Peptides, macrophage activators, and glycosylation methods

Abstract

In one embodiment, the problem that the present invention aims to solve is to produce an artificial peptide whose three-dimensional structure is formed by folding. [Solution] The peptide represented by the following general formula (1) JPEG2026136935000021.jpg17169 [wherein AA0, AA1, AA2, AA3, AA4, X, l, m and n as described in the specification].

Description

[Technical Field]

[0001] This invention relates to peptides, macrophage activators, and methods for adding sugar molecules to amino acids. [Background technology]

[0002] GcMAF (Gc protein-derived macrophage activating factor) is known to convert inactive macrophages (monocytes) into an active form, further enhancing their inherent antitumor, phagocytic, and anti-inflammatory effects. Therefore, there was a trend in the pharmaceutical industry to develop GcMAF as a drug, but progress was slow. The reasons for this include: 1) the difficulty in obtaining a three-dimensional structure that maintains physiological activity because GcMAF is a high-molecular-weight protein; 2) the structural instability of its active site due to degradation by glycohydrolases in the body; and 3) the difficulty in introducing glycohydrolysis-resistant glycosylation structures.

[0003] As described above, artificially producing medium to high molecular weight peptides is difficult because it is challenging to reproduce their three-dimensional structure, and there is a strong demand for its realization. Furthermore, there is a strong desire to develop glycoproteins that are resistant to degradation by glycohydrolases. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cancer Treat. Res. Commun. 2022, 31, 100537 [Non-Patent Document 2] Anticancer Res. 2002, 22, 4297-4300 [Non-Patent Document 3] RU Lemieux, RM Ratcliffe, Can.J. Chem.1979, 57, 1244-1251. [Non-Patent Document 4] O. Kanie, T. Takeda, Y. Ogihara, J. Carbohydr.Chem.1990, 9, 159-165. [Non-Patent Document 5] T.Rosen, IM Lico, DTW Chu, J. Org.Chem.1988, 53, 1580-1582. [Non-Patent Document 6] R.Deziel, Tetrahedron Lett.1987, 28, 4371-4372. [Non-Patent Document 7] JM Richardson, CW Jones, Adv. Synth.Catal.2006, 348, 1207-1216. [Non-Patent Document 8] P.Sjolin, M. Elofsson, J. Kihlberg, J. Org.Chem.1996, 61, 2, 560-565. [Non-Patent Document 9] SC Garman, L. Hannick, A. Zhu, DN Garboczi, Structure, 2002, 10, 425-434. [Non-Patent Document 10] M. Karplus, J. Comput.Chem.1983, 4, 187-217. [Overview of the project] [Problems that the invention aims to solve]

[0005] In one embodiment, the problem to be solved by the present invention is to produce an artificial peptide whose three-dimensional structure is formed by folding. In another embodiment, the problem to be solved by the present invention is to provide an artificial glycopeptide in which the degradation of sugar chains by sugar hydrolases is suppressed. [Means for solving the problem]

[0006] Under these circumstances, the inventors, through diligent research, discovered that in a peptide in which two peptide chains having an α-helix structure are linked via a peptide chain having a turn structure, folding is promoted by positioning two cysteine ​​molecules near the ends of the two α-helix peptide chains and crosslinking the thiol groups derived from these cysteine ​​molecules with disulfide bonds, thereby obtaining a desired three-dimensional structure. The inventors also developed a new method for synthesizing cysteine ​​thioglycosides after considerable trial and error. Furthermore, when glycopeptides were synthesized using this thioglycosidized cysteine, it was found that the resulting glycopeptides having a glycosidic structure exhibited high resistance to degradation by glycohydrolases. The present invention is based on these new findings. Accordingly, the present invention provides the following items.

[0007] Item 1. Peptides represented by the following general formula (1)

[0008] [ka]

[0009] [In the formula, AA0, AA1, AA2, AA3, and AA4 represent amino acids. (AA0) l This shows a peptide chain with a turn structure. (AA1) m and (AA2) n (AA3) indicates peptide chains having the same or different α-helix structure. o and (AA4) p represents a peptide chain consisting of identical or different amino acids. X represents a single bond or a drug-derived group that activates macrophages. l, m, and n are identical or different and represent natural numbers. o and p are identical or different and represent integers.

[0010] Section 2. (AA0) l (AA1) m and (AA2) n The peptide described in item 1, wherein at least one of the constituent amino acids is modified with an N-substituted α-galactosamine.

[0011] Peptides described in item 1, excluding Cys, that are derived from GcMAF.

[0012] Item 4. Does it have the amino acid sequence represented by Sequence ID No. 1? Sequence number 1: Cys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Asp Ala Thr Pro Cys Glu Leu Ala Lys Leu Val Asn Lys Arg Cys or In the amino acid sequence represented by Sequence ID No. 1, one or more amino acids are deleted, substituted, or added (however, the N-terminal and C-terminal Cys in Sequence ID No. 1 are conserved), and The peptide according to claim 1, wherein the peptide contains Cys in addition to the N-terminal and C-terminal Cys in SEQ ID NO: 1, and one of the Cys in addition to the N-terminal and C-terminal Cys is modified with α-galactosamine which may have a substituent on its nitrogen atom.

[0013] Item 5. Macrophage activators containing the peptides described in Item 3.

[0014] Item 6. A pharmaceutical product containing the peptide described in Item 3.

[0015] Item 7. A method for adding a sugar molecule to cysteine, comprising carrying out the reaction shown in the following formula in the presence of a promoter molecule, wherein the promoter molecule contains a hard Lewis acid according to the HSAB rule:

[0016] [ka]

[0017] [In the formula, Y represents a protecting group for hydrogen or a thiol group. P1 represents a protecting group for an amino group. P2 represents a protecting group for a carboxylic acid.]

[0018] [ka]

[0019] This represents a monosaccharide molecule in which the anomeric position is protected by a protecting group P3 (the hydroxyl group in the monosaccharide molecule may be protected. Also, the carbon adjacent to the anomeric carbon may have an amino group or azi group substituted with a protecting group as a substituent). P3 represents a group derived from a hard Lewis base in the HSAB rule. Item 8. A method for producing a cysteine ​​compound with a sugar molecule attached, comprising carrying out a reaction shown in the following formula in the presence of a promoter molecule, wherein the promoter molecule contains a hard Lewis acid according to the HSAB rule:

[0020] [ka]

[0021] [In the formula, Y represents a protecting group for hydrogen or a thiol group. P1 represents a protecting group for an amino group. P2 represents a protecting group for a carboxylic acid.]

[0022] [ka]

[0023] This represents a monosaccharide molecule in which the anomeric position is protected by a protecting group P3 (the hydroxyl group in the monosaccharide molecule may be protected. Also, the carbon adjacent to the anomeric carbon may have an amino group or azide substituted with a protecting group as a substituent). P3 represents a group derived from a hard Lewis base in the HSAB rule.

[0024] Item 9. Cysteine ​​compounds represented by the following formulas

[0025] [ka]

[0026] [In the formula, P1 represents an amino group protecting group. P2 represents a carboxylic acid protecting group.] base

[0027] [ka]

[0028] This represents a monovalent group obtained by removing the hydroxyl group at the anomeric position from a monosaccharide molecule (the hydroxyl group in the monosaccharide molecule portion of the group may be protected. Furthermore, the carbon adjacent to the anomeric carbon may have a substituent such as a protected amino group or azide). [Effects of the Invention]

[0029] In one embodiment, the present invention makes it possible to produce artificial peptides whose three-dimensional structure is formed by folding. In another embodiment, the present invention makes it possible to provide artificial glycopeptides in which the degradation of sugar chains by sugar hydrolases is suppressed. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1. Structure and target structure of the Gc protein. a: Structure of the Gc protein (PDB: 1j78). The box shows the structure responsible for macrophage activation activity with GalNAc bound to T420 (green); b: Close-up of the helix-turn-helix structure (T397-C436). T420 and T418 are highlighted as shown in panel d (green). Differences may exist at this site due to point mutations and glycosylation of the Gc protein. K403 and S430 are highlighted in magenta, indicating that the amino acid side chains of these amino acids are in close proximity to each other; c: Wheel projection of the helix-turn-helix (K403-S430) of the Gc protein. The color coding indicates the characteristics of the amino acids. Multiple hydrophobic amino acid residues form a helix facing each other, which is similar to the leucine zipper structure; d: Sequence mutation of the Gc protein and structure of target compound 1. [Figure 2] Figure 2. Kinetic analysis of compounds 20, 21, and 22. a: Time course study of compound 21 (containing Thr) and compound 22 (containing Ser) in the reaction with chicken αGalNAc-ase. b: Lineweaver-Burk plot of the reaction with compound 21 as a substrate. Closed circles: compound 21. Open circles: compound 22. Closed squares: compound 20. [Figure 3] Figure 3. Synthesis of cyclic compound 1 and its spectral data. a: Compound 27, which has cysteine ​​at both the N-terminus and C-terminus and GalNAc via a thioglycosidic bond, was bonded to a resin and protected, synthesized according to the Fmoc procedure, and this was further converted to cyclic compound 1. b: HPLC chromatograms of released compound 28 and compound 1. c: MS spectrum of compound 1. The molecular ion was observed as a bicharged negative ion. CD spectrum of compound 1. [Figure 4] Figure 4. Production of LPS-induced IL-12 by compound 1. Each error bar represents the mean ± standard deviation of three independent experiments. Statistical significance between groups was determined by one-way ANOVA. ** P < 0.01 vs. LPS-stimulated cells. [Modes for carrying out the invention]

[0031] In this invention, "protein" and "peptide" are used to include oligopeptides and polypeptides. Furthermore, unless otherwise specified, "protein" and "peptide" encompass both modified proteins (such as those modified by sugar chains) and unmodified proteins. This also applies to proteins that are not explicitly stated to be proteins. In this specification, unless otherwise specified, the primary sequence of amino acids is defined as having the N-terminus at the left end and the C-terminus at the right end.

[0032] In the present invention, examples of the amino acid include amino acids having hydrophobic aliphatic side chains such as Ala, Leu, Ile, Met, and Val; amino acids having hydrophobic aromatic side chains such as Phe and Trp; amino acids having polar uncharged side chains such as Tyr, Asn, Cys, Gln, Ser, and Thr; acidic amino acids having charged side chains such as Asp and Glu; basic amino acids having charged side chains such as Arg, His, and Lys; and other amino acids such as Gly and Pro.

[0033] peptide In one embodiment, the present invention provides a peptide represented by the following general formula (1): [Chemical formula] [In the formula, AA0, AA1, AA2, AA3, and AA4 represent amino acids. (AA0) l represents a peptide chain having a turn structure. (AA1) m and (AA2) n represent peptide chains having the same or different α-helix structures. (AA3) o and (AA4) p represent peptide chains composed of the same or different amino acids. X represents a single bond or a group derived from an agent that activates macrophages. l, m, and n are the same or different and represent natural numbers. o and p are the same or different and represent integers.].

