Sulfonated sialic acid oligosaccharide library as well as preparation method and application thereof

Abstract

The invention provides a method for synthesizing a sialic acid oligosaccharide library with various sulfonated structures by a chemical enzyme method, and researches on the binding activity of sialic acid binding immunoglobulin-like lectin (Siglecs). Specifically, sialic acid and 9-azido-sialic acid are used as chemical-like protecting groups through an enzyme-catalyzed sialylation reaction, a specific position of oligosaccharide is protected, and then a sulfonate group is introduced at a preset site by utilizing high efficiency of a chemical reaction. Sialic acid or 9-azido-sialic acid, which is used as a protecting group, is removed by sialyl hydrolase, so that a sulfated oligosaccharide framework structure is obtained. And finally, carrying out enzymatic extension to obtain sulfonated sialic acid oligosaccharides with diversified structures. By means of the strategy, 66 kinds of sulfonated sialic acid oligosaccharides can be rapidly and efficiently prepared, the oligosaccharide chip prepared from the sulfonated sialic acid oligosaccharides is used for conducting systematic high-throughput activity screening on 12 proteins in the Siglecc family, and the high-affinity glycosyl ligands of the Siglecc proteins are found through research.

Description

Technical Field

[0001] This invention relates to the field of oligosaccharide synthesis technology, specifically to a library of sulfonated sialic acid oligosaccharides, its preparation method, and its applications. Background Technology

[0002] Oligosaccharides typically link with proteins or glycolipids to form glycoconjugates. Studies have shown that oligosaccharide conjugates participate in various physiological and pathological regulatory processes and are closely related to life activities. Abnormalities in their regulation are closely related to the occurrence and development of major diseases such as tumors, neurodegenerative diseases, and viral infections. Tumor development is often accompanied by the abnormal expression of related glycoantigens. Glycomolecules are showing increasingly important application prospects as drug targets in disease treatment and detection. Siglecs are a class of sialic acid-binding immunoglobulin-like lectins, and 15 siglec molecules have been identified in the human body. Sialic acid binds to sialic acid-binding immunoglobulin-like lectins (siglecs) on immune cells, inhibiting immune cell activation. The research group of Wang Ben at Zhejiang University found that knocking out Siglec-5 and Siglec-10 on the surface of macrophages can also significantly enhance the anti-solid tumor function of chimeric antigen receptor (CAR)-macrophages.

[0003] Sulfonation is an important modification of oligosaccharides. Sulfonation of sialic acid oligosaccharides plays a crucial role in the interaction between Siglec and oligosaccharide ligands. Related studies have found that sulfonated sialic acid oligosaccharide analogs exhibit significantly higher affinity for Siglec-8 than non-sulfonated oligosaccharides. (June, Spain) The research group used NMR technology to reveal that the sulfonate group is directly involved in the interaction between sialic acid oligosaccharides and Siglec-8. This study demonstrates that sialic acid oligosaccharides with specific sulfonation modifications hold significant drug potential in the treatment of diseases related to the Siglec protein.

[0004] Precise structural analysis of site-specific sulfonated sialic acid oligosaccharides is crucial not only for fundamental research into the mechanism of action of Siglec proteins but also for the development of immunotherapeutic strategies targeting Siglec proteins. Limited synthetic strategies make the acquisition of site-specific sulfonated sialic acid oligosaccharides extremely difficult, and a comprehensive library of sulfonated sialic acid oligosaccharide compounds remains lacking. This hinders research into the interaction mechanisms between Siglec family proteins and potential glycosyl ligands, further impacting the development of Siglec in the field of tumor immunotherapy.

[0005] Therefore, there is an urgent need in this field for a chemically enzymatically sulfonated sialic acid oligosaccharide library, its preparation method, and its applications. Summary of the Invention

[0006] The purpose of this invention is to develop a library of sulfonated sialic acid oligosaccharides, its preparation method, and its applications.

[0007] A first aspect of the present invention provides a sialic acid oligosaccharide library, said oligosaccharide library comprising oligosaccharide members selected from the group consisting of:

[0008] (a) An unsulfonated sialic acid oligosaccharide having the structure shown in formula (Ia):

[0009]

[0010] In the formula,

[0011] Z is either absent, or contains N-acetylneuraminic acid glycosyl, or contains 2-5 N-acetylneuraminic acid monomers as N-acetylneuraminic acid oligosaccharide.

[0012] G1 is galactosyl;

[0013] G2 is selected from the following group: N-acetylglucosinolate, glucosinolate, or N-acetylglucosamine;

[0014] R is selected from the group consisting of: - substituted or unsubstituted C1-C10 alkyl-azides, - substituted or unsubstituted C2-C10 alkenyl-azides; the substitution refers to being substituted by one or more substituents selected from the group consisting of: halogen (fluorine, chlorine, or bromine), hydroxyl, amino, nitro, C1-C3 alkyl, or carboxyl.

[0015] Q can be absent, fucose, or N-acetylneuraminic acid glycosyl;

[0016] (b) Sulfonated sialic acid oligosaccharides, said sulfonated sialic acid oligosaccharides having the structure shown in formula (Ib):

[0017]

[0018] In the formula,

[0019] The definitions of Z, Q, and R are the same as those in formula I;

[0020] G1h is a sulfonated galactosyl group;

[0021] G2h is selected from the following group: sulfonated N-acetylglucosinolate, sulfonated glucosinolate, or sulfonated N-acetylglucosamine.

[0022] In another preferred embodiment, at least one oligosaccharide member in the sialic acid oligosaccharide library is a sulfonated sialic acid oligosaccharide.

[0023] In another preferred embodiment, in the sialic acid oligosaccharide library, at least one oligosaccharide member is a non-sulfonated sialic acid oligosaccharide selected from the group consisting of:

[0024]

[0025]

[0026] in, It is N-acetylglucosinolate;

[0027] It is glucose-based;

[0028] It is galactosyl;

[0029] It is N-acetylgalactosamine;

[0030] It is an N-acetylneuraminic acid group;

[0031] It is fucose;

[0032] The definition of R is as described in the first aspect of this invention.

[0033] In another preferred embodiment, R is a substituted or unsubstituted C1-C10 alkyl azide, wherein the substitution refers to substitution by one or more substituents selected from the group consisting of halogen (fluorine, chlorine, bromine), hydroxyl, amino, nitro, or carboxyl, preferably.

[0034] In another preferred embodiment, G1 and G2 are connected by a glycosidic bond selected from the group consisting of β-1,4 glycosidic bonds or β-1,3 glycosidic bonds.

