Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
  • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
  • C07H5/06—Aminosugars
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof

Definitions

• the present disclosure relates to a solubilizing group for solubilizing poorly water-soluble proteins, and a method for producing solubilized proteins and the like using the solubilizing group.
• protein solubilization there has long been a need for protein solubilization.
• the protein when using a protein as an active pharmaceutical ingredient, it is preferable for the protein to be sufficiently soluble in water in order to improve absorbability when administered systemically (e.g., orally), to increase the concentration of the active ingredient in the formulation, and to achieve an appropriate viscosity in the formulation.
• protein solubilization is still necessary for the analysis of protein folding and complexes between folded proteins and target molecules that cause diseases.
• the number of poorly water-soluble candidate molecules is increasing, and the need for solubilization technology is further increasing.
• Patent Document 1 describes a hydrophilic polylysine tag for solubilizing poorly water-soluble proteins.
• the present disclosure aims to provide a novel means that can be used to solubilize poorly water-soluble proteins.
• Glycans are known to bind to membrane proteins and secretory proteins in vivo and to affect their biological activities in various ways. However, despite their usefulness, their use has been limited due to the difficulty of obtaining them and their high cost. In the course of intensive research aimed at solving the above problems, the present inventors have found that poorly water-soluble proteins can be solubilized by adding a glycan-solubilizing group to the poorly water-soluble protein. Furthermore, they have found that by making the glycan-solubilizing group retain a specific functional group moiety, the glycan-solubilizing group used for solubilization can be separated, recovered, and reused after obtaining the target solubilized protein, thereby completing the present invention.
• a hydrophilic solubilizing group for a protein or peptide comprising: a sugar chain portion and a functional group portion bound to an amino acid side chain and capable of being detached from the side chain of the protein or amino acid in the presence of an acid, the functional group being bound to an amino group or a carboxy group of the sugar chain; Solubilizing groups.
• the solubilizing group according to (1) The sugar chain is characterized in that it consists of four or more sugars. Solubilizing groups.
• the solubilizing group according to (1) The sugar chain is characterized in that it consists of seven or more sugars. Solubilizing groups.
• the solubilizing group according to (1) The sugar chain is characterized in that it consists of 9 or more sugars. Solubilizing groups.
• the solubilizing group according to (1) is a two-antennary complex type glycan, a three-antennary complex type glycan, or a four-antennary complex type glycan, Solubilizing groups.
• the solubilizing group according to (1) The sugar chain is a double-antennary complex sugar chain, Solubilizing groups.
• the solubilizing group according to (8) is a sugar chain selected from the group consisting of a disialo sugar chain, a monosialo sugar chain, an asialo sugar chain, a diglucunac sugar chain, and a dimannose sugar chain. Solubilizing groups.
• the solubilizing group according to (1) The sugar chain has the following formula: [In the formula, R 1 and R 2 are the same or different, and Ac represents an acetyl group.
• the sugar chain is characterized by being represented by Solubilizing groups.
• the solubilizing group according to (1) It has the following structure: Solubilizing groups.
• a method for producing a solubilized protein or peptide comprising the steps of: Attaching the solubilizing group according to claim 1 to a sulfhydryl group, a carboxy group or an amino group of one or more amino acid side chains constituting any protein or peptide; Including, method.
• a method for producing a target protein or peptide using a poorly water-soluble protein or peptide comprising the steps of: The method includes the steps of: binding the solubilizing group according to claim 1 to a sulfhydryl group, a carboxy group, or an amino group of one or more amino acid side chains constituting the poorly water-soluble protein or peptide to solubilize the protein or peptide; and linking the solubilized protein or peptide to another soluble protein or peptide, or a solubilized protein or peptide to obtain a target protein. Manufacturing method.
• the present disclosure provides a novel means that can be used to solubilize poorly water-soluble proteins, etc.
• FIG. 1 shows the results of HPLC purification of the produced sugar chain-solubilizing group 1 .
• FIG. 2 shows the reaction progress of the sugar chain solubilizing group 1 with peptide 2 and the results of HPLC purification of the reaction product, compound 3 .
• FIG. 3 shows the progress of the deprotection reaction of sugar chain solubilizing group 1 from compound 3 and the results of HPLC purification of peptide 2 , which is the product of the reaction.
• FIG. 4 shows the progress of the reaction for removing the SUMO-tagged protein from compound 3 , and the results of HPLC purification of the reaction products, compound 4 and the SUMO-tagged protein.
• FIG. 1 shows the results of HPLC purification of the produced sugar chain-solubilizing group 1 .