[0034] (AA0) l represents a peptide chain having a turn structure. AA0 represents an amino acid, and when l is 2 or more, a plurality of AA0s may be the same or different. l represents a natural number of 1 or 2 or more. l is preferably 2 or more, more preferably 3 or more, more preferably 4 or more, more preferably 5 or more, and more preferably 6 or more. l is preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, and more preferably 6 or less.

[0035] (AA0) lFor example, those having amino acid sequences represented by Pro Asp Ala Thr Pro Cys (SEQ ID NO: 2) and Pro Asp Ala Cys Pro Thr (SEQ ID NO: 3) are preferred. Also, within the range in which a turn structure can be formed, (AA0) l As an example, the amino acid sequence represented by Sequence ID No. 2 may have an amino acid sequence in which one or several (e.g., two) amino acids are substituted, added, or deleted. Also, within the range in which a turn structure can be formed, (AA0) l In these embodiments, the amino acid sequence may have one or more amino acids (for example, two) substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 3. In these embodiments, it is preferable that cysteine ​​in the amino acid sequence represented by SEQ ID NO: 2 or 3 is preserved.

[0036] (AA1) m represents a peptide chain having an α-helix structure. AA1 represents an amino acid, and if m is 2 or more, multiple AA1s may be the same or different. m represents a natural number of 1 or 2 or more. m is preferably 5 or more, more preferably 6 or more, more preferably 7 or more, more preferably 8 or more, more preferably 9 or more, more preferably 10 or more, and more preferably 11 or more. m is preferably 20 or less, more preferably 19 or less, more preferably 18 or less, more preferably 17 or less, more preferably 16 or less, more preferably 15 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, and more preferably 11 or less.

[0037] (AA1) m For example, those having the amino acid sequence represented by Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu (SEQ ID NO: 4) are preferred. Also, within the range in which an α-helix structure can be formed, (AA1) m This could include an amino acid sequence in which one or several (e.g., two) amino acids are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No. 4.

[0038] (AA2) n represents a peptide chain having an α-helix structure. AA2 represents an amino acid, and if n is 2 or more, multiple AA2s may be the same or different. n represents 1 or a natural number of 2 or more. n is preferably 5 or more, more preferably 6 or more, more preferably 7 or more, more preferably 8 or more, and more preferably 9 or more. n is preferably 20 or less, more preferably 19 or less, more preferably 18 or less, more preferably 17 or less, more preferably 16 or less, more preferably 15 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, and more preferably 9 or less.

[0039] (AA2) n For example, those having the amino acid sequence represented by Glu Leu Ala Lys Leu Val Asn Lys Arg (SEQ ID NO: 5) are preferred. Also, within the range in which an α-helix structure can be formed, (AA2) n This could include an amino acid sequence in which one or several (e.g., two) amino acids are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No. 5.

[0040] (AA3) o represents a peptide chain. AA3 represents an amino acid, and if o is 2 or more, multiple AA3s may be the same or different. o represents an integer (0, 1, or 2 or more). Examples of o values ​​include 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, etc. o is preferably 20 or less, preferably 15 or less, preferably 10 or less, preferably 9 or less, and more preferably 7 or less.

[0041] (AA3) o For example, those having the amino acid sequence represented by Glu Asn Thr Phe Thr Glu Tyr (SEQ ID NO: 6) are preferred. Also, within the range in which the effects of the present invention can be obtained, (AA3) oThis could include an amino acid sequence in which one or several (e.g., two) amino acids are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No. 6.

[0042] (AA4) p represents a peptide chain. AA4 represents an amino acid, and if p is 2 or more, multiple AA4s may be the same or different. p represents an integer (0, 1, or 2 or more). Examples of p values ​​include 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, etc. P is preferably 25 or less, preferably 20 or less, preferably 15 or less, preferably 10 or less, and more preferably 9 or less.

[0043] (AA4) p For example, those having the amino acid sequence represented by Asp Phe Ala Ser Asn Cys Cys Ser Ile (SEQ ID NO: 7) are preferred. Also, within the range in which the effects of the present invention can be obtained (AA4) p This could include an amino acid sequence in which one or several (e.g., two) amino acids are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No. 7.

[0044] Furthermore, in one embodiment, the peptide of the present invention is preferably a peptide derived from GcMAF. More specifically, in a preferred embodiment, the peptide of the present invention has the amino acid sequence represented by SEQ ID NO: 1. SEQ ID NO: 1: Cys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Asp Ala Thr Pro Cys Glu Leu Ala Lys Leu Val Asn Lys Arg Cys.

[0045] Furthermore, in a modified embodiment of the GcMAF-derived peptide, the peptide of the present invention may have an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1 (however, the N-terminal and C-terminal Cys in SEQ ID NO: 1 are conserved). There have been no previous reports of an artificial peptide having two GcMAF-derived α-helix structures, in which these GcMAF-derived α-helix structures are linked via a turn structure, and folding such a peptide has been difficult; therefore, this embodiment is also useful from this viewpoint.

[0046] In these embodiments, the peptide of the present invention contains Cys in addition to the N-terminal and C-terminal Cys in SEQ ID NO: 1, and it is preferable that at least one (preferably 1 to 3, more preferably 1 to 2, more preferably 1) of the Cys other than the N-terminal and C-terminal Cys is modified with an N-substituted α-galactosamine. Examples of N-substituted α-galactosamines include N-acetyl-α-galactosamine (αGalNAc), N-(2-azidoacetyl)-α-galactosamine, N-(propioloyl)-α-galactosamine, etc., with N-acetyl-α-galactosamine (αGalNAc) being preferred. When Cys is modified with two or more of these compounds, the multiple N-substituted α-galactosamines may be the same or different. In such embodiments, (AA0) l (AA1) m and (AA2) n Preferably, at least one (preferably 1 to 3, more preferably 1 to 2, more preferably 1) of the amino acids constituting the compound is modified with αGalNAcN-substituted α-galactosamine, (AA0) l It is preferable that at least one (preferably 1 to 3, more preferably 1 to 2, and more preferably 1) of the constituent amino acids is modified with αGalNAcN-substituted α-galactosamine.

[0047] The peptide of the present invention preferably has 50 or fewer constituent amino acid residues, more preferably 40 or fewer, more preferably 35 or fewer, more preferably 30 or fewer, and more preferably 28 or fewer. These embodiments are particularly useful because folding relatively small polypeptides is particularly difficult. The lower limit of the number of constituent amino acids in the peptide of the present invention is not limited, but preferably 20 or more, preferably 21 or more, preferably 22 or more, preferably 23 or more, preferably 24 or more, more preferably 25 or more, preferably 26 or more, preferably 27 or more, and more preferably 28 or more.

[0048] The peptide of the present invention preferably has a molecular weight of 5000 or less, more preferably 4500 or less, and even more preferably 4000 or less. The lower limit of the molecular weight of the peptide of the present invention is not limited, but it is preferably 3000 or more, more preferably 2750 or more, and even more preferably 2500 or more.

[0049] Macrophage activators In one embodiment, the present invention provides a macrophage activator comprising a peptide represented by the general formula (1), which is derived from GcMAF. The structures of the peptides AA0, AA1, AA2, AA3, AA4, l, m, n, o, p, x, etc. in this embodiment are as described above. In the present invention, the macrophage activating ability can be evaluated by the method described in the examples below. In this specification, "the peptide represented by the general formula (1), which is derived from GcMAF" may also be simply referred to as "the GcMAF-derived peptide of the present invention."

[0050] In this embodiment, the GcMAF-derived peptide of the present invention may be used as a macrophage activator itself, or it may be used as a composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.).

[0051] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0052] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, as well as ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid.

[0053] Examples of stabilizers include sodium bisulfite.

[0054] Examples of pH adjusting agents include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.

[0055] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and other quaternary ammonium salts, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0056] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

[0057] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol. Examples of viscosity-concentrating agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0058] Furthermore, the above composition may further contain, in addition to the GcMAF-derived peptide of the present invention, agents known to have preventive and therapeutic effects on diseases that can be treated by macrophage activation. Examples of diseases that can be treated by macrophage activation include cancer, hepatitis (hepatitis B, hepatitis C, etc.), herpes, influenza, pneumonia, tuberculosis, and EB virus infection. Examples of agents known to have preventive and therapeutic effects on cancer include chemotherapeutic agents such as cisplatin, carboplatin, nedaplatin, docetaxel, paclitaxel, fluorouracil, and tegafur; endocrine therapy agents such as tamoxifen, toremifene, and fulvestrant; and molecular targeted drugs such as nivolumab, cetuximab, and isatuximab. Examples of agents known to have preventive and therapeutic effects on hepatitis include interferon, lamivudine, adefovir, vemlidy, entecavir, and tenofovir. Drugs known to have preventive and therapeutic effects on herpes include, for example, acyclovir, vidarabine, and valacyclovir. Drugs known to have preventive and therapeutic effects on influenza include, for example, Tamiflu, Relenza, Rapiacta, and Inavir. Drugs known to have preventive and therapeutic effects on pneumonia include, for example, potassium clavulanate, amoxicillin hydrate, ceftriaxone sodium hydrate, and levofloxacin hydrate. Drugs known to have preventive and therapeutic effects on tuberculosis include, for example, levofloxacin, isoniazid, rifampicin, pyrazinamide, and ethambutol.

[0059] In embodiments of the composition, the content of the GcMAF-derived peptide of the present invention in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc.

[0060] The formulation form is not particularly limited and can include various forms such as oral preparations like tablets, pills, capsules, powders, granules, syrups, and sublingual preparations; and parenteral preparations such as injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, drips, topical preparations (ointments, creams, patches, inhalants), and suppositories. Among the above formulation forms, injections are preferred, for example.

[0061] The amount of the GcMAF-derived peptide of the present invention contained in the formulation cannot be specified in general terms as it varies depending on the route of administration, the patient's age, weight, symptoms, etc. However, it should be an amount that results in a daily dose of approximately 10 to 5000 mg of the GcMAF-derived peptide of the present invention, more preferably approximately 100 to 1000 mg. If administered once a day, this amount should be contained in one formulation; if administered three times a day, one-third of this amount should be contained in one formulation.