[0035] In another preferred embodiment, G1 and Z are connected by a glycosidic bond selected from the group consisting of α-1,3 glycosidic bonds or α-1,6 glycosidic bonds.

[0036] In another preferred embodiment, Z is linked to Z using an α-2,8 glycosidic bond.

[0037] In another preferred embodiment, Q and G2 are connected by a glycosidic bond selected from the group consisting of α-1,3 glycosidic bonds, α-1,4 glycosidic bonds, or α-1,6 glycosidic bonds.

[0038] In another preferred embodiment, the sulfonic acid group is modified at the 6-position of the sugar unit in the sulfonated sialic acid oligosaccharide.

[0039] In another preferred embodiment, the sialic acid oligosaccharide library is selected from sulfonated sialic acid oligosaccharides of the following group:

[0040]

[0041] in, It is N-acetylglucosinolate;

[0042] It is glucose-based;

[0043] It is galactosyl;

[0044] It is N-acetylgalactosamine;

[0045] It is an N-acetylneuraminic acid group;

[0046] It is fucose;

[0047] The definition of R is as described above.

[0048] In another preferred embodiment, the sialic acid oligosaccharide library is selected from the group consisting of non-sulfonated sialic acid oligosaccharides:

[0049]

[0050] in, It is N-acetylglucosinolate;

[0051] It is glucose-based;

[0052] It is galactosyl;

[0053] It is N-acetylgalactosamine;

[0054] It is an N-acetylneuraminic acid group;

[0055] It is fucose;

[0056] The definition of R is as described above.

[0057] In another preferred embodiment, the sialic acid oligosaccharide library is selected from the group consisting of:

[0058]

[0059] in, It is N-acetylglucosinolate;

[0060] It is glucose-based;

[0061] It is galactosyl;

[0062] It is N-acetylgalactosamine;

[0063] It is an N-acetylneuraminic acid group;

[0064] It is fucose;

[0065] The definition of R is as described in the first aspect of this invention.

[0066] In another preferred embodiment, R is -(CH2)3-N3.

[0067] A second aspect of the present invention provides a method for preparing sulfonated sialic acid oligosaccharides from a sialic acid oligosaccharide library as described in the first aspect of the present invention, comprising the steps of:

[0068] (s1) Reaction of the oligosaccharide shown in formula (I) with a first protecting agent containing a protecting group and / or a second protecting agent containing a protecting group, such that the H in the hydroxyl group at a predetermined position on G1 and / or G2 is replaced by the protecting group and / or the protecting group, thereby obtaining an oligosaccharide modified with the protecting group and / or the protecting group.

[0069]

[0070] (s2) An oligosaccharide modified with a protecting group-like group and / or a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group-like group and / or a protecting group; and

[0071] (s3) The sulfonated oligosaccharide modified with a protective group and / or a protecting group is subjected to a deprotection reaction to remove the protective group and / or the protecting group, thereby obtaining a sulfonated sialic acid oligosaccharide.

[0072] In another preferred embodiment, in step (s3), the sulfonated sialic acid oligosaccharide contains neither a protecting group nor a protecting group.

[0073] In another preferred embodiment, the protective group is selected from the group consisting of sialic acid or 9-azido-sialic acid.

[0074] In another preferred embodiment, the protecting group is used to protect the hydroxyl group at position 4 or 6 of G1.

[0075] In another preferred embodiment, the protecting group includes a silyl protecting group.

[0076] In another preferred embodiment, the chlorosilane is selected from the group consisting of tert-butyldimethylchlorosilane or tert-butyldiphenylchlorosilane.

[0077] In another preferred embodiment, the protecting group is used to protect the hydroxyl group at the 6-position of G1.

[0078] In another preferred embodiment, the method includes the steps of:

[0079] Method 1:

[0080] (s1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group to obtain the oligosaccharide modified with the protecting group.

[0081]

[0082] (s2) The oligosaccharide modified with a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group; and

[0083] (s3) The sulfonated oligosaccharide modified with a protecting group is subjected to a deprotection reaction to remove the protecting group, thereby obtaining a sulfonated sialic acid oligosaccharide;

[0084] Method 2:

[0085] (p1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group, thereby obtaining an oligosaccharide modified with a protecting group.

[0086]

[0087] (p2) The oligosaccharide modified with a protecting group reacts with a second protecting agent containing a protecting agent, thereby replacing the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group, to obtain an oligosaccharide modified with a protecting group and a protecting group.

[0088] (p3) The oligosaccharide modified with a protecting group and a protecting group is subjected to a deprotection reaction to remove the protecting group and obtain an oligosaccharide modified with only a protecting group.

[0089] (p4) The oligosaccharide modified with a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group; and

[0090] (p3) The sulfonated and protected oligosaccharide is subjected to a deprotection reaction to remove the protecting group, thereby obtaining sulfonated sialic acid oligosaccharide;

[0091] Method 3:

[0092] (z1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group to obtain the oligosaccharide modified with the protecting group.

[0093]

[0094] (z2) The oligosaccharide containing a fucosylation group is reacted with a third reagent containing a fucosylation group to replace the H in the hydroxyl group at a predetermined position on G2 with the fucosylation group, thereby obtaining an oligosaccharide modified with a fucosylation group and a fucosylation group.

[0095] (z3) Oligosaccharides modified with fucose groups and protecting groups are reacted with sulfonating agents to obtain sulfonated oligosaccharides modified with fucose groups and protecting groups; and

[0096] (z4) The sulfonated oligosaccharide modified with fucose and a protective group is subjected to a deprotection reaction to remove the protective group and / or the protective group, thereby obtaining the sulfonated sialic acid oligosaccharide.

[0097] The protecting group is selected from the group consisting of sialic acid or 9-azido-sialic acid.

[0098] Z, G1, G2, Q, and R are as described in the first aspect of this invention.

[0099] In another preferred embodiment, the step preceding step (s2) further includes step (s2p) fucoidylating the modified protecting group or the oligosaccharide of the protecting group.

[0100] In another preferred embodiment, in step (s1), the protective group is linked to the oligosaccharide shown in formula (I) via sialyl transferase.

[0101] In another preferred embodiment, in step (s1), the reaction equivalent of the protective group is 1 to 5, preferably 1 to 3 equivalents, for example, about 1.5 equivalents.

[0102] In another preferred embodiment, in step (s1), the sialyl transferase is selected from the group consisting of Pd2,6ST, M2,6ST, BtST, PPST, or combinations thereof.