• FIG. 2 shows the reaction progress of the sugar chain solubilizing group 1 with peptide 2 and the results of HPLC purification of the reaction product, compound 3 .
• FIG. 3 shows the progress of the deprotection reaction of sugar chain solub
• FIG. 5 shows the reaction progress of the sugar chain solubilizing group 1 with the IL-6 N-terminal peptide, and the results of HPLC purification of the reaction product, compound 6 .
• FIG. 6 shows the progress of the deprotection reaction of sugar chain solubilizing group 1 from compound 6 , and the results of HPLC purification of the reaction products, compound 5 and sugar chain solubilizing group 1 .
• FIG. 7 shows the progress of the peptide ligation reaction between peptide 6 having a sugar chain solubilizing group 1 and solubilizing peptide 7 , and the results of HPLC purification of compound 8 , which is the reaction product.
• FIG. 8 shows the progress of the desulfurization reaction of compound 8 and the results of HPLC purification of the reaction product, compound 9 .
• FIG. 9 shows an HPLC chart of compound 10, which is the product of the deprotection reaction of sugar chain-solubilizing group 1 from compound 9 .
• FIG. 10 shows the reaction progress of the folding reaction of compound 10 and the HPLC chart of compound 11 , which is the reaction product.
• the present disclosure relates to a hydrophilic solubilizing group for solubilizing a protein (also referred to herein simply as the "solubilizing group according to the present disclosure").
• protein may include proteins or portions thereof (i.e., peptides), as well as derivatives thereof. Proteins are typically poorly water-soluble proteins. Derivatives of proteins refer to derivatives of natural proteins, and include, but are not limited to, for example, those in which some of the amino acids constituting the natural protein are replaced with non-natural amino acids, those in which natural proteins have been subjected to chemical or genetic processing, combinations of fragments of natural proteins, etc.
• proteins include any poorly water-soluble protein that needs to be solubilized, and may include, but are not limited to, interleukins such as IL-1, IL-2, and IL-6, various chemokines, interferons, other cytokines such as lymphokines, transmembrane proteins such as rhodopsin, and fragments thereof.
• the solubilizing group according to the present disclosure is characterized by comprising a sugar chain portion and a functional group portion that is bound to an amino acid side chain and is detachable from the protein or amino acid side chain in the presence of an acid, and is bound to an amino group or carboxy group of the sugar chain.
• the functional group portion may include, but is not limited to, functional group portions such as triphenylmethanol, methoxybenzyl, phenacyl, and diphenylmethyl.
• the functional group portion may further include a carboxy group, an amino group, a hydroxyl group, or the like for bonding with the glycan portion.
• acids that can be used in the present disclosure include, but are not limited to, inorganic acids such as hydrochloric acid and sulfuric acid, boron trifluoride diethyl ether (BF3.OEt2), dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST), trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tripropylsilyl trifluoromethanesulfonate, dimethylethylsilyl trifluoromethanesulfonate, tribenzylsilyl trifluoromethanesulfonate, trinaphthylsilyl trifluoromethanesulfonate or tribenzylmethylsilyl trifluoromethanesulfonate, silver trifluoromeromethanesulfonate, silver trifluorome
• the solubilizing group is composed of a glycan portion and a triphenylmethanol portion bonded to an amino group or a carboxy group of the glycan. In a more specific embodiment of the present disclosure, the solubilizing group is composed of a triphenylmethanol portion bonded to a carboxy group of the glycan.
• a "glycan” refers to a compound formed by linking one or more unit sugars (monosaccharides and/or derivatives thereof). When two or more unit sugars are linked, the unit sugars are bonded to each other by dehydration condensation through a glycosidic bond.
• glycans include, but are not limited to, a wide range of glycans, including monosaccharides and polysaccharides contained in living organisms (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid, and complexes and derivatives thereof), as well as glycans decomposed or derived from complex biomolecules such as decomposed polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids.
• the glycan may be linear or branched.
• sugar chain also includes derivatives of sugar chains
• examples of sugar chain derivatives include, but are not limited to, sugars that constitute the sugar chains, such as sugars having a carboxy group (for example, aldonic acids whose C-1 position has been oxidized to become a carboxylic acid (for example, D-gluconic acid obtained by oxidizing D-glucose), and uronic acids whose terminal C atom has become a carboxylic acid (D-glucuronic acid obtained by oxidizing D-glucose)), sugars having an amino group or a derivative of an amino group (for example, an acetylated amino group) (for example, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc.), sugars having both an amino group and a carboxy group (for example, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.),
• the type of unit sugar constituting the glycan is not particularly limited as long as it can impart sufficient solubility to the poorly water-soluble protein depending on the solubility of the protein.
