synthesis of specific structure heparin oligosaccharides and the exploration of new functions are research hotspots in the field. The backbone structure of heparin oligosaccharides is formed by repeating the basic building block of disaccharide composed of hexuronic acid and glucosamine. The hydroxyl groups connected to the second position of hexuronic acid and the third and sixth positions of glucosamine can be modified by sulfation. The N position of glucosamine can exist in the form of naked amino group (-NH2) or be modified by acetylation / sulfation.

[0049] Hexuronic acid includes two conformations of glucuronic acid and iduronic acid. Iduronic acid is formed from glucuronic acid by epimerization reaction. Its spatial structure is highly variable, which is helpful for the interaction between sugar chain and protein and the physiological function. Therefore, in the process of artificial synthesis of heparin oligosaccharide analogues, it is usually necessary to introduce iduronic acid and modify specific hydroxyl sites by sulfation to endow the sugar chain with physiological functions.

[0050] As described above, the chemical enzyme method is the main method for synthesizing heparin oligosaccharide analogs, which consists of three parts of monosaccharide block synthesis, sugar chain extension, and sugar chain modification. Among them, the extension and modification of the sugar chain are realized by using specific sugar chain extension enzyme and a series of sulfation enzymes, and the synthesis product usually has a high proportion of iduronic acid and a high degree of sulfation, and has 2-O, 6-O and 3-O sulfation modification. However, the traditional chemical enzyme method needs to go through a series of sugar chain extension, enzyme modification reactions, involves a series of intermediate product separation and purification, the process is complicated, the cost is high and the reaction is difficult to control.

[0051] Therefore, the present application improves from three aspects of substrate selection, catalytic reaction process optimization and fine regulation and control, and provides a synthesis method of heparin oligosaccharide analogs.

[0052] Among them, the heparin oligosaccharide analogs of the present application include the following structure of oligosaccharide:

[0053]

[0054] Among them, R is H or SO3H, R' is H, Ac or SO3H, and m is an integer of 2-10.

[0055] The synthesis method of heparin oligosaccharide analogs provided by the present application comprises:

[0056] Step S1: enzymatic hydrolysis of African giant snail polysaccharide to obtain a first substrate comprising at least one of 6-12 sugars;

[0057] Step S2: sulfation modification based on the first substrate to obtain a heparin oligosaccharide analog,

[0058] Among them, the structure of the African giant snail polysaccharide is as follows:

[0059]

[0060] Among them, n is an integer of 10-90, i.e. African giant snail is a polysaccharide composition.

[0061] In step S1, the present application selects African giant snail polysaccharide as the starting substrate for the reaction. African giant snail polysaccharide is a glycosaminoglycan similar in structure to heparin polysaccharide found in African giant snails, which is a polysaccharide molecule formed by the large number of repeating arrangement of disaccharide units composed of 2-O-iduronic acid (IdoA2S) and N-acetylglucosamine (GlcNAc). Since all hexuronic acids in the sugar chain are iduronic acid with 2-O sulfation, the whole sugar chain structure is uniform.

[0062] The Lymnaea natalensis polysaccharide can be subjected to enzymatic hydrolysis by heparin lyase to degrade it into a series of oligosaccharide fragments, so as to obtain the first substrate including at least one of 6-12 saccharides. That is, the first substrate can be an oligosaccharide composition including one, two, three or four of hexaose, octaose, decaose and dodecaose, and can further include disaccharide, tetraose and other saccharides. The first substrate can also be a single hexaose, octaose, decaose or dodecaose. These single oligosaccharides can be obtained by separating and enriching the oligosaccharide fragments obtained by enzymatic hydrolysis by using molecular exclusion chromatography. For example, the Lymnaea natalensis polysaccharide can be subjected to enzymatic hydrolysis to obtain oligosaccharide fragments, and the obtained oligosaccharides can be separated by size and the octasaccharides can be enriched by using molecular exclusion chromatography, so as to obtain the following octasaccharides as the first substrate:

[0063]

[0064] It can be understood by those skilled in the art that the selection of the first substrate can be adjusted according to the actual application needs, as long as it includes at least one of 6-12 saccharides capable of exerting physiological functions, and the present application does not make specific limitations.

[0065] In step S2, the sulfation modification based on the first substrate means that the sulfation modification can be directly performed on the substrate, or the first substrate can be further reacted to generate other substrates and then the sulfation modification is performed.