[0103] In another preferred embodiment, the Pd2,6ST is derived from Photobacterium damsela.

[0104] In another preferred embodiment, the M2,6ST is an A200Y / S232Y mutant derived from Photobacterium damsela.

[0105] In another preferred embodiment, the BtST is derived from Bibersteinia trehalosi.

[0106] In another preferred embodiment, the PPST is derived from Photobacterium phosphoreum.

[0107] In another preferred embodiment, in step (s1), the protecting group in Mg2+ Modifications are performed while the object exists.

[0108] In another preferred embodiment, in step (s1), Mg 2+ The concentration is 5–100 mM, preferably 10–80 mM, more preferably 10–50 mM, for example, about 20 mM.

[0109] In another preferred embodiment, in step (s1), the amount of sialidyltransferase is 0.05 to 1 mg, more preferably 0.05 to 0.5 mg, more preferably 0.05 to 0.3 mg, for example, about 0.1 mg.

[0110] In another preferred embodiment, in step (s1), the modification is carried out at a pH of 6 to 9, preferably 7 to 9, for example, about 8.5.

[0111] In another preferred embodiment, in step (s1), the modification is performed at 20–40°C, more preferably 25–40°C, more preferably 30–40°C, for example, about 37°C.

[0112] In another preferred embodiment, in step (s1), the reaction time for the modification is 0.1 to 24 hours, more preferably 1 to 12 hours, for example, about 2 hours.

[0113] In another preferred embodiment, in step (s1), the protecting group is substituted in an alkaline solution.

[0114] In another preferred embodiment, in step (s1), the reaction equivalent of the protecting group is 1 to 20, more preferably 5 to 15, more preferably 8 to 13, for example, about 10 equivalents.

[0115] In another preferred embodiment, in step (s1), the base is selected from the group consisting of pyridine, triethylamine, diethylamine, ammonia, or combinations thereof.

[0116] In another preferred embodiment, in step (s1), the protective base is modified at 0–40°C, preferably 10–30°C, more preferably 20–30°C, for example, about 25°C.

[0117] In another preferred embodiment, in step (s1), the reaction time for the modification is 0.1 to 24 hours, more preferably 1 to 12 hours, for example, about 2 hours.

[0118] In another preferred embodiment, in step (s2), the sulfonating agent is reacted in an alkaline solution, the sulfonating agent containing a sulfonation donor.

[0119] In another preferred embodiment, in step (s2), the sulfonation donor is selected from the group consisting of sulfur trioxide / pyridine, sulfur trioxide / diethylamine, or combinations thereof.

[0120] In another preferred embodiment, in step (s2), the reaction equivalent of the sulfonation donor is 1 to 20, more preferably 3 to 15 equivalents, more preferably 5 to 13 equivalents, for example, 8 equivalents.

[0121] In another preferred embodiment, in step (s2), the base is selected from the group consisting of pyridine, triethylamine, diethylamine, ammonia, or combinations thereof.

[0122] In another preferred embodiment, in step (s2), the sulfonation reaction temperature is 0–40°C, more preferably 10–30°C, more preferably 20–30°C, for example, about 25°C.

[0123] In another preferred embodiment, in step (s2), the sulfonation reaction time is 0.1 to 24 hours, more preferably 1 to 12 hours, for example, about 2 hours.

[0124] In another preferred embodiment, in step (s3), the deprotecting group is obtained by sialic acid hydrolysis catalyzed by sialic acid hydrolase.

[0125] In another preferred embodiment, in step (s3), the sialic acid hydrolase comprises NanA, preferably NanA derived from Streptococcus penumoniae.

[0126] In another preferred embodiment, in step (s3), the amount of sialic acid hydrolase is 0.01 to 1 mg, more preferably 0.05 to 0.5 mg, for example, about 0.1 mg.

[0127] In another preferred embodiment, in step (s3), the removal of the protective group is carried out at a pH of 6 to 9, preferably at a pH of 6.5.

[0128] In another preferred embodiment, in step (s3), the reaction temperature for removing the protective group is 0–40°C, more preferably 10–40°C, more preferably 20–40°C, for example, about 37°C.

[0129] In another preferred embodiment, in step (s3), the reaction time for removing the protective group is 0.1 to 24 hours, more preferably 1 to 12 hours, for example, about 1 hour.

[0130] In another preferred embodiment, in step (3), the deprotection group is catalytically hydrolyzed TBS under acidic conditions.

[0131] In another preferred embodiment, in step (3), the acid is selected from the group consisting of acetic acid, hydrochloric acid, or combinations thereof.

[0132] In another preferred embodiment, in step (3), the concentration of the acid is 0.1 to 10 M, more preferably 0.5 to 5 M, more preferably 0.5 to 3 M, for example, 1 M.

[0133] In another preferred embodiment, in step (3), the deprotection is carried out at 0–40°C, preferably 10–30°C, more preferably 20–30°C, for example, about 25°C.

[0134] In another preferred embodiment, in step (3), the reaction time for deprotection is 0.1 to 24 hours, more preferably 0.5 to 12 hours, for example, about 0.5 hours.

[0135] In another preferred embodiment, in step (s2p), the fucosylation is performed by fucotransferase catalyzing the linking of fucose to the oligosaccharide.

[0136] In another preferred embodiment, in step (s2p), the fucosyltransferase comprises Hp1,3 / 4FuT, preferably Hp1,3 / 4FuT derived from Helicobacter pylori.

[0137] In another preferred embodiment, in step (s2p), the amount of fucosyltransferase is 0.01 to 1 mg, more preferably 0.05 to 0.5 mg, for example, about 0.1 mg.

[0138] In another preferred embodiment, in step (s2p), the fucosylation is performed on Mg 2+ It takes place in the presence of [something].

[0139] In another preferred embodiment, in step (s2p), Mg 2+ The concentration is 5–100 mM, preferably 10–80 mM, more preferably 10–50 mM, for example, about 10 mM.

[0140] In another preferred embodiment, in step (s2p), the fucosylation is carried out at a pH of 6 to 9, preferably at a pH of 7.5.

[0141] In another preferred embodiment, in step (s2p), the fucosylation is carried out at 10–40°C, more preferably 20–40°C, more preferably 25–40°C, for example, about 37°C.

[0142] In another preferred embodiment, in step (s2p), the reaction time for fucosylation is 0.1 to 24 h, more preferably 1 to 12 h, for example, about 1 h.