• the glycan according to the present disclosure contains at least two mannose units.
• the glycan in the present disclosure may be a glycan that exists in the body as a complex carbohydrate (glycopeptide (or glycoprotein), proteoglycan, glycolipid, etc.), or a glycan that does not exist in the body as a complex carbohydrate.
• Glycosylation that exists as complex carbohydrates in the body is preferred, for example, when the solubilizing group according to the present disclosure is attached to a poorly water-soluble protein to solubilize it and administer it to the body.
• glycans include N-linked glycans and O-linked glycans, which are glycans that are bound to peptides (or proteins) in the body as glycopeptides (or glycoproteins), and preferably N-linked glycans are used.
• N-linked glycans include high mannose, complex, and hybrid types, and preferably complex types.
• the complex type sugar chain used in the present disclosure is, for example, a sugar chain represented by the following general formula: [In the formula, R 1 and R 2 are the same or different, Ac represents an acetyl group. Examples of such sugar chains include those represented by the following formula:
• the glycan is not limited in number as long as it contains enough sugars to impart sufficient solubility to the poorly water-soluble protein, but it is preferable that the glycan is composed of 4 or more sugars, for example 5 or more, 7 or more, 9 or more, or 11 or more sugars.
• the glycan is a glycan consisting of 4 to 11, 7 to 11, for example 9 sugars.
• the glycan is a biantennary complex-type glycan.
• a complex-type glycan is characterized by containing two or more types of monosaccharides and having the basic structure shown below and a lactosamine structure represented by Gal ⁇ 1-4GlcNAc.
• the double-antennary complex-type glycan refers to a basic structure in which a single glycan chain consisting of 0 to 3 sugars is bound to each of the two mannoses at the terminals.
• double-antennary complex-type glycans include the disialo glycan shown below: Monosialoglycan, Asialoglycan, Diglucunac glycan, Dimannose glycan, In one embodiment of the present disclosure, the biantennary complex-type glycan is an asialo glycan.
• the complex-type glycan of the present disclosure includes, in addition to the biantennary complex-type glycan, a triantennary complex-type glycan (a triantennary complex-type glycan) and a quadrantennary complex-type glycan (a quadrantennary complex-type glycan).
• a triantennary or quadrantennary complex-type glycan includes a trisialoglycan represented by the following structural formula: Tetrasialoglycan represented by the following structural formula
• the triple-antennary complex-type glycan and quadruple-antennary complex-type glycan also include glycans that have lost one or more sugars from the non-reducing end of these trisialo- or tetrasialo-glycans.
• the complex-type glycan of the present disclosure also includes those to which fucose is attached.
• the complex-type glycan to which fucose is attached is a fucose-containing complex-type glycan represented by the following structural formula:
• sugar chains that have lost one or more sugars from the non-reducing end of these fucose-containing complex-type sugar chains can also be included.
• disialo glycan include those shown in the above chemical formula, as well as those with a different bond pattern from the examples shown in the chemical formula, and such glycans are also included in the glycans of this disclosure.
• examples of such glycans include disialo glycan or monosialo glycan in which sialic acid and galactose are bonded by an ( ⁇ 2 ⁇ 3) bond.
• a sugar chain in which the carboxy group of the sialic acid has been modified can also be used.
• the modification of the carboxy group of the sialic acid is preferably a group that can eliminate the negative charge of the carboxy group or convert it to a positive charge, and examples of such groups include alkylamino groups having 1 to 30 carbon atoms, benzyl groups, amino groups, and aminoethylamino groups.
• the negative charge of the carboxy group of the sialic acid can be eliminated (benzyl groups, amino groups, etc.) or converted to a positive charge (aminoethylamino group, etc.), thereby aiming to control the blood clearance and in vivo distribution of the glycosylated polypeptide.
• the high mannose glycan used in the present disclosure is a glycan in which two or more mannoses are further bound to the basic structure of the complex glycan described above. Since high mannose glycans are bulky, binding a high mannose glycan to a peptide can increase stability in blood.
• a glycan containing 5 to 9 mannoses such as a mammalian high mannose glycan
• Examples of high mannose glycans preferably used in the present disclosure include: High mannose-5 (M-5) High mannose-9 (M-9) etc. can be mentioned.