[0066] Among them, the sulfation modification can be performed by using 6-O-sulfotransferase or 3-O-sulfotransferase according to different substrate types, or 6-O-sulfotransferase (6-OST) and 3-O-sulfotransferase (3-OST) can be used at the same time.

[0067] The 6-O-sulfotransferase and the 3-O-sulfotransferase can be free enzymes or immobilized enzymes.

[0068] In a specific embodiment, the 6-O-sulfotransferase and the 3-O-sulfotransferase are immobilized enzymes.

[0069] The immobilized enzyme can be obtained by immobilizing the enzyme on a solid carrier by using the physical or chemical methods known in the art. The solid carrier can use various types of carriers known in the art, such as polymer microspheres, nanoparticles, etc.

[0070] In a specific embodiment, the immobilized enzyme is an enzyme immobilized by using polymer magnetic microspheres.

[0071] Figure 1An exemplary process of immobilizing sulfotransferase onto magnetic beads to obtain immobilized sulfotransferase is shown. The N-hydroxysuccinimide (NHS) magnetic beads (magnetic agarose microspheres) are a kind of pre-activated polymer magnetic microspheres, and the surface is modified with NHS groups, which can be coupled with proteins or polypeptides containing amino groups.

[0072] After immobilizing the sulfotransferase, the catalytic activity of the enzyme does not change significantly compared with the free enzyme, and the enzyme activity can be maintained for more than 1 year. Through immobilization, the modified enzymes can be reused, greatly reducing the cost of catalytic reaction, and laying the foundation for the fine control of subsequent catalytic reaction.

[0073] In a specific embodiment, the sulfation modification on the basis of the first substrate to obtain the heparin oligosaccharide analog means directly performing sulfation modification on the first substrate to obtain the heparin oligosaccharide analog. The sulfation modification is performed by using 6-O-sulfotransferase 1 (6-OST-1).

[0074] Specifically, the sulfation modification reaction can be performed under the reaction conditions known in the art for 6-O-sulfotransferase 1. For example, the reaction conditions generally require a buffer salt system (MES, HEPES buffer salt, etc.), divalent metal ions (calcium ions, magnesium ions, manganese ions, etc.); the enzyme and substrate ratio can be adjusted, and the reaction can be promoted to the positive direction by increasing the enzyme amount, such as setting the molar ratio of the substrate to the enzyme to 5:1; the reaction temperature depends on the source of the enzyme, and is generally 37 degrees Celsius or room temperature; the reaction time can be adjusted according to the needs, and if complete sulfation is desired, the reaction time can be adjusted to more than 4 hours; the sulfation reaction particularly requires a sulfonic acid group donor (PAPS, etc.).

[0075] In a specific embodiment, the sulfation modification on the basis of the first substrate to obtain the heparin oligosaccharide analog includes:

[0076] Step S21: hydrazinolysis of the first substrate to obtain a second substrate;

[0077] Step S22: sulfation modification of the second substrate to obtain a heparin oligosaccharide analog.

[0078] The hydrazinolysis reaction in step S21 can be performed under the hydrazinolysis reaction conditions known in the art.

[0079] Taking the octasaccharide as the first substrate as an example, the second substrate obtained by hydrazinolysis of the first substrate is as follows:

[0080]

[0081] In step S22, the second substrate is subjected to sulfation modification using 6-O-sulfotransferase 1.

[0082] Specifically, the sulfation modification reaction can be carried out under reaction conditions known in the art for 6-O-sulfotransferase 1. For example, the reaction conditions generally require a buffer salt system (MES, HEPES buffer salt, etc.), divalent metal ions (calcium ions, magnesium ions, manganese ions, etc.); the enzyme and substrate ratio can be adjusted, and by increasing the amount of enzyme, the reaction can be driven to the positive side, such as the molar ratio of substrate to enzyme can be set to 5:1; the reaction temperature depends on the source of the enzyme, and is generally 37 degrees Celsius or room temperature; the reaction time can be adjusted according to the needs, if you want to achieve complete sulfation, you can adjust the reaction time to more than 4 hours as appropriate; sulfation reaction requires a sulfonic acid group donor (PAPS, etc.).