[0143] In a third aspect, the present invention provides an oligosaccharide chip, the oligosaccharide chip comprising a solid support and a sialic acid oligosaccharide library or a sialic acid oligosaccharide member thereof as described in the first aspect of the present invention, modified on the surface of the solid support.

[0144] In another preferred embodiment, the solid support is a glass plate, preferably a glass plate activated with N-hydroxysuccinimide (NHS).

[0145] In another preferred embodiment, the oligosaccharide chip is prepared by the following method:

[0146] The oligosaccharide members from the sialic acid oligosaccharide library, as described in the first aspect of the present invention, are printed onto a glass plate to obtain the oligosaccharide chip.

[0147] In another preferred embodiment, a non-contact microarray printer is used for printing.

[0148] In a fourth aspect, the invention provides the use of oligosaccharides from the sialic acid oligosaccharide library as described in the first aspect of the invention for preparing a control for determining the binding activity of immunoglobulin-like lectin (Siglec).

[0149] In another preferred embodiment, the Siglec is selected from the group consisting of: Siglec-1, Siglec-2, Siglec-3, Siglec-4, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-15, or combinations thereof.

[0150] In a fifth aspect, the present invention provides a sialic acid oligosaccharide, said sialic acid oligosaccharide comprising oligosaccharides selected from the group consisting of:

[0151]

[0152]

[0153] in, It is N-acetylglucosinolate;

[0154] It is glucose-based;

[0155] It is galactosyl;

[0156] It is N-acetylgalactosamine;

[0157] It is an N-acetylneuraminic acid group;

[0158] It is fucose;

[0159] The definition of R is as described in the first aspect of this invention.

[0160] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0161] Figure 1 The diagram shows the binding activity of sulfonated sialic acid molecules 1-66 with human Siglec-1 to Siglec-11 and Siglec-15. * The relative fluorescence value of the molecule with the best binding activity to each Siglec protein was defined as 100%.

[0162] Figure 2 The 1H NMR spectra of sulfonated backbone molecules 1-16 are shown. 1 H NMR).

[0163] Figure 3 A schematic diagram of the preparation method of sulfonated sialic acid molecules 1-66 is shown.

[0164] Figure 4 A schematic diagram of the structure of sulfonated sialic acid molecules 1-66 is shown. Detailed Implementation

[0165] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a sialic acid oligosaccharide library, comprising both unsulfonated and sulfonated sialic acid oligosaccharides. The inventors also developed a method for preparing the sialic acid oligosaccharide library of this invention, which allows for the modification of sulfonic acid groups at specific sites on the oligosaccharides. Experiments show that different oligosaccharides in the sialic acid oligosaccharide library of this invention exhibit different binding activities for immunoglobulin-like lectins (Siglecs). Based on this characteristic, the oligosaccharides of this invention can be used to prepare control standards for determining immunoglobulin-like lectin binding activity.

[0166] The inventors have developed a chemical enzymatic method for synthesizing a library of sulfonated sialic acid oligosaccharides with diverse structures, and studied the binding activity of sialic acid with Siglecs (immunoglobulin-like lectins). Specifically, the inventors used sialic acid and 9-azido-sialic acid as "chemical protecting groups" in an enzymatically catalyzed sialylation reaction to protect specific positions of the oligosaccharides, and then utilized the high efficiency of the chemical reaction to introduce sulfonate groups at predetermined sites. Subsequently, sialic acid hydrolysase removed the sialic acid or 9-azido-sialic acid as "protecting groups" to obtain the sulfonated oligosaccharide backbone structure. Finally, enzymatic extension yielded structurally diverse sulfonated sialic acid oligosaccharides. Using this strategy, 66 sulfonated sialic acid oligosaccharides can be rapidly and efficiently prepared. Oligosaccharide chips prepared from these sulfonated sialic acid oligosaccharides were used for systematic high-throughput activity screening of 12 proteins in the Siglec family, and the study revealed high-affinity glycosyl ligands for Siglec proteins. Based on this, the present invention was completed.

[0167] the term

[0168] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0169] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0170] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0171] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0172] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0173] As used in this article, It refers to N-acetylglucosinolates (GlcNAc).

[0174] As used in this article, Refers to glucose (Glc).

[0175] As used in this article, It refers to galactosyl (Gal).

[0176] As used in this article, It refers to N-acetylgalactosamine (GalNAc).

[0177] As used in this article, Refers to N-acetylneuraminic acid (Neu5Ac).

[0178] As used in this article, It refers to fucosylate (Fuc).

[0179] As used in this article, R is preferred.

[0180] As used herein, the term "S" indicates that the corresponding sugar unit has been sulfonated, for example, The “6S” indicates that the 6th position of N-acetylglucosamine in this oligosaccharide is modified by a sulfonic acid group.

[0181] The term "C1-C10 alkyl" refers to a straight-chain or branched alkyl group having 1-10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0182] The term "C2-C10 alkenyl" refers to a straight-chain or branched alkenyl group having 1 to 10 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.

[0183] The term "C1-C10 alkyl azide" refers to C1-C10 alkyl groups linked to azide groups. For example, the structural formula of a C3 alkyl azide is:

[0184] Sialic acid oligosaccharides

[0185] Sialic acid oligosaccharides are a term in biochemistry and molecular biology referring to oligosaccharides containing sialic acid. Sialic acid is a class of acidic amino sugars with nine carbon atoms and a pyranose structure, widely distributed in nature. It is also known as neuraminic acid, and its systematic name is 5-amino-3,5-dideoxy-D-glycerol-D-galactonanoneose. The most common sialic acids include N-acetylneuraminic acid (Neu5Ac), N-hydroxyacetylneuraminic acid (Neu5Gc), and 3-deoxy-D-glycerol-D-galactonanoneose (KDN).

[0186] sulfonation

[0187] Sulfonation refers to the process of introducing a sulfonic acid group (-SO3H) into a molecule through a chemical reaction. In biophysics, sulfonation specifically refers to the oxidation of the free sulfhydryl group (-SH) of cysteine ​​in proteins to form sulfonic acid (-SO3H). This typically occurs during the higher oxidation processes of reactive cysteine ​​and is a reversible process in mammals. Furthermore, in organic synthesis, sulfonation is often used to directly introduce sulfonic acid groups into organic molecules, such as using sulfonate ions provided by sulfuric acid to electrophilically substitute carbon-based aromatic hydrocarbon structures, forming the functional unit benzenesulfonic acid.