[0062] As described above, according to the present invention, the peptide represented by general formula (1), which is derived from GcMAF, has macrophage-activating activity. Therefore, according to the present invention, the peptide represented by general formula (1), which is derived from GcMAF, can be used for the prevention and treatment of diseases that can be treated by macrophage activation. Accordingly, in one embodiment, the present invention provides a pharmaceutical product comprising the peptide represented by general formula (1), which is derived from GcMAF, preferably the peptide represented by general formula (1), for the prevention and treatment of diseases that can be treated by macrophage activation. The pharmaceutical product of the present invention is expected to attack cancer cells by macrophage activation. Furthermore, the pharmaceutical product of the present invention is expected to enhance resistance to infectious diseases in the target population (especially elderly people with noticeable weakened immunity). Accordingly, diseases that can be prevented and / or treated by the pharmaceutical product of the present invention include, for example, cancer, hepatitis (hepatitis B, hepatitis C, etc.), herpes, influenza, pneumonia, tuberculosis, EB virus infection, etc. Furthermore, details regarding the structure and method of use of the peptide represented by general formula (1) in the embodiments of the pharmaceutical product of the present invention are the same as those described for macrophage activators. The pharmaceutical product of the present invention is administered to mammals and the like. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cattle, horses, and sheep.

[0063] The macrophage activator of the present invention may be applied to living organisms or used in vitro. In such embodiments, for example, macrophages can be activated by acting on them in vitro with the GcMAF-derived peptide of the present invention. The macrophage activation effect can be measured and evaluated, for example, by the method described in the examples of this application.

[0064] Method for preparing cysteine ​​compounds In one embodiment, the present invention provides a method for adding a sugar molecule to cysteine, comprising carrying out a reaction represented by the following formula in the presence of a promoter molecule, wherein the promoter molecule comprises a hard Lewis acid according to the HSAB rule: [ka] [In the formula, Y represents a protecting group for hydrogen or a thiol group. P1 represents a protecting group for an amino group. P2 represents a protecting group for a carboxylic acid. Compound 3 represents a monosaccharide molecule in which the anomeric position is protected by the protecting group P3 (the hydroxyl group in the monosaccharide molecule may also be protected. Furthermore, the carbon adjacent to the carbon at the anomeric position may have an amino group or azide substituted with a protecting group as a substituent). P3 represents a group derived from a hard Lewis base in the HSAB rule.]

[0065] In the formula, Y represents a protecting group for hydrogen or a thiol group. Examples of thiol protecting groups include triphenylmethyl group (Trt), 4-methylbenzyl group (Bzl(4Me)), tert-butyl group, and N-(acetyl)aminomethyl group (Acm). P1 represents a protecting group for an amino group. Examples of amino protecting groups include 9-fluorenylmethyloxycarbonyl group (Fmoc) and tert-butoxycarbonyl group (Boc). P2 represents a protecting group for a carboxylic acid. Examples of carboxylic acid protecting groups include allyl group (-CH2-CH=CH2), benzyl group, trichloroethyl group, methoxymethyl group, pentenyl group, and trimethylsilylethyl group. Compound 3 represents a monosaccharide molecule in which the anomeric position is protected by the protecting group P3 (in this specification, compound 3 may be referred to as a monosaccharide molecule-derived compound). Examples of monosaccharides include glucose, fructose, xylose, ribose, mannose, fucose, and sialic acid. Examples of monosaccharide skeletons include pyranose and furanose, with pyranose being preferred.

[0066] In the present invention, the carbon atom at the anomeric position (anomeric position) of the monosaccharide molecule-derived compound is protected. Examples of protecting group P3 include a fluorine atom, an imidate group such as -OC(=NH)-CCl3, a phosphate ester leaving group, a phosphite ester leaving group, and a thioimidate leaving group. In the present invention, it is preferable to use a hard base as the protecting group P3. Using a hard base as the protecting group P3 is preferable because it facilitates the elimination reaction of the protecting group P3 from the sugar molecule in the presence of a promoter, which is a molecule containing a hard Lewis acid. In the present invention, it is preferable that the protecting group P3 is bonded to the carbon atom at the anomeric position of the monosaccharide molecule in the monosaccharide molecule-derived compound 3. Furthermore, in the monosaccharide molecule-derived compound, the hydroxyl group in the monosaccharide molecule portion may also be protected. Examples of protecting groups include acyl groups, alkyloxybenzyl groups such as methoxybenzyl groups, carbonate protecting groups such as alloc and troc groups, and silyl protecting groups such as tert-butyldimethylsilyl groups. Examples of acyl groups include acetyl groups, propionyl groups, butyryl groups, 4-methylpentanoyl groups, and hexanoyl groups. In addition, the carbon adjacent to the anomeric carbon may have an amino group substituted with a protecting group as a substituent. As a protecting group for amino groups, a protecting group that provides α-selectivity in the glycosylation reaction is preferred. Examples of protecting groups for amino groups include acyl groups, allyloxycarbonyl groups, and halogenoalkoxycarbonyl groups. Examples of alkenyloxycarbonyl groups include alkenyloxycarbonyl groups in which the alkenyl portion is a linear or branched hydrocarbon group having a carbon-carbon double bond. Examples of the number of carbon atoms in the alkenyl portion of an alkenyloxycarbonyl group include 2-6 carbon atoms, 2-4 carbon atoms, and 2-3 carbon atoms. Examples of the number of carbon atoms in an alkenyloxycarbonyl group include 3-7 carbon atoms, 3-5 carbon atoms, and 3-4 carbon atoms. Non-limiting examples of alkenyloxycarbonyl groups include allyloxycarbonyl, (buto-2-en-1-yloxy)carbonyl, (pento-2-en-1-yloxy)carbonyl, (hexo-2-en-1-yloxy)carbonyl, etc.Examples of halogenoalkoxycarbonyl groups include halogenoalkoxycarbonyl groups in which the halogenoalkoxy portion is a linear or branched saturated hydrocarbon group substituted with a halogen. Examples of halogens include fluorine, chlorine, bromine, and iodine, with chlorine being preferred. Examples of the number of halogens include 1 or more, 1 to 7, 1 to 5, and 1 to 3. The alkoxy portion may also be a perhalogenoalkoxy group in which all hydrogens are substituted with halogens. Examples of the number of carbon atoms in the halogenoalkoxy portion include 1 to 6, 1 to 4, and 1 to 3. Examples of the number of carbon atoms in the halogenoalkoxycarbonyl group include 2 to 7, 2 to 5, and 2 to 4. Examples of halogenoalkoxycarbonyl groups include (2,2,2-trichloroethoxy)carbonyl group, perfluoroethoxycarbonyl group, (3,3,3-trichloropropoxy)carbonyl group, (4,4,4-tribromobutoxy)carbonyl group, (5,5,5-trichloropentoxy)carbonyl group, and (6,6,6-trichlorohexyloxy)carbonyl group. In addition, the carbon adjacent to the anomeric carbon may have an azi group (-N3) as a substituent.

[0067] The method of the present invention is characterized in that the promoter molecule contains a hard Lewis acid according to the HSAB rule (hard acid, hard base, soft acid, soft base rule). In the present invention, hard acids and bases are poorly polarized, while soft acids and bases are easily polarized. As for hard Lewis acids, H + Examples of hard bases include TiCl4, ZnCl2, AlCl3, FeCl3, SnCl4, SnCl2, Cp2ZrCl2, Cp2Zr(OTf)2, Cp2HfCl2, Cp2Hf(OTf)2, BF3, etc. Examples of soft Lewis acids include CuBr2, AuCl, Ag2CO3, AgOTf, HgCl2, etc. Examples of hard bases include F - Cl - , OH - SO4 2- NH3, NO 3- Examples include -C(=NH)-. Examples of soft bases include CN. - , I- S2 - 5CN - CO is one example.

[0068] Examples of promoter molecules containing hard Lewis acids include Cp2Hf(OTf)2, TMSOTf(Trimethylsilyl Trifluoromethanesulfonate), Cp2Zr(OTf)2, and Cp2Hf(OClO-4). 2、 Examples include Cp2Zr(OClO-4)2. In the present invention, the concentration of the promoter molecule is not particularly limited, but can be set in the range of 0.01 to 5 M, preferably 0.05 to 0.5 M, and more preferably 0.1 to 0.3 M. The ratio of compound 2 to compound 3 used is also not particularly limited, but can be set in the range of 1 to 5 mol, preferably 1 to 2 mol, and preferably 1 mol, of compound 3 to 1 mol of compound 2. The reaction temperature is also not particularly limited, but can be set in the range of -100 to 0°C, preferably -50 to -10°C, and preferably -20°C. The reaction time is also not particularly limited, but can be set in the range of 2 to 48 hours, preferably 5 to 36 hours, and preferably 8 to 24 hours. The cysteine ​​compound 8 obtained by the above reaction may be isolated and purified as appropriate based on known methods. Furthermore, the cysteine ​​compound 8 is useful as a raw material for the synthesis of glycated peptides, etc.

[0069] Specific embodiments of the present invention will be described in more detail below using examples, but the present invention is not limited to these examples. [Examples]

[0070] Introduction Many secreted proteins are modified post-translation, but glycosylation is the most complex. Protein function may be regulated by attached glycans, and it is known that parts of the glycan structure play a role in delivering proteins to specific sites through specific interactions. Among the various glycoproteins, some are used as pharmaceuticals, and others are currently undergoing clinical trials. However, current knowledge of glycan synthesis in cells is limited, so there are problems with quality control of the glycan portion, and the direction of reconstructing diverse glycans into homogeneous ones is being considered. On the other hand, there is also the direction of introducing polyethylene glycol (PEG) into protein drugs. Since polyethylene glycol is stable in the bloodstream, it can improve drug efficacy. The future direction will likely be to create homogeneous and stable glycan-binding proteins. Glycans are generally cleaved from the non-reducing end by exo-glycosylhydrase. This suggests that simple modification of terminal glycans improves the stability of the glycan portion, and consequently the entire drug molecule. The idea of ​​stabilizing interglycosidic bonds can be achieved by replacing the exoanomeric oxygen with an electron-withdrawing substituent such as 1) its conjugated sulfur atom, 2) a carbon atom, or 3) a fluorine atom. To illustrate the concept of glycosidase-resistant glycopeptides and proteins as future pharmaceuticals, we focused on Gc proteins that are O-glycosylated at only one site. Gc proteins, consisting of 458 amino acids and 51.2 kDa, are albumin relatives and are known to have multiple functions, including the transport of vitamin D and its metabolites, actin scavenging, macrophage activation, and antitumor activity through angiogenesis inhibition. Interestingly, it has been reported that only GalNAc-modified Gc proteins are involved in macrophage activation. Therefore, this particular molecule is called GcMAF. Based on our concept described above and the structure of GcMAF, we decided to synthesize a substructure of a Gc protein containing thioglycosidally linked GalNAc. Specifically, we considered replacing the glycosidase-resistant glycoside from Thr to Cys. Because GcMAF has a large molecular weight and is difficult to synthesize, we investigated the molecular structure of the protein based on reported crystallographic data and a reported functional knowledge base. After extensive investigation, we concluded that one of the helix-turn-helix (HTH) structures that presents GalNAc is important for macrophage activation (Figure 1a-c). The target glycopeptide 1 is a relatively short peptide consisting of 28 amino acids corresponding to aa403 to aa430 of the Gc protein, and its structural motif is helix-turn-helix (HTH). The target molecule is chemically mutated as Lys403Cys, Thr420Cys, and Ser430Cys (Figure 1d). (Figure 1d) The substitution of Thr420 with Cys confers glycosidase resistance to the molecule, and a series of GalNAc-amino acids were evaluated as substrates for GalNAc-ase. Important findings regarding the glycosylation reaction for the synthesis of GalNAc-Cys precursors are also described below.