[0083] In one specific embodiment, the sulfation modification on the basis of the first substrate to obtain heparin oligosaccharide analogs includes:

[0084] Step S21: hydrazinolysis of the first substrate to obtain a second substrate;

[0085] Step S22: catalyzing the second substrate using N-site sulfotransferase to obtain a third substrate;

[0086] Step S23: sulfation modification of the third substrate to obtain heparin oligosaccharide analogs.

[0087] The hydrazinolysis reaction in step S21, and the reaction catalyzed by N-site sulfotransferase in step S22 can be carried out under the hydrazinolysis reaction, N-site sulfotransferase catalysis reaction conditions known in the art.

[0088] Taking the octasaccharide described above as the first substrate as an example, the third substrate obtained is as follows:

[0089]

[0090] In step S23, the third substrate is subjected to sulfation modification, which can be carried out using 6-O-sulfotransferase 1, or 3-O-sulfotransferase 5 (3-OST-5), or both 6-O-sulfotransferase 1 and 3-O-sulfotransferase 5.

[0091] Specifically, the sulfation modification reaction can be carried out under the reaction conditions known in the art for 6-O-sulfotransferase 1, 3-O-sulfotransferase 5. For example, the reaction conditions generally require a buffer salt system (MES, HEPES buffer salt, etc.), divalent metal ions (calcium ions, magnesium ions, manganese ions, etc.); the enzyme and substrate ratio can be adjusted, and by increasing the enzyme amount, the reaction can be driven to the positive direction, for example, the molar ratio of substrate to enzyme can be set to 5:1; the reaction temperature depends on the source of the enzyme, and is generally 37 degrees Celsius or room temperature; the reaction time can be adjusted according to the degree of sulfation requirement; the sulfation reaction particularly requires a sulfonic acid group donor (PAPS, etc.).

[0092] In one specific embodiment, the sulfation modification on the basis of the first substrate to obtain the heparin oligosaccharide analogue includes any two or three of the following:

[0093] directly sulfating the first substrate to obtain the heparin oligosaccharide analogue;

[0094] hydrazinolysis of the first substrate to obtain a second substrate, and sulfating the second substrate to obtain the heparin oligosaccharide analogue;

[0095] hydrazinolysis of the first substrate to obtain a second substrate, catalyzing the second substrate using an N-site sulfotransferase to obtain a third substrate, and sulfating the third substrate to obtain the heparin oligosaccharide analogue.

[0096] Further, the synthesis method of the present application can be carried out using a microfluidic system, for example, step S2 can be carried out using a microfluidic system.

[0097] Using microfluidic technology can achieve fine control of catalytic reactions, including control of key factors such as reaction time and reaction sequence. When the amount of oligosaccharide required for synthesis is small, it can be achieved through a digital droplet microfluidic module.

[0098] Specifically, in the digital droplet microfluidic module, the oligosaccharide substrate, the buffer salt required for the enzyme catalysis process, and the PAPS can be dissolved in droplets of a specific size. The droplets are placed on a modular metal plate and controlled by an external variable electric field. At this time, the droplets are mainly driven by the electrostatic force in the electric field, thereby realizing controllable programmed movement. In addition, a specific module on the metal plate can be controlled by magnetic force through an electromagnetic switch. When the switch is in the open state, magnetic beads containing modification enzymes can be fixed in the area and react with the substrate in the droplets in the area. The sequential catalysis of different enzymes can be realized by controlling the movement of the droplets. Since the ratio of enzymes and substrates in the system can be regulated in a wide range, the sulfation of all modifiable sites can be completed in a shorter time; or by accurately controlling the reaction time, partial sulfation of the substrate can be achieved. In addition, by programming the trajectory of the droplets, the automated synthesis of specific structure products can be realized.

[0099] Figure 2 The sulfation reaction based on liquid microfluidic technology is shown. Under this reaction system, the size of the droplets can be adjusted according to the required product amount and substrate concentration, so that they occupy different cell numbers, and the droplets move horizontally or vertically by a variable electric field. The droplets contain a buffer salt system, an oligosaccharide from African giant snail (first substrate), and a sulfogroup donor (such as 3'-phosphoadenosine-5'-phosphosulfate, etc.). The dark gray area in the figure is a controllable magnetic module. When the electromagnetic switch is in the open state, magnetic beads with sulfotransferase fixed therein can be fixed in the area, and when the droplets move to the position, the sulfation reaction of the substrate can be carried out. During the reaction, the reaction solution can be introduced into the mass spectrometry detection system through a capillary to realize real-time monitoring of the reaction process.