[0188] The oligosaccharides of the present invention

[0189] As used herein, the terms "molecule of the present invention," "disaccharide molecule of the present invention," "oligosaccharide of the present invention," and "disaccharide backbone molecule of the present invention" are used interchangeably and all refer to oligosaccharide members in the sialic acid oligosaccharide library of the present invention, wherein the oligosaccharide members are selected from the following group:

[0190] (a) An unsulfonated sialic acid oligosaccharide having the structure shown in formula (Ia):

[0191]

[0192] In the formula,

[0193] Z is either absent, or contains N-acetylneuraminic acid glycosyl, or contains 2-5 N-acetylneuraminic acid monomers as N-acetylneuraminic acid oligosaccharide.

[0194] G1 is galactosyl;

[0195] G2 is selected from the following group: N-acetylglucosinolate, glucosinolate, or N-acetylglucosamine;

[0196] R is selected from the group consisting of: - substituted or unsubstituted C1-C10 alkyl-azides, - substituted or unsubstituted C2-C10 alkenyl-azides; the substitution refers to being substituted by one or more substituents selected from the group consisting of: halogen (fluorine, chlorine, or bromine), hydroxyl, amino, nitro, C1-C3 alkyl, or carboxyl.

[0197] Q can be absent, fucose, or N-acetylneuraminic acid glycosyl;

[0198] (b) Sulfonated sialic acid oligosaccharides, said sulfonated sialic acid oligosaccharides having the structure shown in formula (Ib):

[0199]

[0200] In the formula,

[0201] The definitions of Z, Q, and R are the same as those in formula I;

[0202] G1h is a sulfonated galactosyl group;

[0203] G2h is selected from the following group: sulfonated N-acetylglucosinolate, sulfonated glucosinolate, or sulfonated N-acetylglucosamine.

[0204] In a preferred embodiment, at least one oligosaccharide member in the sialic acid oligosaccharide library is a sulfonated sialic acid oligosaccharide.

[0205] In a preferred embodiment, at least one oligosaccharide member in the sialic acid oligosaccharide library is a non-sulfonated sialic acid oligosaccharide selected from the group consisting of:

[0206]

[0207]

[0208] in, It is N-acetylglucosinolate;

[0209] It is glucose-based;

[0210] It is galactosyl;

[0211] It is N-acetylgalactosamine;

[0212] It is an N-acetylneuraminic acid group;

[0213] It is fucose;

[0214] The definition of R is as described in the first aspect of this invention.

[0215] This invention discloses 66 oligosaccharide molecules that can be used to prepare control standards for determining the binding activity of immunoglobulin-like lectins (Siglec).

[0216] The method of the present invention

[0217] In this invention, the terms "method of the present invention" and "method for preparing sialic acid oligosaccharides of the present invention" are used interchangeably and both refer to methods capable of preparing oligosaccharide members in the sialic acid oligosaccharide library of the present invention.

[0218] The method of the present invention includes the following steps:

[0219] (s1) Reaction of the oligosaccharide shown in formula (I) with a first protecting agent containing a protecting group and / or a second protecting agent containing a protecting group, such that the H in the hydroxyl group at a predetermined position on G1 and / or G2 is replaced by the protecting group and / or the protecting group, thereby obtaining an oligosaccharide modified with the protecting group and / or the protecting group.

[0220]

[0221] (s2) An oligosaccharide modified with a protecting group-like group and / or a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group-like group and / or a protecting group; and

[0222] (s3) The sulfonated oligosaccharide modified with a protective group and / or a protecting group is subjected to a deprotection reaction to remove the protective group and / or the protecting group, thereby obtaining a sulfonated sialic acid oligosaccharide.

[0223] In a preferred embodiment, the method of the present invention includes the following steps:

[0224] Method 1:

[0225] (s1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group to obtain the oligosaccharide modified with the protecting group.

[0226]

[0227] (s2) The oligosaccharide modified with a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group; and

[0228] (s3) The sulfonated oligosaccharide modified with a protecting group is subjected to a deprotection reaction to remove the protecting group, thereby obtaining a sulfonated sialic acid oligosaccharide;

[0229] Method 2:

[0230] (p1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group, thereby obtaining an oligosaccharide modified with a protecting group.

[0231]

[0232] (p2) The oligosaccharide modified with a protecting group reacts with a second protecting agent containing a protecting agent, thereby replacing the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group, to obtain an oligosaccharide modified with a protecting group and a protecting group.

[0233] (p3) The oligosaccharide modified with a protecting group and a protecting group is subjected to a deprotection reaction to remove the protecting group and obtain an oligosaccharide modified with only a protecting group.

[0234] (p4) The oligosaccharide modified with a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group; and

[0235] (p3) The sulfonated and protected oligosaccharide is subjected to a deprotection reaction to remove the protecting group, thereby obtaining sulfonated sialic acid oligosaccharide;

[0236] Method 3:

[0237] (z1) The oligosaccharide shown in formula (I) is reacted with a first protecting agent containing a protecting group to replace the H in the hydroxyl group at a predetermined position on G1 and / or G2 with the protecting group to obtain the oligosaccharide modified with the protecting group.

[0238]

[0239] (z2) The oligosaccharide containing a fucosylation group is reacted with a third reagent containing a fucosylation group to replace the H in the hydroxyl group at a predetermined position on G2 with the fucosylation group, thereby obtaining an oligosaccharide modified with a fucosylation group and a fucosylation group.

[0240] (z3) Oligosaccharides modified with fucose and protecting groups are reacted with sulfonating agents to obtain sulfonated oligosaccharides modified with fucose and protecting groups.

[0241] (z4) The sulfonated oligosaccharide modified with fucose and a protective group is subjected to a deprotection reaction to remove the protective group and / or the protective group, thereby obtaining the sulfonated sialic acid oligosaccharide.

[0242] The protecting group is selected from the group consisting of sialic acid or 9-azido-sialic acid.

[0243] Z, G1, G2, Q, and R are as described in the first aspect of this invention.

[0244] In a preferred embodiment, the method includes the method shown in formula (II), comprising the steps of:

[0245]

[0246] The disaccharide molecules (1 & 2) are fitted with the protecting group R2 under the action of sialyl transferase to obtain the skeletal oligosaccharide A; and

[0247] Sulfonation (oligosaccharide C) at predetermined sites was performed using a chemical method, and the "protective group" of oligosaccharide C was removed by sialic acid hydrolase to obtain sulfonated oligosaccharides 5 & 8.