[0071] Results and Discussion Synthesis of the synthetic blocks Fmoc-Thr(α-GalNAc4)-OH (17), Fmoc-Ser(α-GalNAc4)-OH (18), and Fmoc-Cys(α-GalNAc4)-OH (19) First, we decided to synthesize glycopeptides using the Fmoc solid-phase synthesis method. To facilitate the synthesis, it was necessary to synthesize glycosylated Cys, but the synthesis of GalNAc-Cys has not been reported. There are various methods for synthesizing thioglycosides, such as nucleophilic attack of thiols on the oxocarbenium ion or nucleophilic attack of mixed thioacetals (anomeric thiols) on the leaving group position. The former is considered most suitable for our synthetic strategy because it allows the use of appropriately protected cysteine, and is also advantageous for subsequent peptide synthesis. Before synthesizing the series of GalNAc-amino acids, a series of glycosyl donors were synthesized. Since α-bonding is required for GalNAcization, 2-azide derivatives were used. Compound 2 was prepared according to the azid chromatography method and used as a starting material for the synthesis of glycosyl bromide 3, fluoride 4, and imidate 6.

[0072] [ka]

[0073] Referring to Non-Patent Document 3, compound 2 was treated with LiBr in acetonitrile at room temperature to produce compound 3. When bromide 3 was reacted with silver fluoride in acetonitrile at room temperature, compound 4 was obtained in a 72% concentration. 1 From the coupling constants of 1H-NMR, it was found that bromide 3 underwent α-configuration exclusively, and fluoride 4 underwent β-formation, indicating that the fluorination reaction proceeded via substitution. Meanwhile, the nitroester of compound 2 was removed, and compound 5 was obtained in NaNO2 and dioxane at 70°C for 5 hours in 56% yield. In the presence of trichloroacetonitrile and potassium carbonate, the conversion of compound 5 to imidate 6 proceeded almost quantitatively. Since a series of glycosyl donors were obtained, we attempted to glycosylate the protected cysteine ​​compound 7.

[0074] [ka]

[0075] Concerns arose that glycosyl bromide 3 might not be successfully converted under standard glycosylation conditions. Therefore, the coupling of compound 3 with Fmoc-Cys-OAll (7) in the presence of a silver salt was first investigated. Repeated chromatography yielded thioglycoside 9, but the yield did not exceed 30%. This low yield can be explained by the hard-and-soft acid-base (HSAB) concept, where silver, acting as a soft Lewis acid, coordinates to the bromine atom to form an oxocarbenium ion, while simultaneously coordinating to the nucleophilic sulfur atom, inhibiting the glycosylation reaction. Therefore, glycosyl fluoride 4 was selected, and hafnosene dichloride-AgOTf was chosen for its activation. The hard acid-and-hard base combination should selectively proceed in the presence of the thiol group of compound 7, a soft base, and the resulting glycosyl cation and soft acid could be a good combination for glycosylation. The reaction proceeded at -20°C, yielding compound 9 in 64% yield. Glycosylation with Imidate 6 using TMSOTf yielded better results, with a yield of 78%. Since TMSOTf used in this glycosylation reaction can be used to remove the trityl group of compound 8, a precursor of compound 7, we attempted to directly glycosylate compound 8 with Imidate 6, referring to Patent Document 4, and obtained compound 8 in a sufficient yield of 52%. The protected form is more stable than the thiol form, and the shorter route is useful. Next, other GalNAcylated protected amino acids such as compounds 12 and 13 were synthesized. In the standard glycosylation reaction using the OH group as a nucleophile, the silver salt worked without problems. Therefore, Fmoc-Thr-OAll (compound 10) and Fmoc-Ser-OAll (compound 11) were coupled with bromide 3 in the presence of silver perchlorate to obtain glycosylated compounds 12 (86%) and 13 (87%), respectively. The protecting groups of the resulting series of protected GalN3 amino acids (compounds 8, 12, and 13) were sequentially removed as follows. First, the 2-azide group was reduced and simultaneously acetylated using thioacetic acid in pyridine. Next, the allyl group was deprotected by the action of a homogeneous Pd0 catalyst, referring to Non-Patent Literature 5. After removing the aryl ester, referring to Non-Patent Literature 6 and 7, the reaction mixture was treated with QuadraPure, which has thiourea function, and further purified by column chromatography to obtain compounds 17-19 useful for peptide synthesis. A portion of the sample (compounds 17-19) was treated with hydrazine to remove the acetyl group and obtain compounds 20-22 for enzyme assays. 1 According to 1H-NMR analysis, racemization did not occur.

[0076] [ka]

[0077] Hydrolytic enzyme assays of Fmoc-Thr(α-GalNAc)-OH (compound 21), Fmoc-Ser(α-GalNAc)-OH (compound 22), and Fmoc-Cys(α-GalNAc)-OH (compound 20) (Figure 2) Thioglycosides are known to have weak inhibitory activity against glycosidases, but the important thing is whether the compound has the potential to enhance the effect of a drug when used as such, regardless of whether it has inhibitory activity or not. Hydrolytic enzymesTo confirm stability against α-GalNAc-ase, we investigated a series of compounds 20-22 as substrates for chicken α-GalNAc-ase. The crystal structure of α-GalNAc-ase (1KTB) (Non-Patent Literature 9) suggested the presence of canyon-like channels connected to the catalytic site that could accommodate bulky groups; therefore, we investigated the enzymatic reaction without removing the Fmoc group. Furthermore, the sides of the canyon are composed of acidic amino acids, which may prevent released Fmoc-amino acids from remaining there and exhibiting product inhibition. First, compounds 21 and 22 were accepted as substrates as expected, but compound 20 was not (Figure 2a). Furthermore, time-course studies revealed that the inhibitory effect of compounds 21 and 22 on the hydrolysis products was negligibly small. Regarding the substrate activity of compounds 21 and 22, compound 21 was twice as good a substrate as 22 (Figure 2a). Next, we investigated the inhibitory activity of compound 20 against α-GalNAc-ase and found that compound 20 was a weak inhibitor. Regardless of its inhibitory activity, the important fact here is that compound 20 was resistant to α-GalNAc-ase.

[0078] Design and Synthesis of GalNAc Peptides Before beginning glycopeptide synthesis, preliminary calculations were performed to design miniature MAFs. Since GalNAc residues are necessary for macrophage activation, the HTH motif of the Gc protein was considered preferable for the GcMAF substructure, as shown in Figure 1d (sequences 1-4). While molecules corresponding to those with GalNAc bound to Thr420 have been reported to have potent macrophage-activating effects, Gc proteins have minor variants resulting from point mutations in the gene, and the nature of post-translational modifications such as glycosylation sites is also ambiguous. To roughly evaluate the stability and structure of the glycopeptide corresponding to the GcMAF substructure (Lys403-Ser430) (see Figure 1b), we began investigating the conformation of the peptide corresponding to the Lys403-Ser430 sequence using a combination of force field, single amino acid potential (SAAP), and Monte Carlo simulation. The calculations started with the expanded conformation of a 28-amino acid peptide in an aqueous environment with a dielectric constant of 0.6 and a temperature of 300-650 K. In the initial stages of the investigation, the peptide was synthesized, but although the CD spectrum adopted an α-helix in trifluoroethanol, it did not show the presence of α-helices or β-sheets. The folding process of chemically synthesized peptide molecules is quite different from that of natural phenomena. Folding of chemically synthesized peptides begins when the full-length peptide is exposed to an aqueous medium after deprotection and purification, whereas natural folding begins with peptide synthesis in the endoplasmic reticulum in the presence of various chaperones. Therefore, we were concerned that the folding state of the synthesized compound might differ from that of the crystalline structure, as suggested by SAAP calculations. To restrict the folding of a 28-amino acid GalNAcylated peptide, we considered introducing disulfide bonds at the N-terminus and C-terminus. The distance between the γ-carbon of Lys403 and the alcoholic oxygen of Ser430 is approximately 3.3 Å in the crystalline structure (Figure 1b), and based on the shape of the interatomic space, we considered chemically mutating both Lys403 and Ser430 to create disulfide bonds such that the bond length of the two cysteine ​​disulfide bonds (approximately 2.1 Å) is compromised throughout the molecule, thereby restricting the folding as observed in the crystalline structure of Gc proteins. Based on the preliminary calculation results and considerations above, we decided to synthesize GalNAc-modified cyclic peptide 1, which corresponds to Lys403-Ser430 and includes GalNAc-bound Thr420Cys, Lys403Cys, and Ser430Cys. As shown in Scheme 4, the Fmoc protocol was applied to the synthesis of compound 1. A C-terminal trityl-protected Cys molecule bonded to a chlorotrityl polystyrene resin was used for the synthesis. The Fmoc-protected amino acids (4 eq.) were coupled using DCC in NMP as the solvent in the presence of HOBt, except for the coupling of the GalNAc-Cys moieties (17, 2 eq.). Piperidine was used to deprotect the Fmoc group in all cycles. After 27 reaction cycles, the protected resin conjugate 23 was obtained.