[0100] In one specific embodiment, the method for synthesizing heparin oligosaccharide analogs of the present application comprises: subjecting the African giant snail polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars; and subjecting the first substrate to sulfation modification to obtain the heparin oligosaccharide analogs; wherein the sulfation modification is performed by 6-O-sulfotransferase and / or 3-O-sulfotransferase, and the 6-O-sulfotransferase and the 3-O-sulfotransferase are immobilized enzymes; and the reaction for the sulfation modification is performed by using a microfluidic system.

[0101] In one specific embodiment, the method for synthesizing heparin oligosaccharide analogs of the present application comprises: subjecting the African giant snail polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars; and subjecting the first substrate to sulfation modification to obtain the heparin oligosaccharide analogs; wherein the sulfation modification is performed by 6-O-sulfotransferase 1, and the 6-O-sulfotransferase 1 is an immobilized enzyme; and the reaction for the sulfation modification is performed by using a microfluidic system. In one specific embodiment, the method for synthesizing heparin oligosaccharide analogs of the present application comprises: subjecting the African giant snail polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars; and subjecting the first substrate to sulfation modification to obtain the heparin oligosaccharide analogs; wherein the sulfation modification is performed by 6-O-sulfotransferase 1, and the 6-O-sulfotransferase 1 is an immobilized enzyme; and the reaction for the sulfation modification is performed by using a microfluidic system.

[0102] In one embodiment, the method for synthesizing heparin oligosaccharide analogs of the present application comprises: subjecting the Achatina polygona polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars; subjecting the first substrate to hydrazinolysis to obtain a second substrate; subjecting the second substrate to sulfation modification to obtain the heparin oligosaccharide analogs; wherein the sulfation modification is performed by 6-O-sulfotransferase 1, which is an immobilized enzyme; and the reaction for sulfation modification is performed using a microfluidic system.

[0103] In one embodiment, the method for synthesizing heparin oligosaccharide analogs of the present application comprises: subjecting the Achatina polygona polysaccharide to enzymatic hydrolysis to obtain a first substrate comprising at least one of 6-12 sugars; subjecting the first substrate to hydrazinolysis to obtain a second substrate; subjecting the second substrate to catalysis using N-sulfotransferase to obtain a third substrate; subjecting the third substrate to sulfation modification to obtain the heparin oligosaccharide analogs; wherein the sulfation modification is performed by 6-O-sulfotransferase 1 and / or 3-O-sulfotransferase 5, which are immobilized enzymes; and the reaction for sulfation modification is performed using a microfluidic system.

[0104] The present application also provides an apparatus for performing the above-mentioned method. The apparatus can be any known apparatus suitable for the above-mentioned method.

[0105] In one embodiment, the apparatus comprises a chip, which comprises:

[0106] a sample inlet channel for introducing at least one of the first substrate, the second substrate, and the third substrate;

[0107] a reaction pool immobilized with 6-O-sulfotransferase 1 and optionally 3-O-sulfotransferase 5 for sulfation modification of at least one of the first substrate, the second substrate, and the third substrate to obtain the heparin oligosaccharide analogs;

[0108] a sample outlet channel for flowing out the synthesized heparin oligosaccharide analogs to achieve real-time monitoring of the product structure and regulation of the reaction process.

[0109] The sample inlet channel can introduce one, two, or three of the first substrate, the second substrate, and the third substrate. The sample introduction speed and amount can be controlled according to the amount of the synthesized heparin oligosaccharide analogs and the substrate concentration required. When more than two substrates are required to be introduced, the substrates can be introduced sequentially or simultaneously.

[0110] The reaction pool is fixed with 6-O-sulfotransferase 1 and various substrates, sulfonic acid group donors and buffers required for sulfonic acid modification. Since 3-O-sulfotransferase 5 can also catalyze the third substrate for sulfonic acid modification, the reaction pool can be further fixed with 3-O-sulfotransferase 5. The fixed 6-O-sulfotransferase 1 and 3-O-sulfotransferase 5 in the reaction pool can be achieved by using the above-mentioned immobilization method and immobilization material. In a specific embodiment, 6-O-sulfotransferase 1 and 3-O-sulfotransferase 5 are fixed by magnetic beads.