• preferred glycans include, for example, glycans that exist in the human body as glycoproteins bound to proteins (for example, glycans described in "FEBS LETTERS Vol. 50, No. 3, February 1975") and that have the same structure (glycans with the same types of constituent sugars and their binding patterns) or that have lost one or more sugars from the non-reducing end thereof, such as the glycans described below.
• a solubilizing group according to the present disclosure has the structure:
• the solubilizing group according to the present disclosure can be produced according to a method well known to those skilled in the art. For example, it can be produced by dehydration condensation between a reactive group (e.g., amino group) of a glycosylated amino acid having a desired glycochain (e.g., glycosylated asparagine, glycosylated cysteine) and a reactive group (e.g., carboxy group) of a functional group portion according to the present disclosure.
• a reactive group e.g., amino group
• a glycosylated amino acid having a desired glycochain e.g., glycosylated asparagine, glycosylated cysteine
• a reactive group e.g., carboxy group
• 4-(diphenylhydroxymethyl)benzoic acid is activated and reacted with a glycosylated amino acid such as glycosylated asparagine to obtain a solubilizing group containing triphenylmethanol as a functional group portion.
• the reaction time may vary depending on the reagents and solvents used, but may be 1 minute to 48 hours, preferably 10 minutes to 24 hours, for example, 6 hours.
• the reaction temperature may vary depending on the reagents and solvents used, but may be -78°C to 300°C, preferably -78°C to 150°C, for example, room temperature.
• Reagents capable of activating the carboxyl group of the functional group portion include, but are not limited to, carbodiimide-based condensing agents such as 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD), triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM), thionyl chloride, lower alkyl haloformates such as ethyl chloroformate, sulfuric acid, etc.
• the above reagents may be used alone or in combination.
• the solvent that can be used when producing the solubilizing group according to the present disclosure is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, tert-butyl alcohol, 2-methoxyethanol, and 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP); ethers such as diethyl ether, diphenyl ether, tetrahydrofuran, and 1,2-dimethoxyethane; aromatic hydrocarbons such as chlorobenzene, toluene, and xylene; saturated hydrocarbons such as cyclohexane and hexane; amides such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP); halogenated hydrocarbons such as dichloromethane and carbon tetrachloride; acetonitrile, dimethyl sulfoxide, pyridine, formic acid, acetic acid, trifluoroacetic acid, hydro
• the present disclosure relates to a protein or peptide that includes one or more of the solubilizing groups.
• the protein or peptide of the present disclosure typically has increased solubility in water due to the attachment of the solubilizing groups.
• the site at which the solubilizing group is bonded to the protein or peptide is not particularly limited, but typically, the solubilizing group can be bonded to a sulfhydryl group, a carboxyl group, or an amino group in the side chain of an amino acid that constitutes the protein or peptide.
• the solubilizing group according to the present disclosure is bonded to a sulfhydryl group in the side chain of an amino acid that constitutes the protein or peptide.
• solubilizing groups that the protein or peptide according to the present disclosure has per molecule can be changed as appropriate depending on the size and solubility of the protein or peptide, the desired degree of solubility, and the like, and is not particularly limited, but may be one solubilizing group per 20 to 30 amino acid residues.
• the introduction of the solubilizing group according to the present disclosure into a protein or peptide can be carried out according to methods well known to those skilled in the art.
• the solubilizing group according to the present disclosure can be introduced into a protein or peptide by forming a disulfide bond between a sulfhydryl group of a cysteine in the protein or peptide to be solubilized and the solubilizing group according to the present disclosure.
• the reaction can be carried out in an appropriate solvent, such as, but not limited to, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) or trifluoroethanol.
• HFIP 1,1,1,3,3,3-hexafluoroisopropanol
• a catalyst may be used if necessary, and although not limited to this, for example, trifluoroethanol can be used.
• the present disclosure relates to a method for producing a soluble protein or peptide (also referred to in the present disclosure as the "method for producing a soluble protein, etc., according to the present disclosure”).
• the method for producing a soluble protein, etc., according to the present disclosure includes a step of binding a solubilizing group according to the present disclosure to a sulfhydryl group, a carboxy group, or an amino group in one or more amino acid side chains constituting any protein or peptide (also referred to in the present disclosure as the "binding step").
• the binding step can be carried out in an appropriate solvent, for example, but not limited to, 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP), trifluoroethanol, or the like, by mixing the target protein with the solubilizing group according to the present disclosure.
• HFIP 1,1,1,3,3,3-hexafluoro-isopropanol
• the reaction may be carried out using a catalyst as necessary, for example, but not limited to, trifluoroethanol, or the like.