[0079] [ka]

[0080] After peptide synthesis was complete, all amino acid protecting groups were removed under reducing conditions using TFA. During this process, glycopeptide 24 was also released from the support resin. The remaining acetyl groups on GalNAc were removed (hydrazine treatment), and simultaneously, disulfide bonds were formed in the presence of DMSO. Compound 1 was obtained in 8.4% yield by HPLC purification using an acetonitrile gradient. (Figure 3b) Compound 1 is [M+2H] 2+ The divalent ion was observed at m / z 1671.07 (Figure 3c). The CD spectrum of compound 1 suggested the presence of a partial α-helix structure (Figure 3d). Next, referring to Non-Patent Literature 10, the structure of compound 1 synthesized based on the CHARMM36 force field was calculated using the atomic coordinates of the crystalline state as the initial structure. The N-terminal and C-terminal amino acid side chains at positions 403 and 430 were replaced with the Cys side chain linked via a disulfide bond in the presence of GalNAc at position 420. After optimizing the geometry, water molecules were added to minimize the energy, and finally, the molecular structure was searched for by CHARMM at 300 K under periodic boundary conditions. The suggested structure of compound 1 was shown to employ an HTH motif and was consistent with the CD spectrum. Having successfully synthesized cyclic glycopeptide 1, we further investigated its macrophage-activating effect. Specifically, we performed the following method. J774.1 cells were stimulated for 24 hours with compound 1 (10 or 20 μg / mL), lipopolysaccharide (LPS) (500 ng / mL), or compound 1 + LPS, and interleukin-12p40 in the supernatant was quantified. More specifically, the mouse macrophage-like cell line J774.1 was first obtained from the Japan Research Bioresource Collection. The cells were cultured in RPMI1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Compound 1 was dissolved in PBS at a concentration of 1 mg / mL. LPS derived from E. coli 0111:B4 was purchased from Sigma-Aldrich and dissolved in the medium at a concentration of 100 μg / mL. J774.1 cells (5 × 10⁶) 5 Compound 1 was added to a 1.5 mL tube (600 μL) at a final concentration of 0, 10, or 20 μg / mL. The cells were incubated at 37°C for 30 minutes with gentle rotation, after which LPS was added to the tube at a final concentration of 500 ng / mL. The cells were then seeded into the wells of a 48-well culture plate and cultured at 37°C for 24 hours. After 24 hours, the culture supernatant was collected and IL-12p40 in the supernatant was quantified using a commercially available ELISA kit (BD). The results are shown in Figure 4. As shown in Figure 4, IL-12 secretion was not observed when cells were treated with compound 1 alone, but LPS treatment induced IL-12 secretion. When cells were co-stimulated with compound 1, LPS-induced IL-12 secretion increased significantly in a peptide concentration-dependent manner. These results indicate that compound 1 alone does not have the ability to induce IL-12 production, but enhances LPS-induced IL-12 production. IL-12 is involved in the cytotoxic activity of innate killer cells, and furthermore, IL-12 is required for angiotensin's anti-angiogenic activity. Since GcMAF inhibits angiogenesis, compound 1 may be useful as an antitumor agent.

[0081] conclusion GcMAF is a trace component transiently produced from Gc proteins and has been shown to be effective in various cancer treatments. Despite playing a crucial role in macrophage activation, its activity is lost by hydrolysis of GalNAc. Overcoming this problem by introducing glycosides that are stable against enzymatic hydrolysis could lead to a common approach in drug discovery. Anticipating future general drug development, we focused on the design and chemical synthesis of a cyclic glycopeptide, which is considered to be the minimal structure of the active domain of GcMAF. The glycan was thioglycosidically linked to the backbone peptide in the form of GalNAc-Cys. To stabilize the conformation of the relatively small protein, the N-terminal and C-terminal amino acids were chemically mutated to Cys to form a CC bond. During the synthesis process, thioglycosylation of the GalNAc precursor was achieved based on the concepts of hard and soft acids and bases. More precisely, glycosyl fluoride and imidate were used as donors and activated by corresponding hard acidic conditions for coupling with the protected Cys thiol group (soft base). The synthesized glycopeptide was shown to possess a partial α-helix structure. Furthermore, when GalNAc-Cys was investigated as a substrate for GalNAc-ase, it was shown to be a slow substrate. We successfully demonstrated an overall framework for the development of novel glycoprotein drugs. The synthesized glycopeptide is the smallest of the active glycoproteins.

[0082] Materials and methods Compounds 2 and 3 were prepared according to the procedure described in Non-Patent Document 16. The ys(Trt)-Trt(2-Cl)-resin was purchased from Watanabe Chemical Industry Co., Ltd. (Hiroshima, Japan). Suitablely protected amino acid synthons for peptide synthesis are commercially available from various companies and were used as is. CH2Cl2 was distilled over CaH2 before use. Thin-layer chromatography (TLC) was performed using Merck Millipore 105715, Kieselgel 60 F. 254Visualization was performed using a 0.25mm thick plate (Merck Ltd., Tokyo, Japan). Visualization was performed using ultraviolet light and 1% Ce(SO4)2-1.5%(NH4)6MoO 24 A 10% H2SO4 solution or a 10% H2SO4 solution was used, followed by heating. Silica gel column chromatography was performed using a Wakogel C-300 (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). 1 The 1H NMR (500 MHz) spectrum was recorded in a deuterated solvent using an AVANCE 500 spectrometer (Bruker Biospin GmbH, Bremen, Germany) with Me4Si (0.00 ppm) as the internal standard. 1313C NMR (125 MHz) spectra were recorded on the same spectrometer using Me4Si (0.00 ppm) or a solvent peak (CDCl3; 77.0 ppm or CD3OD; 49.0 ppm) as an internal standard. Amino acid composition was determined using a LaChrom amino acid analyzer (Hitachi, Tokyo, Japan) after hydrolysis with 6M HCl aqueous solution at 150°C for 2 hours in a vacuum-sealed tube. High-resolution mass spectra (HRMS) were recorded using an AccuTOF LC-plus 4G (JEOL, Tokyo, Japan). Ional mass measurements of glycopeptides were performed using a MALDI-TOF-MS S3000 (JEOL, Tokyo, Japan). Analytical HPLC was performed using a Separr C18G (4.6 × 150 mm, RIKEN, Tokyo, Japan) under linear gradient conditions (from 10% CH3CN containing 0.1% TFA to 40% CH3CN containing 0.1% TFA) at room temperature for 25 minutes. The flow rate was 1 mL / min. Detection was performed at 220 nm. Glycopeptide isolation was performed using an Inertsil® ODS-SP (10 × 250 mm, GL Science, Tokyo, Japan) HPLC under linear gradient conditions (from 20% CH3CN containing 0.1% TFA to 40% CH3CN containing 0.1% TFA) over 20 minutes at room temperature. The flow rate was 2.5 mL / min. Detection was performed at 235 nm. Far-ultraviolet CD spectra were recorded using a Jasco J-710 spectrophotometer (Jasco, Tokyo, Japan) with 0.15 mg / mL of protein in 50 mM phosphate buffer (pH 7.0) at 20°C in a 1 mm cuvette. Chicken N-acetylgalactosaminidase (α-GalNAc-ase, EC 3.2.1.49) and p-nitrophenyl α-DN-acetylgalactosaminide (pNP-α-GalNAc) were obtained from Merck KgaA, Darmstadt. A Prominence HPLC system equipped with CBA-20A, DGU-20A3, LC-20AD, TOSO UV-8020, and SSC-2120 was used for the enzymatic reaction analysis. A reversed-phase column (Inertsil C8, particle size 5 μm, inner diameter 2.1 nm × length 150 mm, GL Sciences Inc., Japan) was placed in an oven at 44.5 °C. A 50% acetonitrile aqueous solution containing 0.1% formic acid and 0.02% trifluoroacetic acid was used under isocratic conditions. The pump was operated at a flow rate of 0.2 mL / min, and the injection volume was 10 μL. The Fmoc-supported substrate and product were detected at 262 nm.

[0083] 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosylfluoride (Compound 4) 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosylbromide (compound 3, 2.87 g, 7.36 mmol) was dissolved in CH3CN (20 mL), to which silver fluoride (2.80 g, 22.1 mmol) was added. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. The reaction mixture was filtered with ELISA using a Celite pad, the organic solution was washed with saturated NaHCO3 and NaCl, dried over Na2SO4, and concentrated using a rotary evaporator. Silica gel column chromatography of the resulting syrup was performed using 5:1 toluene-ELISA as the eluent to obtain compound 4 (1.76 g, 72%). R f = 0.41 (toluene:.'' = 4:1). 1 H NMR (500 MHz, CDCl3): δ5.30 (m, 1H, H-4), 5.05 (dd, 1H, J 1,2 = 7.6, J 1,F = 51.7 Hz, H-1), 4.78 (ddd, 1H, J 3,F = 0.8, J 3,4= 3.2, J 2,3 = 10.9 Hz, H-3), 4.12 (dd, 1H, J 5,6a = 6.6, J gem = 11.2 Hz, H-6a), 4.11 (dd, 1H, J 5,6b = 6.5 Hz, H-6b), 3.91 (m, 1H, H-5), 3.76 (ddd, 1H, J 2,F = 12.7 Hz, H-2), 2.11, 2.01, and 1.99 (each s, 3H, 3×Ac). HRMS (ESI): m / z calcd for C 12 H 16 FN3O7Na + ([M+Na] + ) 356.0865, found 356.2149.

[0084] 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α- and β-D-galactopyranose (Compound 5) To a solution of compound 2 (0.504 g, 1.34 mmol) in 1,4-dioxane (20 mL), NaNO2 (0.508 g, 7.37 mmol) was added, and the resulting mixture was stirred at 70 °C for 5 hours under a nitrogen atmosphere. The reaction mixture was diluted with SiO2, washed with water and saturated NaCl, dried on Na2SO4, and concentrated. The resulting residue was passed through a silica gel column with 2:1 toluene-SiO2 as the eluent to obtain compound 5 (0.359 g, 81%). R f = 0.49 (toluene-Ifylene = 1:1) 1 H NMR (500 MHz, CDCl3): α:β = 1.4:1; α-anomer, δ5.47 (dd, 1H, J 3,4 = 3.2, J 4,5 = 1.3 Hz, H-4), 5.43 (d, 1H, J 1,2 = 3.3 Hz, H-1), 4.47 (dt, 1H, J 5,6a = J 5,6b= 6.6 Hz, H-5), 4.15-4.07 (m, overlapped with H-6s of β-anomer, H-6s), 3.75 (dd, 1H, H-2), 2.16, 2.07 and 2.06 (each s, 3H, 3×Ac). β-anomer, δ5.35 (dd, 1H, J 3,4 = 3.4, J 4,5 = 1.0 Hz, H-4), 4.83 (dd, 1H, J 2,3 = 10.8 Hz, H-3), 4.71 (d, 1H, J 1,2 = 7.9 Hz), 4.15-4.07 (m, overlapped with H-6s of α-anomer, H-6s), 3.92 (dt, J 5,6s = 6.4 Hz, H-5), 3.67 (dd, 1H, H-2), 2.17, 2.07 and 2.06 (each s, 3H, 3×Ac). HRMS (ESI): m / z calcd for C 12 H 17 N3O8Na + ([M+Na] + ) 354.0908, found 354.0900.