[0111] In a specific embodiment, the chip is a microfluidic chip.

[0112] Those skilled in the art can understand that the reaction conditions such as substrates, buffers, reaction time and reaction temperature used in the reaction pool can be adjusted according to specific circumstances.

[0113] The device can further include a first substrate generating unit for hydrolyzing Lissachatina megasculpta polysaccharide to generate a first substrate. It can be understood that the first substrate generating unit can include various devices for hydrolyzing Lissachatina megasculpta polysaccharide, separating, purifying, desalting and the like of the hydrolysis product.

[0114] Further, the device can further include a second substrate generating unit for hydrazinolysis of the first substrate to obtain a second substrate.

[0115] Further, the device can further include a third substrate generating unit for catalyzing the second substrate to obtain a third substrate, for example, N-site sulfotransferase can be used to convert the second substrate into the third substrate.

[0116] Further, the device can further include a mass spectrometry detection system to realize real-time monitoring of the reaction process.

[0117] The synthesis method of the present application uses Lissachatina megasculpta oligosaccharide as a substrate, which saves the processes of sugar chain extension, epimerization modification and 2-O sulfonic acid modification, greatly reduces the number of reaction steps, saves time and cost, and avoids the common problem of incomplete reaction in the process of epimerization and 2-O sulfonic acid modification; by using immobilized sulfotransferase, the enzyme can be reused; through the use of droplet microfluidic technology, the enzyme catalysis process can be finely controlled, the reaction efficiency can be improved, and the automatic synthesis of specific structure products can be realized.

[0118] EMBODIMENT

[0119] EMBODIMENT 1

[0120] 1.1 Preparation of substrate

[0121] African giant snail polysaccharide is extracted from African giant snail. The shell of African giant snail is removed, and the rest is extracted with acetone to remove fat. The defatted sample is crushed and dissolved in 0.05M sodium carbonate buffer, and alkaline protease is added for treatment for 48 hours. Trichloroacetic acid is added to a final concentration of 12.5% to precipitate the protein, and after standing for 10 minutes, centrifugation is performed at 8000g for 20 minutes, and the supernatant is recovered. The supernatant is passed through a DAED ion chromatography column, and polysaccharides are adsorbed with the stationary phase using 0.05M sodium chloride, and then eluted using a 0.05-1M sodium chloride gradient. The eluate is desalted by G10 molecular exclusion chromatography, and high-purity African giant snail polysaccharide is obtained after freeze-drying.

[0122] African giant snail polysaccharide is partially enzymatically degraded using heparin lyase II, and the specific conditions of the enzymatic degradation are as follows:

[0123] Enzymatic degradation buffer: 0.1M sodium acetate, 0.01M calcium acetate, pH = 7; enzyme to substrate ratio: 50μg substrate / 1mIU heparinase II; enzymatic degradation time: 2 hours; enzymatic degradation temperature: 35 degrees Celsius.

[0124] After the enzymatic degradation is completed, the obtained oligosaccharide is separated by size using Bio-Gel P2 molecular exclusion chromatography (mobile phase 0.2M ammonium bicarbonate, flow rate 0.1mL / min), and octasaccharide is enriched therein, and after repeated freeze-drying and desalting, the structure of the obtained octasaccharide is

[0125] ΔHexA-GlcNAc-(IdoA2S-GlcNAc-)3

[0126] (Substrate 1), considering the mechanism of action of heparin lyase, the structure at the non-reducing end of the sugar chain is an unsaturated hexuronic acid. In order to obtain a more abundant substrate structure, the octasaccharide structure is hydrazinolysed, which will remove the acetyl group on the glucosamine, and the obtained structure is

[0127] ΔHexA-GlcNH2-(IdoA2S-GlcNH2-)3

[0128] (Substrate 2). The hydrazinolysis conditions are as follows: the substrate 1 is dissolved in a solution containing 1% (w / w) hydrazine sulfate, heated at 97°C for 4 hours; after the reaction is cooled, it is placed in an ice water bath to evaporate most of the hydrazine, and the residual hydrazine is removed by repeated freeze-drying; desalination is performed using a PD-10 chromatography column, and freeze-drying is performed to obtain high-purity substrate 2. The substrate 2 is modified using N-sulfotransferase (NST), and the enzyme modification reaction is carried out in a buffer salt system (50mM Hepes, pH 7.4; 0.1M KCl; 10mM MgCl2; 5mM CaCl2 and 10mM MnCl2), and a sulfonate donor (such as 3'-phosphoadenosine-5'-phosphosulfate, etc.) is added to achieve N-sulfation, and the reaction time is 24 hours, and the obtained structure is

[0129] △HexA-GlcNS-(IdoA2S-GlcNS-)3

[0130] (Substrate 3).