[0248] Alternatively, a chemical method can be used to install a protecting group R3 at a predetermined site on oligosaccharide A (oligosaccharide B). Sialidase removes the protecting group of oligosaccharide B to obtain oligosaccharide D. Oligosaccharide D is then sulfonated at a predetermined site using a chemical method (oligosaccharide E). The TBS protecting group of oligosaccharide E is removed by acid to obtain a disaccharide backbone molecule with a sulfonated site (6 & 9).

[0249] In the formula, R is a C1 to C10 alkyl azide;

[0250] R1 is a hydroxyl group (OH) or an N-acetamide group (NHAc);

[0251] R2 is sialic acid (Neu5Ac) or 9-azido-sialic acid (9-N3-Neu5Ac);

[0252] R3 is a silane protecting group.

[0253] In a preferred embodiment, the method includes the method shown in formula (III), comprising the steps of:

[0254]

[0255] The disaccharide molecules (3 & 4) are fitted with a protecting group R2 under the action of sialyl transferase to yield the oligosaccharide F; and

[0256] Sulfonation of oligosaccharide F at predetermined sites (oligosaccharide H) was carried out using a chemical method, and sialidase removed the "protective group" of oligosaccharide H to obtain sulfonated oligosaccharides 11 & 14.

[0257] Alternatively, a protecting group R3 (oligosaccharide G) can be installed at a predetermined site of oligosaccharide F using a chemical method. The protecting group of oligosaccharide G can be removed by sialic acid hydrolase to obtain oligosaccharide I. Oligosaccharide I can be sulfonated at a predetermined site using a chemical method (oligosaccharide J). The TBS protecting group of oligosaccharide J can be removed by acid to obtain a disaccharide backbone molecule (12 & 15) with sulfonated site.

[0258] In the formula, R is a C1 to C10 alkyl azide;

[0259] R1 is a flat OH or an upright OH;

[0260] R2 is sialic acid (Neu5Ac) or 9-azido-sialic acid (9-N3-Neu5Ac);

[0261] R3 is a silane protecting group.

[0262] In a preferred embodiment, the method includes the method shown in formula (IV), comprising the steps of:

[0263]

[0264] Disaccharide molecules (1&2 and 3&4) are fitted with a protecting group R2 under the action of sialyl transferase to obtain oligosaccharide K or M. Oligosaccharide K or M is then disulfonated (oligosaccharide L or N) using a chemical method. Sialidase removes the "protecting group-like group" to obtain new disulfonated backbone molecules 7&10 and 13&16.

[0265] In the formula, R is a C1 to C10 alkyl azide;

[0266] R1 is a hydroxyl group (OH) or an N-acetamide group (NHAc);

[0267] R2 is 9-azido-sialic acid (9-N3-Neu5Ac);

[0268] R3 is a hydroxyl group (OH) with an equatorial or axial bond.

[0269] In a preferred embodiment, the method includes the method shown in formula (V), comprising the steps of:

[0270]

[0271] Disaccharide molecules (1 & 5) undergo fucosylation under the action of fucosyltransferase to yield fucosyl oligosaccharides 17 & 19; and / or

[0272] Oligosaccharide F is fucosylated to obtain oligosaccharide O, which is then sulfonated chemically to form oligosaccharide P. The TBS protecting group of oligosaccharide P is then removed by acid to obtain a fucose backbone molecule 20 with specific sulfonation sites; and / or

[0273] Oligosaccharide 17 of fucose is fitted with a "protective group" under the action of sialyltransferase to obtain oligosaccharide Q. Oligosaccharide Q is sulfonated by chemical method (oligosaccharide R), and the "protective group" is removed by sialyl hydrolase to obtain disulfonated fucose backbone molecule 21.

[0274] In the formula, R is a C1 to C10 alkyl azide;

[0275] R1 is hydrogen (H) or sulfonate (SO3H);

[0276] R2 is fucose;

[0277] R3 is a silane protecting group;

[0278] R4 is 9-azido-sialic acid (9-N3-Neu5Ac).

[0279] In a preferred embodiment, the method includes the method shown in formula (VI), comprising the steps of:

[0280]

[0281] The backbone molecules 1-22 prepared according to the method described above are subjected to sialylation to obtain α2,3 / 6 monosialylated oligosaccharides 32-54; and / or

[0282] Oligosaccharides 32-49 were subjected to α2,8 sialylation to obtain disialialylated oligosaccharides 55-66.

[0283] In another preferred embodiment, the sialylation reaction refers to the linkage of sialic acid to the backbone molecules 1-22 via catalysis by sialyltransferase.

[0284] In another preferred embodiment, the sialyl transferase is as described above.

[0285] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0286] Example 1: Synthesis of skeleton molecules 1-4

[0287]

[0288] Backbone molecule 2 was synthesized using propyl azide substrates U and V via a known route (J. Am. Chem. Soc. 2005, 127, 50, 17618–17619). Backbone molecules 1 and 3 were synthesized using U as a substrate with β1,4-galactosyltransferase from Neisseria meningitides and β1,4-galactosyltransferase from Chromobacterium violaceum (Cvβ3GalT) (Chem. Commun. 2010, 46, 6066-6068. Bioorgan. Med. Chem. 2016, 24, 1696-1705.). Backbone molecule 4 was synthesized using V as a substrate with β1,3-galactosyltransferase from Drosophila melanogaster (Nat. Commun. 2022, 13, 2398).

[0289] Example 2: "Protective Base" Installation Strategy

[0290]

[0291] Substrate molecules 1-4 (1.0 eq), Neu5Ac or 9-N3-Neu5Ac (1.5 eq), and cytidine triphosphate (CTP, 2 eq) were dissolved in Tris-HCl aqueous solution (20 mM, 20 mM MgCl2, pH 8.5). Sialate synthase (NmCSS) and sialate transferases (Pd2,6ST, M2,6ST, BtST, or PPST) derived from Neisseria meningitidis were added to the above reaction solution. The reaction was carried out at 37 °C, and the reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O, 10:3:2, v / v). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation, purified by molecular sieve and ion exchange resin to obtain target compounds 23-26.

[0292] Example 3 Sulfonation reaction

[0293]

[0294] Substrate molecules 69, 71, 73, and 75 (1.0 eq) were dissolved in a mixed solution of N,N-dimethylformamide (DMF) and triethylamine (TEA) (9:1, V:V). Sulfur trioxide / pyridine (SO3 / Py, 8 eq) was added under ice bath conditions, and the reaction was carried out at 0°C for 15 min. The reaction solution was then brought to room temperature, and the reaction continued for 1–2 h. The reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O, 10:3:2, v / v). After the reaction was complete, an equal volume of ethanol and saturated sodium bicarbonate aqueous solution were added to quench the reaction. The supernatant was collected by centrifugation and purified using molecular sieves and ion exchange resin to obtain target compounds 70, 72, 74, and 76.