[0085] 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α- and β-D-galactopyranosyltrichloroacetoimidate (Compound 6) Compound 5 (128 mg, 0.386 mmol) dissolved in CH2Cl2 (1.5 mL) was stirred at 0°C for 3 hours in the presence of K2CO3 (64.0 mg, 0.463 mmol) and CCl3CN (0.16 mL, 1.54 mmol). The reaction mixture was diluted with siRNA, washed with water and saturated NaCl, dried over Na2SO4, and concentrated to dryness. The residue (185 mg) was used in the next glycosylation reaction without further purification. R f = 0.63 (toluene:alkyl = 1:1) 1 H NMR (500 MHz, CDCl3): α:β = 1:2.9; β-anomer, δ5.71 (d, 1H, J1,2 = 8.5 Hz, H-1), 5.41 (dd, 1H, J 3,4 = 3.3, J 4,5 = 1.0 Hz, H-4), 4.92 (dd, 1H, J 2,3 = 10.8 Hz, H-3), 4.18 - 4.15 (m, 2H, H-6s), 4.04 (m, 1H, H-5), 3.96 (dd, 1H, H-2), 2.19, 2.08, and 2.04 (each s, 3H, 3×Ac); α - anomer, δ6.52 (d, 1H, J 1,2 = 3.5 Hz, H-1), 5.55 (broad dd, 1H, H-4), 5.39 (dd, 1H, J 2,3 = 10.8, J 3,4 = 3.3 Hz, H-3), 4.42 (broad t, H-5), 4.10 - 4.05 (overlapped with H-5(β), H-6s), 2.18, 2.09, and 2.02 (each s, 3H, 3×Ac).

[0086] Cysteine ​​compounds S1 - (3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-Nα - (fluoren-9-ylmethoxylcarbonyl)cysteine ​​allyl ester (compound 9) Method 1: Cp2HfCl2 (0.119 g, 0.314 mmol) and AgOTf (0.130 g, 0.628 mmol) were added to a mixture of fluoride 4 (0.105 g, 0.314 mmol), Fmoc - Cys - OAll (Compound 7, 0.116 g, 0.314 mmol), and CaSO4 (1.5 g) in CH2Cl2 (1.5 mL) under a nitrogen atmosphere at -20 °C. The resulting mixture was stirred at that temperature for 24 hours and filtered through a celite pad using EtOAc. The solution was washed with saturated NaHCO3 and NaCl, dried over Na2SO4, and filtered. After concentration, the obtained residue was purified by silica gel column using toluene:EtOAc 4:1 as the eluent to give Compound 9 (0.139 g, 64%). Method 2: TMSOTf (0.034 mL, 0.187 mmol) was added to a mixture of imidate 6 (0.223 g, 0.469 mmol) and Fmoc-Cys-OAll (Compound 7, 0.187 g, 0.468 mmol) in CH2Cl2 (4.0 mL) at -20 °C under a nitrogen atmosphere. After completion of the reaction, the mixture was washed with saturated NaHCO3 and NaCl, dried over Na2SO4, and filtered. After concentration, the resulting residue was purified by a silica gel column to give Compound 9 (0.254 g, 78%). Method 3: TMSOTf (0.105 mL, 0.58 mmol) was added to a mixture of imidate 6 (0.229 g, 0.48 mmol) and Fmoc-Cys(Trt)-OAll (Compound 8, 0.527 g, 0.82 mmol) in CH2Cl2 (3.0 mL) at -20 °C under a nitrogen atmosphere in the presence of MS4A (Molecular Sieves 4A). The reaction mixture was stirred at 0 °C for 3 hours. After filtration through a Celite pad, the mixture was treated by the method described in Method 1 to give Compound 9 (17.3 mg, 52%). R f = 0.30 (toluene:EtOAc = 4:1). 1 1H NMR (500 MHz, CDCl3): δ 7.79 - 7.30 (m, 8H, aromatic Hs of Fmoc), 6.08 (d, 1H, J = 8.4 Hz, amide-NH), 5.92 (m, 1H, OCH2CHC=CH2), 5.41 (broad d, 1H, J 3,4 = 3.3 Hz, H-4), 5.36 (m, 1H, OCH2CHC=CH2a), 5.33 (d, 1H, J 1,2 = 3.5 Hz), 5.29 (m, 1H, OCH2CHC=CH2b), 5.04 (dd, 1H, J 2,3= 11.1 Hz, H-3), 4.72 (ddd, 1H, J = 3.6, 5.2, 8.4 Hz, Cys α-proton), 4.68 (d, 2H, J = 5.7 Hz, OCH2CHC=CH2), 4.49-4.41 (m, 3H, H-5 and OCOCH2), 4.25-4.20 (m, H-2 and OCOCH2CH), 4.15 (dd, 1H, J 5,6a = 5.2, J 6a,6b = 11.4 Hz, H-6a), 4.03 (dd, 1H, J 5,6b = 7.5 Hz, H-6b), 3.32 (dd, 1H, J = 5.2, 14.5 Hz, SCH2), 3.04 (dd, 1H, J = 3.6 Hz, SCH2), 2.19, 2.06, and 2.00 (each s, 3H, 3×Ac). 13 C NMR (125 MHz, CDCl3): δ 128.0, 127.2, 125.1, 120.2, 120.0, 86.1 (C-1), 69.5 (C-3), 67.6, 67.5, 67.3, 67.2, 62.2, 57.8, 53.9, 53.0, 47.5, 35.0, and 20.6×3). HRMS (ESI): m / z calcd for C 33 H 36 N4O 11 SNa + ([M+Na] + ) 719.1993, found 719.1988.

[0087] S 1 -(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)cysteine ​​allyl ester (14) To a stirred solution of compound 9 (127.5 mg, 0.183 mmol) in Pyr (pyridine) (0.5 mL), AcSH (0.207 μL, 2.74 mmol) was partially added while stirring, and the mixture was stirred at room temperature for 48 hours. After the reaction was complete, the mixture was diluted with SiO2, washed with 10% KHSO4, saturated NaHCO3, and NaCl, and dried over Na2SO4. After concentration, the residue was purified by silica gel column chromatography using toluene:SiO2 = 2:1 as the eluent to obtain compound 14 (69.1 mg, 53%). R f = 0.23 (toluene: Depositphotos = 2:1). 1 H NMR (500 MHz, CDCl3): δ7.80-7.30 (m, 8H, aromatic Hs of Fmoc), 6.08 (d, 1H, J = 8.6 Hz, amide-NH), 5.90 (m, 1H, m, OCH2CHC=CH2), 5.58 (d, 1H, J = 8.4 Hz, amide-NH), 5.37 (overlapped with H-1, H-4), 5.36 (d, 1H, J 1,2 = 5.2 Hz), 5.35 (m, 1H, OCH2CHC=CH2a), 5.28 (m, 1H, OCH2CHC=CH2b), 4.95 (dd, 1H, J 2,3 = 11.8, J 3,4 = 3.2 Hz, H-3), 4.82 (ddd, 1H, H-2), 4.72 (ddd, 1H, J = 3.5, 4.7, 8.4 Hz, Cys α-proton), 4.71-4.62 (OCH2CHC=CH2), 4.49 (dd, 1H, J = 7.1, 10.6 Hz, CO2CH2a), 4.45-4.41 (m, 2H, CO2CH2b and CO2CH2CH), 4.22 (broad t, H-5), 4.19 (dd, 1H, J 5,6a = 5.1, J 6a,6b = 11.3 Hz, H-6a), 3.99 (dd, 1H, J 5,6b= 7.7 Hz, H-6b), 3.35 (dd, 1H, J = 5.2, 14.5 Hz, SCH2), 3.02 (dd, 1H, J = 3.5 Hz, SCH2), 2.20, 2.02, 1.99 and 1.98 (each s, 3H, 3×Ac). 13 C NMR (125 MHz, CDCl3): δ 131.2, 127.7, 127.0, 125.1, 120.2, 119.4, 87.6 (C-1), 68.3, 68.2, 67.3, 66.8, 66.4, 62.0, 54.4, 48.4, 47.2, 36.2, 23.4, 20.8 × 3, and 20.6). HRMS (ESI): m / z calcd for C 35 H 40 N2O 12 SNa + ([M+Na] + ) 735.2194, found 735.2190.

[0088] S1-(2-Acetamidoo-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)cysteine ​​(Compound 17) Compound 14 (120.2 mg, 0.169 mmol) dissolved in THF (3 mL) was mixed with Pd(PPh3)4 (9.41 mg, 8.43 mmol) and N-methylaniline (0.193 mL, 1.690 mmol). The reaction mixture was stirred at room temperature for 14 hours and then concentrated. The resulting residue was purified by silica gel column chromatography using CH2Cl2:MeOH = 9:1 as the eluent. Since the compound was found to contain a palladium species, the yellow syrup-like substance was passed through a Quadra Pure TU column to obtain compound 17 (105.3 mg, 93%). R f = 0.10 (CH2Cl2:MeOH = 9:1). 1H NMR (500 MHz, CDCl3): δ7.74-7.24 (m, 8H, aromatic Hs of Fmoc), 6.63 (d, 1H, J = 8.1 Hz, amide-NH), 6.34 (d, 1H, J = 9.5 Hz, amide-NH), 5.42 (d, 1H, J 1,2 = 5.1 Hz, H-1), 5.31 (broad d (dd), 1H, H-4), 4.90-4.74 (m, 2H, H-3 and CαH), 4.80 (ddd, 1H, J 2,3 = 11.7 Hz, H-2), 4.43-4.29 (m, 3H, ArCHCH2), 4.24 (dd, 1H, J 5,6a = 6.9, J 6a,6b =11.0 Hz, H-6a), 4.18 (broad t, 1H, H-5), 3.99 (dd, 1H, J 5,6a = 6.3 Hz, H-6b), 3.31 (dd, 1H, J = 4.6, J =15.2 Hz, SCH2), 2.99 (dd, 1H, J = 2.20 Hz, SCH2) and 2.14-1.92 (m, 12H, -NHCOCH3and -OCOCH3× 3). HRMS (ESI): m / z calcd for C 32 H 36 N2O 12 SNa + ([M+Na] + ) 695.1881, found 695.1884.