[0131] Specifically, the preparation process of substrate 1, substrate 2 and substrate 3 is shown in the following formula:

[0132]

[0133] 1.2 Magnetic Bead Immobilization of Sulfotransferase

[0134] To enhance the biological activity of heparin oligosaccharide analogs, 6-O-sulfotransferase (6-OST) and 3-O-sulfotransferase (3-OST) are typically used to modify glucosamine with 6-O and 3-O sulfonations. 6-OST has three distinct isoforms, all with similar crystal structures and catalytic activities, and all exhibit broad substrate selectivity. They can catalyze the 6-O sulfonation of glucosamine with acetyl, sulfo, and exposed amino groups at the N-position. 6-OST-1 was selected for magnetic bead immobilization. 3-OST has multiple isoforms, each with distinct substrate selectivity. Considering the structure of the African giant snail oligosaccharide, 3-OST-5 was selected for immobilization. The sulfotransferases were immobilized using NHS magnetic beads. The immobilization reaction conditions are as follows.

[0135] Washing buffer: 1 mM hydrochloric acid; Coupling buffer: 100 mM 2-morpholinoethanesulfonic acid, coupling at room temperature for 2 hours; Blocking buffer: 3 M ethanolamine, blocking at room temperature for 2 hours; Storage buffer: 1x PBS.

[0136] 1.3 Sulfonation reaction based on liquid microfluidics technology

[0137] Use Figure 2 The droplet microfluidic system shown controls the sulfonation reaction. In this reaction system, the droplet size can be adjusted to occupy different numbers of cells depending on the desired product amount and substrate concentration. The droplet's lateral or vertical movement is manipulated by a variable electric field. The droplet contains a buffer salt system (50mM Hepes, pH 7.4; 0.1M KCl; 10mM MgCl2; 5mM CaCl2; and 10mM MnCl2), oligosaccharides from giant African snails, and a sulfo group donor (such as 3'-phosphoadenosine-5'-phosphosulfate). The dark gray area in the figure represents a controllable magnetic module. When the electromagnetic switch is turned on, magnetic beads immobilized with sulfotransferase are fixed to this area. When the droplet moves to this position, the substrate sulfonation reaction proceeds. During the reaction, the reaction solution can be introduced into a mass spectrometry detection system via a capillary tube to enable real-time monitoring of the reaction process.

[0138] By controlling the route of the droplet, catalytic reactions of different substrates with different enzymes can be achieved. By controlling the reaction time, the sulfation of the product can be controlled to different degrees. After sufficient reaction, the products obtained after incubation of each substrate with different sulfotransferases are shown in Table 1.

[0139] Table 1. Synthesis of products with different structures

[0140]

[0141] For example, by programming, the following specific structure products can be synthesized automatically.

[0142] Incubation of substrate 3 with 6-OST-1, with a substrate to enzyme molar ratio of 10:1 and an incubation time of 20 minutes, can achieve conversion of 50% of GlcNS in the substrate to GlcNS6S;

[0143] Incubation of substrate 3 with 6-OST-1, with a substrate to enzyme molar ratio of 10:1 and an incubation time of 8 hours, can achieve conversion of 100% of GlcNS in the substrate to GlcNS6S;

[0144] By programmed control of the route of the droplet, incubation of substrate 3 with 6-OST-1 for 8 hours, and then incubation of the product with 3-OST-5 for 20 hours, the product

[0145] ΔHexA2S-GlcNS6S / GlcNS3S6S-(IdoA2S-GlcNS6S / GlcNS3S6S-)3.

[0147] Comparative Example 1

[0148] Instead of using the polysaccharide of African giant snail as the substrate, K5 octasaccharide (-GlcA-GlcNAc-)4, which is frequently reported in the literature, was used as the substrate. The reaction flow for preparing substrate 3 is shown in the following formula. It can be seen that, compared with the synthesis method of the present application, using K5 octasaccharide as the substrate requires additional epimerization and 2-O sulfation reactions, and introduces a separation and purification process (as shown in the last two steps of the following formula), increasing the complexity of the synthesis route.