[0295] Example 4: TBS Protective Base Mounting Reaction

[0296]

[0297] Substrate molecules 23-26 (1.0 eq) were dissolved in pyridine. Tert-butyldimethylchlorosilane (TBSCl, 10 eq) was added under ice bath conditions, and the reaction was carried out at 0°C for 15 min. The reaction solution was then brought to room temperature, and the reaction continued for 1-2 h. The reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O, 10:3:2, v / v). After the reaction was complete, an equal volume of ethanol and triethylamine were added to quench the reaction. The supernatant was collected by centrifugation and purified by molecular sieve and ion exchange resin to obtain target compounds 77, 79, 81, and 83.

[0298] Example 5 Protecting group removal reaction

[0299]

[0300] Substrate molecules 70, 72, 74, and 76 (1.0 eq) were dissolved in an aqueous sodium acetate solution (50 mM, pH 6.5), and sialidase NanA (0.1 mg / mmol substrate) was added. The reaction was carried out at 37 °C for 1–2 h, and the reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O, 10:3:2, v / v). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was purified by molecular sieve and ion exchange resin to obtain target compounds 5, 8, 11, and 14.

[0301] Example 6 Fucosylation reaction

[0302]

[0303] Substrate molecules 1 and 5 (1.0 eq) and fucose guanosine diphosphate (1.5 eq) were dissolved in Tris-HCl aqueous solution (20 mM, 10 mM MgCl2, pH 7.5). Fucosyltransferase Hpα1,3 / 4FucT was added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O, 10:3:2, v / v). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation. The supernatant was purified by molecular sieve and ion exchange resin to obtain target compounds 17 and 19.

[0304] Example 7: Sialization reaction

[0305]

[0306] Substrate molecules 5, 14, and 38 (1.0 eq) and cytidine monophosphate sialic acid (CMP-Neu5Ac, 1.5 eq) were dissolved in Tris-HCl aqueous solution (20 mM, 5 mM MgCl2, pH 8.5). Sialyltransferases (Pd2,6ST, M2,6ST, BtST, or PPST) were added to the above reaction solution, and the reaction was carried out at 37 °C. The reaction progress was monitored by thin-layer chromatography (TLC) (EtOAc / MeOH / H2O / HOAc, 4:2:1:0.2, v / v). After the reaction was completed, an equal volume of ethanol was added to quench the reaction, and the supernatant was collected by centrifugation, purified by molecular sieve and ion exchange resin to obtain target compounds 27, 38, 47, and 59.

[0307] Example 8: Oligosaccharide Chip Fabrication

[0308] 1-66 sugar molecules (0.5 mg) were dissolved in deionized water, and palladium hydroxide (0.3 mg, 20% wt water) was added. The mixture was reacted at room temperature under hydrogen atmosphere for 2 hours. TLC analysis showed that the reaction was complete. The solid was filtered off through a 0.22 μm filter membrane, and the mixture was lyophilized to obtain aminoated sugar molecules. Sixty-six aminoated sugar molecules were then printed onto an N-hydroxysuccinimide (NHS) activated glass plate using a Scienion sciflexarray S1 non-contact microarray printer (equipped with a Scienion PDC80 nozzle). Oligosaccharide chips were formed on Slide H, Schott Inc. Sixty-six aminoglycoside molecules were dissolved in sodium phosphate buffer (250 mM, pH 8.5) at a concentration of 100 μM. Four chips were printed in a group (spot volume ~400 pL, temperature 20 °C, humidity 50%). Each plate contained 14 subarrays (2x7), with each subarray displaying 288 dots (18x16). After printing, the slides were incubated overnight in a saturated NaCl chamber (providing 75% relative humidity). The plates were then blocked with 5 mM ethanolamine in Tris-HCl buffer (pH 9, 50 mM) for 1 h at 50 °C. The plates were rinsed with deionized water, dried, and stored in a desiccator.

[0309] Example 9: Oligosaccharide chip detection of protein binding activity

[0310] The prepared oligosaccharide chip was used to systematically screen the activity of 12 Fc-tagged Siglec proteins, identifying high-affinity glycosyl ligands for each Siglec protein. The basic experimental procedure is as follows:

[0311] First, the recombinant human Siglec-Fc chimeric antibody was premixed with commercially available Alexa Fluor 647-conjugated goat anti-human IgG at a 1:2 ratio and incubated for 10 minutes to ensure all Siglec-Fc chimeric antibodies were labeled. Then, the Siglec-Fc labeled with the secondary antibody was prepared into a solution of a specific concentration (e.g., 10 μg / mL) using TSM binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.05% Tween, 1% BSA). Subsequently, 100 μL of the prepared protein solution was added to an incubator containing a glass chip and incubated at room temperature for 1 hour. After incubation, the protein solution in the incubator was discarded, and the sample was washed sequentially with TSM wash buffer (soaking for 2 minutes), TSM buffer (soaking for 2 minutes), and deionized water. The sample was then centrifuged, dried, and the chip was scanned.

[0312] After incubation, the glass chips were washed and dried, then scanned using a GenePix 4000B microarray scanner at an appropriate excitation wavelength (5 μm resolution). Different gain and PMT values ​​were used during scanning to ensure all signal intensities remained within the linear range of the scanner detector and that no signal saturation occurred. Images were analyzed and fluorescent spots were integrated using GenePix Pro 7 software (version 7.2.29.2, Molecular Devices). The integrated data were analyzed using an Excel macro (http: / / zenodo.org / record / 5146251). The two spots with the highest and lowest total fluorescence intensity values ​​from the six replicates of each glycan binding result were removed, and the mean and standard deviation were calculated using the remaining four spot values. Finally, the binding data were integrated with the chip-fixed sulfosyl sialic acid oligosaccharide structure information, and a binding histogram was generated using GraphPad Prism software (version 5.0).