[0089] S1-(2-Acetamido-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)cysteine ​​(Compound 20) Compound 17 (25.2 mg, 37.5 μmol) was dissolved in THF-H2O (3:1) (2 mL), and hydrazine (0.04 mL) was added at room temperature. The reaction mixture was stirred for 48 hours. The resulting residue was separated by Sep-pak(C). 18The compound was placed in a cartridge, washed with water, and eluted with MeOH. After evaporation, the crude compound was purified using a silica gel column and eluted with CH2Cl2-MeOH-AcOH = 3:1:0.04 to obtain 20 (14.4 mg, 70%). 1 H NMR (500 MHz, methanol-d4) δ:7.72-7.20 (m, 8H, Ar), 5.50 (d, 1H, J 1,2 = 4.5 Hz, 1-H), 4.35 (dd, 1H, 11.2 Hz, H-2), 4.31 (broad ddd, 1H, H-5), 4.23 (d, 2H, J CH,CH2 = 7.2 Hz, ArCHCH2), 4.14 (t, 1H, ArCHCH2), 4.09 (t, 1H, CαH), 3.79 (broad t, 1H, H-4), 3.70 (dd, 1H, J 5,6a = 6.9, J 6a,6b = 11.5 Hz, H-6a), 3.64 (dd, 1H, J 5,6b = 5.0 Hz, H-6b), 3.57 (dd, 1H, J = 2.9, J = 11.4 Hz, H-4), 3.02 (dd, 1H, J CαH,CβHa = 7.8, J CβHa,CβHb = 14.0 Hz, CβHa), 3.00 (dd, 1H, J CαH,CβHb = 3.9 Hz, CβHb) and 1.87 (s, 3H, NHCOCH3). HRMS (ESI): m / z calcd for C 32 H 36 N2O 12 SNa + ([M+Na] + ) 569.1564, found 569.4052.

[0090] threonine compound O 1 -(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)threonine allyl ester (compound 12) Compound 10 (Fmoc-Thr-OAll, 0.30 g, 0.761 mmol) and Ag2ClO4 (0.21 g, 1.0 mmol) were stirred in CH2Cl2 (5 mL) at room temperature in the presence of MS 4A (1 g). To this solution, bromide 3 (0.2 g, 0.5 mmol) dissolved in CH2Cl2 (2.5 mL) was slowly added using a syringe over 40 minutes at 0°C. The reaction mixture was filtered through a Celite pad, the organic layer was washed with saturated NaHCO3 and NaCl, dried on Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography (toluene:siRNA = 2:1) to obtain compound 12 (0.46 g, 0.66 mmol, 86%). R f = 0.43 (Hexane:IQ = 1:1). 1 H NMR (500 MHz, CDCl3): δ7.79-7.15 (m, 8H, aromatic Hs of Fmoc), 5.95 (m, 1H, OCH2CHC=CH2), 5.67 (d, 1H, J = 9.4 Hz, amide-NH), 5.47 (broad d, 1H, J 3,4 = 3.3 Hz, H-4), 5.38 (m, 1H, OCH2CHC=CH2a), 5.30 (dd, 1H, J 2,3 = 11.4 Hz, H-3), 5.28 (m, 1H, OCH2CHC=CH2b), 5.05 (d, 1H, J 1,2 = 3.6 Hz, H-1), 4.70 (d, 2H, OCH2CHC=CH2), 4.52-4.46 (m, 2H, OCHCH3and Thr α-proton), 4.43 (dd, 1H, J 5,6a = 7.5, J 6a,6b = 10.6 Hz, H-6a), 4.36 (dd, 1H, J 5,6b= 7.5 Hz, H-6b), 4.31-4.25 (m, 3H, H-5 and CO2CH2CH), 4.10 (CO2CH2), 3.37 (dd, 1H, H-2), 2.15, 2.08, 2.05 (each s, 3H, 3×Ac), and 1.36 (d, 3H, J = 6.3 Hz, Thr CH3). 13 C NMR (125 MHz, CDCl3): δ 131.3, 127.6, 127.1, 125.4, 120.0, 119.3, 99.5 (C-1), 76.9, 68.3, 67.6, 67.4, 66.6 × 2, 62.0, 58.8, 57.6, 47.0, 20.8 × 3, and 18.6). HRMS (ESI): m / z calcd for C 34 H 38 N4O 12 Na + ([M+Na] + ) 717.2378, found 717.2394.

[0091] O 1 -(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)threonine allyl ester (compound 15) Compound 15 was obtained in 79% of the same manner as described for the synthesis of compound 14. R f = 0.29 (hexane:SiO = 1:2). 1 H NMR (500 MHz, CDCl3): δ7.80-7.15 (m, 8H, aromatic Hs of Fmoc), 5.88 (m, 1H, OCH2CHC=CH2), 5.78 (d, 1H, J = 9.8 Hz, amide-NH), 5.59 (d, 1H, J = 9.8 Hz, amide-NH), 5.39 (broad d, 1H, J 3,4 = 2.9 Hz, H-4), 5.36 (m, 1H, OCH2CHC=CH2a), 5.31 (m, 1H, OCH2CHC=CH2b), 5.09 (dd, 1H, J 2,3= 11.5 Hz, H-3), 4.88 (d, 1H, J 1,2 = 3.5 Hz), 4.67 (m, 1H, OCH2aCHC=CH2), 4.51-4.52 (m, 2H, OCH2bCHC=CH2, H-2), 4.51-4.42 (m, 3H, NHCO2CH2CH and Thr α-proton), 4.31-4.26 (m, 2H, NHCO2CH2CH and OCHCH3), 4.23 (broad t, H-5), 4.11 (dd, 1H, J 5,6a = 5.6, J 6a,6b = 11.1 Hz, H-6a), 4.08 (dd, 1H, J 5,6b = 7.6 Hz, H-6b), 2.17, 2.04, 2.01×2 (each s, 3H, 3×Ac), and 1.34 (d, 3H, J = 6.5 Hz, Thr CH3). HRMS (ESI): m / z calcd for C 36 H 42 N2O 13 Na + ([M+Na] + ) 733.2579, found 733.2569.

[0092] O 1 -(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)threonine (compound 18) Compound 18 was obtained in the same manner as described for the synthesis of 85% compound 17. R f = 0.49 (CH2Cl2:MeOH:AcOH = 9:1:0.05). 1 H NMR (500 MHz, acetone-d6): δ7.77-7.19 (m, 8H, aromatic Hs of Fmoc), 5.25 (broad d, 1H, J 3,4 = 3.4 Hz, H-4), 4.92 (dd, 1H, J 2,3 = 11.8 Hz, H-3), 4.89 (d, 1H, J 1,2= 3.7 Hz), 4.40-4.23 (m, 6H, NHCO2CH2CH, Thr α-proton, H-2, H-5), 4.15 (t, 1H, J = 6.3 Hz, OCHCH3), 4.00 (dd, 1H, J 5,6a = 5.4, J 6a,6b = 11.1 Hz, H-6a), 3.97 (dd, 1H, J 5,6b = 7.5 Hz, H-6b), 2.00, 1.88, 1.77, 1.75 (each s, 3H, 4×Ac), and 1.25 (d, 3H, Thr CH3). HRMS (ESI): m / z calcd for C 33 H 38 N2O 13 Na + ([M+Na] + ) 693.2266, found 693.2253.

[0093] O 1 -(2-Acetamido-2-deoxy-α-D-galactopyranosyl)-Nα -(fluoren-9-ylmethoxylcarbonyl)threonine (compound 21) Compound 21 was obtained in a quantitative yield (9.60 mg) using the same method as described for the synthesis of compound 20. R f = 0.32 (CHCl3:MeOH:AcOH = 4:1:0.5). 1 H NMR (500 MHz, methanol-d4) δ:7.73-7.19 (m, 8H, Ar), 4.77 (d,1H, J = 3.99 Hz, H-1), 4.41 (dd, 1H, J CH,CH2a = 6.81, J CH2a,CH2b = 10.87 Hz, ArCHCH2), 4.36 (dd, 1H, J CH,CH2b= 6.47, ArCHCH2b) ,4.26 (ddd, 1H, H-5, J = 6.42, 6.87 Hz), 4.19-4.10 (m, 3H, H-2, CαH, ArCHCH2), 3.79-3.76 (m, 2H, H-4, CβH), 3.66-3.57 (m, 3H, H-3, 6), 1.90 (s, 3H, NHCOCH3) and 1.43 (d, 3H, CγH3, J = 6.38Hz). HRMS (ESI): m / z calcd for C 27 H 32 N2NaO 10 ([M+Na] + ) 567.1949 found 567.4162.

[0094] Serine compounds O 1 -(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)serine allyl ester (compound 13) Compound 13 was obtained in 87% quantity using donor 3 and serine derivative 12 in the same manner as described for the synthesis of compound 11. R f = 0.38 (toluene: = 3:2). 1 H NMR (500 MHz, CDCl3): δ7.79-7.31 (m, 8H, aromatic Hs of Fmoc), 5.95 (m, 2H, OCH2CHC=CH2, amide-NH), 5.47 (broad d, 1H, J 3,4 = 2.9 Hz, H-4), 5.37 (m, 1H, OCH2CHC=CH2a), 5.33-5.28 (m, 2H, OCH2CHC=CH2b, H-3), 5.05 (d, 1H, J 1,2= 3.6 Hz, H-1), 4.75-4.68 (m, 2H), 4.60 (m, 1H, Ser α proton), 4.42 (d, 2H, J= 7.3 Hz, H6s), 4.26 (broad t, 1H, H-5), 4.19 (t, 1H, OCOCH2CH),), 4.14 (dd, 1H, J = 3.1 and 10.5 Hz, Ser-OCH2aCH), 4.04 (d, 2H, OCOCH2CH), 4.03 (dd, 1H, J = 3.2 Hz, Ser-OCH2bCH), 3.63 (dd, 1H, J 1,2 = 3.6, J 2,3 = 11.1 Hz, H-2), 2.15, 2.07, and 1.98 (each s, 3H, 3×Ac). HRMS (ESI): m / z calcd for C 33 H 36 N4O 12 Na + ([M+Na] + ) 703.2222, found 703.2220.

[0095] O 1 -(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)serine allyl ester (16) Compound 16 was obtained from compound 13 in a 73% concentration using the same method as described for the synthesis of compound 14. R f = 0.18 (toluene:alkyl = 5:1). 1 H NMR (500 MHz, CDCl3): δ7.80-7.12 (m, 8H, aromatic Hs of Fmoc), 6.53 (d, 1H, J = 8.4 Hz, amide-NH), 5.89 (m, 1H, OCH2CHC=CH2), 5.37-5.27 (m, 3H, OCH2CHC=CH2, H-4), 5.09 (dd, 1H, J 2,3 = 11.5, J 3,4 = 2.9 Hz, H-3), 4.81 (d, 1H, J 1,2= 3.2 Hz), 4.68-4.61 (m, 2H, OCH2CHC=CH2), 4.61-4.42 (m, 4H), 4.24 (t, 1H, J = 6.7 Hz), 4.13 (broad t, 1H, H-5), 4.08 (dd, 1H, J 5,6a = 5.7, J 6a,6b = 11.4 Hz, H-6a), 4.03 (dd, 1H, J 5,6b = 7.5 Hz, H-6b), 3.93 (broad s, 2H), 2.16, 2.00 × 2, and 1.93 (each s, 3H, 4×Ac). HRMS (ESI): m / z calcd for C 35 H 40 N2O 13 Na + ([M+Na] + ) 719.2423, found 719.2415.