[0149]

Claims

1. A heparin oligosaccharide analog, comprising an oligosaccharide having the following structure: wherein R is H or SO3H, R' is H, Ac or SO3H, and m is an integer of 2-10.

2. A method for synthesizing a heparin oligosaccharide analog, comprising: enzymatically hydrolyzing a polysaccharide of giant African snail to obtain a first substrate comprising at least one of 6-12 sugars; Based on the first substrate, sulfonation modification is performed to obtain a heparin oligosaccharide analogue, The structure of the African giant snail polysaccharide is shown below: n is an integer from 10 to 90.

3. The method according to claim 2, wherein the sulfonation modification is performed using 6-O-sulfotransferase and / or 3-O-sulfotransferase. The method according to claim 2 , wherein the 6-O-sulfotransferase and the 3-O-sulfotransferase are immobilized enzymes. The method according to claim 4 , wherein the immobilized enzyme is an enzyme immobilized using polymer magnetic microspheres.

6. The method according to any one of claims 2 to 5, wherein performing sulfonation modification on the first substrate to obtain the heparin oligosaccharide analogue refers to directly performing sulfonation modification on the first substrate to obtain the heparin oligosaccharide analogue. The method according to claim 6 , wherein the sulfonation modification is performed using 6-O-sulfotransferase 1.

8. The method according to any one of claims 2 to 5, wherein performing sulfonation modification on the first substrate to obtain a heparin oligosaccharide analog comprises: subjecting the first substrate to hydrazinolysis to obtain a second substrate; The second substrate is modified by sulfonation to obtain a heparin oligosaccharide analog. The method according to claim 8 , wherein the sulfonation modification is performed using 6-O-sulfotransferase 1.

10. The method according to any one of claims 2 to 5, wherein performing sulfonation modification on the first substrate to obtain a heparin oligosaccharide analog comprises: subjecting the first substrate to hydrazinolysis to obtain a second substrate; The second substrate is catalyzed by an N-sulfotransferase to obtain a third substrate; The third substrate is subjected to sulfonation modification to obtain a heparin oligosaccharide analog. The method according to claim 10 , wherein the sulfonation modification is performed using 6-O-sulfotransferase 1 and / or 3-O-sulfotransferase 5.

12. The method according to any one of claims 2 to 11, wherein the sulfonation modification performed on the first substrate to obtain the heparin oligosaccharide analogue comprises any two or three of the following: directly performing sulfonation modification on the first substrate to obtain a heparin oligosaccharide analog; performing hydrazinolysis on the first substrate to obtain a second substrate, and performing sulfonation modification on the second substrate to obtain a heparin oligosaccharide analog; The first substrate is subjected to hydrazinolysis to obtain a second substrate, the second substrate is catalyzed by N-sulfotransferase to obtain a third substrate, and the third substrate is subjected to sulfonation modification to obtain a heparin oligosaccharide analog.

13. The method according to any one of claims 2 to 12, wherein the sulfonation modification reaction is carried out using a microfluidic system.

14. The method of any one of claims 2-13, wherein the heparin oligosaccharide analog is the heparin oligosaccharide analog of claim 1.

15. An apparatus for performing the method of any one of claims 2-14, comprising a chip comprising: a sample inlet channel for loading at least one of the first substrate, the second substrate, the third substrate; a reaction cell immobilized with 6-O-sulfotransferase 1 and optionally 3-O-sulfotransferase 5 for sulfation modification of at least one of the first substrate, the second substrate, the third substrate to obtain the heparin oligosaccharide analog; a sample outlet channel for flowing out the synthesized heparin oligosaccharide analog to enable real-time monitoring of product structure and reaction process regulation.

16. The apparatus of claim 15, wherein the apparatus further comprises: a first substrate generation unit for hydrolyzing Lissotestacea polysaccharide to generate the first substrate; optionally a second substrate generation unit for hydrazinolysis of the first substrate to obtain the second substrate; optionally a third substrate generation unit for catalyzing the second substrate to obtain the third substrate.

17. The apparatus of claim 15, wherein the chip is a microfluidic chip.

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