[0313] like Figure 1As shown, 66 molecules exhibit strong binding activity with Siglec-1, -2, -3, -4, -7, -8, -9, -10, and -11, while their binding activity with Siglec-5, -6, and -15 is weak. Sulfonation enhances the affinity of sugar molecules for Siglec proteins to some extent. Sulfonated molecules at the Gal-6 position (39, 40, 45, 46, 48, 49, 50, 52, 53, and 54) bind strongly to Siglec-1, with molecule 50 showing the strongest binding, indicating that Gal-6 sulfonation modification has a significant impact on Siglec-1 binding. Sulfonated sialic acid molecule 27 exhibits the strongest binding activity with Siglec-2. Disulfonated β1,3Gal backbone molecules (46 and 49) bind strongly to Siglec-8, but poorly to Siglec-3, suggesting that besides sialic and sulfonic acid groups, the backbone structure also affects the binding of Siglec to ligands. The binding strength of molecules 44 and 47 to Siglec-9 is significantly stronger than that of 38 and 41, indicating that Siglec-9 prefers the α-2,3-sialylated Galβ1,3GlcNAc and Galβ1,3GalNAc skeletal structures. The disulfonated molecule 40 exhibits the strongest binding activity to Siglec-3, even stronger than the fucose-modified molecule 53, suggesting that fucose does not contribute to the binding activity between the molecule and Siglec-3. Sulfonation at the GalNAc-6 position significantly improves the binding activity between the molecule and Siglec-4 (47 vs. 35), while sulfonation at the Gal-6 position has no contribution (47 vs. 35). Replacing the sulfonic acid group (49) at the GalNAc-6 position with sialic acid (50) significantly reduces the binding activity between the molecule and Siglec-4. The GlcNAc, Glc, and GalNAc structures within the 6-position sulfonation significantly enhance the binding activity of α-2,6-sialic acid molecules with Siglec-10 (27, 28, 29, and 30 vs. 23, 24, 25, and 26). The effect of sulfonation on the binding activity of α-2,3-sialic acid molecules with Siglec-10 is related to the skeletal structure. For example, sulfonated Galβ1,4GlcNAc molecule 32 does not contribute to the binding activity of the molecule with Siglec-10 (38, 39, and 40), while sulfonated Galβ1,3GlcNAc molecule 34 significantly enhances the binding activity of the molecule with Siglec-10 (44, 45, and 46). Siglec-11 only shows strong binding with molecules 40 and 53, indicating that Siglec-11 has relatively strict requirements on the skeletal structure and sulfonation modification of the sugar ligand.

[0314] The structure, starting materials, and preparation methods of the above oligosaccharides are shown in Table 1.

[0315] Table 1

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325] Note: Class I refers to compounds with a skeleton of Galβ1,4GlcNAcproN3; Class II refers to compounds with a skeleton of Galβ1,3GlcNAcproN3; Class III refers to compounds with a skeleton of Galβ1,3GlcNAcproN3; Class II refers to compounds with a skeleton of Galβ1,3GalNAcproN3.

[0326] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A library of sialic acid oligosaccharides, characterized in that, The oligosaccharide library includes oligosaccharide members selected from the group consisting of: (a) An unsulfonated sialic acid oligosaccharide having the structure shown in formula (Ia): In the formula, Z is either absent, or contains N-acetylneuraminic acid glycosyl, or contains 2-5 N-acetylneuraminic acid monomers as N-acetylneuraminic acid oligosaccharide. G1 is galactosyl; G2 is selected from the following group: N-acetylglucosinolate, glucosinolate, or N-acetylglucosamine; R is selected from the group consisting of: - substituted or unsubstituted C1-C10 alkyl-azides, - substituted or unsubstituted C2-C10 alkenyl-azides; the substitution refers to being substituted by one or more substituents selected from the group consisting of: halogen (fluorine, chlorine, or bromine), hydroxyl, amino, nitro, C1-C3 alkyl, or carboxyl. Q can be absent, fucose, or N-acetylneuraminic acid glycosyl; (b) Sulfonated sialic acid oligosaccharides, said sulfonated sialic acid oligosaccharides having the structure shown in formula (Ib): In the formula, The definitions of Z, Q, and R are the same as those in formula I; G1h is a sulfonated galactosyl group; G2h is selected from the following group: sulfonated N-acetylglucosinolate, sulfonated glucosinolate, or sulfonated N-acetylglucosamine.

2. The sialic acid oligosaccharide library as described in claim 1, characterized in that, The sialic acid oligosaccharide library is selected from the following group of sulfonated sialic acid oligosaccharides: in, It is N-acetylglucosinolate; It is glucose-based; It is galactosyl; It is N-acetylgalactosamine; It is an N-acetylneuraminic acid group; It is fucose; The definition of R is as described in claim 1.

3. A method for preparing sulfonated sialic acid oligosaccharides from the sialic acid oligosaccharide library as described in claim 1, characterized in that, Including the following steps: (s1) Reaction of the oligosaccharide shown in formula (I) with a first protecting agent containing a protecting group and / or a second protecting agent containing a protecting group, such that the H in the hydroxyl group at a predetermined position on G1 and / or G2 is replaced by the protecting group and / or the protecting group, thereby obtaining an oligosaccharide modified with the protecting group and / or the protecting group. (s2) An oligosaccharide modified with a protecting group-like group and / or a protecting group is reacted with a sulfonating agent to obtain a sulfonated oligosaccharide modified with a protecting group-like group and / or a protecting group; and (s3) The sulfonated oligosaccharide modified with a protective group and / or a protecting group is subjected to a deprotection reaction to remove the protective group and / or the protecting group, thereby obtaining a sulfonated sialic acid oligosaccharide.

4. The method as described in claim 3, characterized in that, The step preceding step (s2) further includes step (s2p) fucoidylating the modified protecting group or the oligosaccharide of the protecting group.

5. The method as described in claim 3, characterized in that, In step (s1), the protective group is linked to the oligosaccharide shown in formula (I) via sialyl transferase.

6. The method as described in claim 3, characterized in that, In step (s1), the protecting group is substituted in an alkaline solution.

7. The method as described in claim 3, characterized in that, In step (s2), the sulfonating agent is reacted in an alkaline solution, and the sulfonating agent contains a sulfonation donor.

8. An oligosaccharide chip, characterized in that, The oligosaccharide chip includes a solid support and a sialic acid oligosaccharide library or a sialic acid oligosaccharide member thereof as described in claim 1, which is modified on the surface of the solid support.

9. The use of the oligosaccharides in the sialic acid oligosaccharide library as described in claim 1, characterized in that, Used to prepare a reference standard for determining the binding activity of immunoglobulin-like lectin (Siglec).

10. A sialic acid oligosaccharide, characterized in that, The sialic acid oligosaccharides include oligosaccharides selected from the group consisting of: in, It is N-acetylglucosinolate; It is glucose-based; It is galactosyl; It is N-acetylgalactosamine; It is an N-acetylneuraminic acid group; It is fucose; The definition of R is as described in claim 1.

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