[0096] O 1 -(2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)serine (compound 19) Compound 19 was obtained from compound 16 in 88% concentration using the same method as described for the synthesis of compound 17. R f = 0.30 (CH2Cl2:MeOH:AcOH = 9:1:0.1). 1 H NMR (500 MHz, acetone-d6): δ7.89-7.33 (m, 8H, aromatic Hs of Fmoc), 5.34 (dd, 1H, J 3,4 = 3.4, J 4,5 = 1.2 Hz, H-4), 4.99 (dd, 1H, J 2,3 = 11.6 Hz, H-3), 4.85 (d, 1H, J 1,2= 3.5 Hz), 4.40 (m, 1H, Ser α proton), 4.47-4.33 (m, 4H, H-2, NHCO2CH2CH, H-5), 4.27 (broad t, 1H, NHCO2CH2CH), 4.13 (dd, 1H, J 5,6a = 5.9, J 6a,6b = 11.1 Hz, H-6a), 4.08-4.02 (m, 3H, Ser CH2, H-6b), 2.13, 1.95, 1.91, and 1.88 (each s, 3H, 4×Ac). HRMS (ESI): m / z calcd for C 32 H 36 N2O 13 Na + ([M+Na] + ) 679.2110, found 679.2103.

[0097] O 1 -(2-acetamido-2-deoxy-α-D-galactopyranosyl)-Nα-(fluoren-9-ylmethoxylcarbonyl)serine (compound 22) Compound 22 was obtained in an amount of 82% (14.0 mg) by the same method as described for the synthesis of compound 20. 1 H NMR (500 MHz, Methanol-d4) δ:7.72-7.19 (m, 8H, Ar), 4.72 (d, 1H, H-1, J 1,2 = 3.57 Hz), 4.35-4.27 (m, 3H, ArCH2CH2, H-5), 4.17-4.12 (m, 2H, ArCHCH2, -OCHCH2), 3.83 (dd, 1H, J 2,3 = 10.76 Hz, H-2), 3.80-3.76 (m, 2H, ArCHCH2, H-4) 3.71 (broad t, 1H, CαH), 3.65 (dd, 1H, H-3, J = 3.09 Hz), 3.63 (dd, 1H, H-6a, J = 6.85 Hz), 3.58 (dd, 1H, H-6b) and 1.86 (s, 3H, -NHCOCH3). HRMS (ESI): m / z calcd for C 20 H 30 N2O 10 Na + ([M+Na] + ) 553.1793, found 553.4509.

[0098] Synthesis of glycopeptides (1) H-Cys(Trt)-Trt(2-Cl)-resin (0.71 mmol / g, 76.1 mg, 0.0540 mmol) was swollen with 1-methyl-2-pyrrolidinone (NMP) for 30 minutes. Fmoc-Arg(Pbf)-Obt, prepared by mixing Fmoc-Arg(Pbf)-OH (0.216 mmol), 1M N,N-dicyclohexylcarboimide (DCC) NMP solution (0.324 mL), and 1M 1-hydroxybenzotriazole (HOBt) NMP solution (0.324 mL) at room temperature for 30 minutes, was added to the reaction vessel, and the mixture was vortexed at 50 °C. The resin was washed with NMP and 50% DCM in CM3Oh solution, treated with 10% Ac2O-5% DIEA in NMP solution for 5 minutes, and washed with NMP. The resin was treated with a 20% piperidine NMP solution for 5 minutes and 15 minutes. After washing with NMP, the peptide elongation reaction was carried out similarly. For amino acid coupling, compound 17, a cys molecule containing 4 GalNAc residues, was used in a 0.108 mmol dose. After the extension reaction was complete, the resin was treated with a 20% piperidine NMP solution for 5 minutes and 15 minutes, washed with NMP, MeOH-CH2Cl2 (1:1), then CH2Cl2, and dried in vacuum to obtain H-Cys(Trt)-Lys(Boc)-Lys(Boc)-Leu-Ala-Glu(tBu)-Arg(Pbf)-Leu-Lys(Boc)-Ala-Lys-(Boc)-Leu-Pro-Asp(tBu)-Ala-Cys(GalNAc4)-Pro-Thr(tBu)-Glu(tBu)-Leu-Ala-Lys(Boc)-Leu-Val-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Cys(Trt)-Trt(2-Cl)-resin (compound 23, 187.4 mg). A portion of the resin 23 (30.5 mg) was treated at room temperature for 2 hours with a 500 μL cocktail containing 82.5% TFA, 5% H2O, 5% thioanisole, 5% phenol, and 2.5% triisopropylsilane (TIS). The cleaved and deprotected peptide 24 was precipitated with an excess of diethyl ether, the supernatant was removed twice, the precipitate was dried under vacuum, and stored with the resin. The crude peptide was treated at room temperature for 24 hours with 2 mL of 6 M aqueous urea solution containing 2% hydrazine and 10% DMSO to remove acetyl groups and form disulfide bonds. After the reaction was complete, the reaction mixture was separated by RP-HPLC (Separ C18G, 150 x 4.6 mm, RIKEN, 0.1% TFA) and filtration. The mixture was purified by RP-HPLC using an Inertsil ODS-SP (10 x 250 mm) column (GL Sciences, Tokyo, Japan) with a linear gradient of acetonitrile containing 0.1% TFA. After lyophilization, glycopeptide 1 (1.06 μmol, yield 8.4%) was obtained. Holding time: 15.5 minutes. MALDI-TOF mass: m / zcalcd for C 144 H 256 N 42 O 42 S3 2+ ([M(1) + 2H] 2+ ) 1670.92, found: 1671.07. Amino acid analysis: Asp 1.93 , Thr 1.17 , Glu 1.97 , Pro 1.65 , Ala4, Val 0.95 , Leu 4.67 Lys 5.56 Arg 1.95 .

[0099] Enzyme assay of compounds 20-22 The synthesized compounds 20-22 were assayed together with pNP-α-GalNAc for chicken α-GalNAc-ase. A typical enzymatic reaction was performed as follows: A reaction mixture containing compound 21 at various concentrations was incubated in 100 μL of citrate buffer (pH 4.0) with α-GalNAc-ase (0.5 unit / mL at 10 μL) at 37°C for 30 minutes. The reaction conditions were determined by preliminary experiments using pNP-α-GalNAc as a substrate. After 30 minutes of incubation, the mixture was heated at 65°C for 15 minutes. 10 μL of the mixture was subjected to HPLC to analyze the reaction process and quantify the amount of Fmoc-protected amino acids released. Each reaction was performed three times. First, the time course for compounds 20-22 was examined, revealing that compound 21 was twice as good a substrate as compound 22. On the other hand, compound 20 was not hydrolyzed. The Km value of Fmoc-Thr(α-GalNAc)-OH(compound 21) was 312 μM, and the Vmax was 1.3 μM / min. The inhibitory activity of compound 20 against the enzyme was investigated using compound 21 as a substrate. The results showed that compound 20 is insufficient as an inhibitor of this enzyme.

Claims

1. The peptide represented by the following general formula (1) 【Chemistry 1】 [wherein, AA ４ , ｐ , AA １ , AA ２ , AA ３ and AA ４ represents an amino acid. (AA 0 ) ｌ represents a peptide chain having a turn structure. (AA １ ) ｍ and (AA ２ ) ｎ represent peptide chains having the same or different α-helix structures. (AA ３ ) ｏ and (AA ４ ) ｐ represent peptide chains composed of the same or different amino acids. X represents a single bond or a group derived from an agent that activates macrophages. l, m, and n are the same or different and represent natural numbers. o and p are the same or different and represent integers.].

2. (AA 0 ) ｌ (AA １ ) ｍ and (AA ２ ) ｎ The peptide according to claim 1, wherein at least one of the amino acids constituting it is modified with N-substituted-α-galactosamine.

3. The peptide according to claim 1, wherein the peptide is derived from GcMAF, excluding Cys.

4. Does it have the amino acid sequence represented by SEQ ID NO: 1? Sequence number 1: Cys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Asp Ala Thr Pro Cys Glu Leu Ala Lys Leu Val Asn Lys Arg Cys or In the amino acid sequence represented by Sequence ID No. 1, one or more amino acids are deleted, substituted, or added (however, the N-terminal and C-terminal Cys in Sequence ID No. 1 are conserved), and The peptide according to claim 1, wherein the peptide contains Cys in addition to the N-terminal and C-terminal Cys in SEQ ID NO: 1, and one of the Cys in addition to the N-terminal and C-terminal Cys is modified with α-galactosamine which may have a substituent on its nitrogen atom.

5. A macrophage activator comprising the peptide described in claim 3.

6. A pharmaceutical product comprising the peptide described in claim 3.

7. A method for adding a sugar molecule to cysteine, comprising carrying out the reaction shown in the following formula in the presence of a promoter molecule, wherein the promoter molecule contains a hard Lewis acid according to the HSAB rule: 【Chemistry 2】 [In the formula, Y represents a hydrogen or thiol protecting group. P １ P indicates a protecting group for the amino group. ２ This indicates a protecting group for carboxylic acids. 【Transformation 3】 The anomeric position is protected by the protective group P ３ This shows a monosaccharide molecule protected by a protecting group (the hydroxyl group in the monosaccharide molecule may be protected. Also, the carbon adjacent to the anomeric carbon may have an amino group or azi group substituted with a protecting group as a substituent). P ３ This indicates a group derived from a hard Lewis base in the HSAB rule.

8. A method for producing a cysteine ​​compound with a sugar molecule attached, comprising carrying out the reaction shown in the following formula in the presence of a promoter molecule, wherein the promoter molecule contains a hard Lewis acid according to the HSAB rule: 【Chemistry 4】 [In the formula, Y represents a hydrogen or thiol protecting group. P １ P indicates a protecting group for the amino group. ２ This indicates a protecting group for carboxylic acids. 【Transformation 5】 The anomeric position is protected by the protective group P ３ This shows a monosaccharide molecule protected by a protecting group (the hydroxyl group in the monosaccharide molecule may be protected. Also, the carbon adjacent to the anomeric carbon may have an amino group or azide substituted with a protecting group as a substituent). P ３ This indicates a group derived from a hard Lewis base in the HSAB rule.

9. Cysteine ​​compounds represented by the following formula 【Transformation 6】 [In the formula, P １ P indicates a protecting group for the amino group. ２ This indicates a protecting group for carboxylic acids. base 【Transformation 7】 This represents a monovalent group obtained by removing the hydroxyl group at the anomeric position from a monosaccharide molecule (the hydroxyl group in the monosaccharide molecule portion of the group may be protected. Furthermore, the carbon adjacent to the anomeric carbon may have a substituent such as a protected amino group or azide).

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