• the reaction time in the binding step may vary depending on the reagents and solvents used, but may be 1 minute to 48 hours, preferably 10 minutes to 24 hours, for example, 6 hours.
• the reaction temperature may vary depending on the reagents and solvents used, but may be -10°C to 50°C, preferably -10°C to 40°C, for example, room temperature.
• the target into which the soluble group according to the present disclosure is introduced does not necessarily have to be a poorly water-soluble protein.
• the scope of the present disclosure also includes a method in which the soluble group according to the present disclosure is introduced into a soluble protein that has been chemically or genetically prepared as a fusion protein with a water-soluble peptide, and then the water-soluble peptide is removed to obtain the desired soluble protein.
• the present disclosure relates to a method for producing a target protein using a poorly water-soluble protein or peptide (also referred to in the present disclosure as the "method for producing a protein according to the present disclosure”).
• the method for producing a protein according to the present disclosure includes a step of solubilizing the poorly water-soluble protein or peptide by binding a solubilizing group according to the present disclosure to a sulfhydryl group, a carboxyl group, or an amino group in one or more amino acid side chains constituting the protein or peptide (also referred to in the present disclosure as the "solubilization step"), and a step of linking the solubilized protein or peptide to another soluble protein or peptide, or a solubilized protein or peptide, to obtain the target protein (also referred to in the present disclosure as the "linking step").
• the poorly water-soluble protein or peptide to be solubilized in the solubilization step is a poorly water-soluble protein or peptide that constitutes a part of the target protein.
• the poorly water-soluble protein or peptide to be solubilized in the solubilization step may be one type or multiple types.
• the solubilization step can be carried out in a suitable solvent, such as, but not limited to, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), trifluoroethanol, or the like, by mixing the target protein with the solubilizing group according to the present disclosure.
• HFIP 1,1,1,3,3,3-hexafluoroisopropanol
• the reaction may be carried out in the presence of a catalyst, if necessary, such as, but not limited to, trifluoroethanol.
• the reaction time in the solubilization step may vary depending on the reagents and solvents used, but may be 1 minute to 48 hours, preferably 10 minutes to 24 hours, for example 6 hours.
• the reaction temperature may vary depending on the reagents and solvents used, but may be -10°C to 50°C, for example room temperature.
• the linking step is a method of linking the protein or peptide solubilized in the solubilization step with an inherently soluble protein or peptide (also referred to as a "soluble protein or peptide" in this disclosure) or with another poorly water-soluble protein or peptide solubilized by the method of this disclosure or a method other than the method of this disclosure.
• Solubilization by a method other than the method of this disclosure includes, but is not limited to, chemical or genetic preparation as a fusion protein with a water-soluble peptide.
• the linking of the soluble protein or peptide/soluble protein or peptide can be performed using any ligation method known to those skilled in the art.
• the linking step is performed using a native chemical ligation method (NCL method).
• NCL method is a chemoselective reaction between a first peptide having an ⁇ -carboxythioester moiety at the C-terminus and a second peptide having a cysteine residue at the N-terminus (e.g., WO 96/34878), and the reaction conditions and the method for preparing the first and second peptides are also known to those skilled in the art (e.g., WO 2013/047372).
• the linking of the soluble protein or peptide/soluble protein or peptide may be performed via a peptide linker or the like.
• the method for producing a protein according to the present disclosure can further include, after obtaining the target protein, removing and recovering the solubilizing group according to the present disclosure. That is, depending on the configuration of the target protein, it may be possible to maintain solubility by linking it to a water-soluble peptide or protein, even in the absence of the solubilizing group according to the present disclosure. In such a case, it would be extremely beneficial to be able to remove and recover the solubilizing group from the target protein and repeatedly use the glycan-containing solubilizing group, which is difficult and expensive to obtain.
• the method for producing a protein according to the present disclosure further includes a step of removing the solubilizing group from the target protein in the presence of an acid, and a step of recovering the solubilizing group.
• Acids that can be used in the protein production method according to the present disclosure include, but are not limited to, inorganic acids such as hydrochloric acid and sulfuric acid, boron trifluoride diethyl ether (BF3.OEt2), dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST), trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tripropylsilyl trifluoromethanesulfonate, dimethylethylsilyl trifluoromethanesulfonate, tribenzylsilyl trifluoromethanesulfonate, trinaphthylsilyl trifluoromethanesulfonate, tribenzylmethylsilyl trifluoromethane
• the removed solubilizing group can be recovered by a method well known to those skilled in the art.
• the method for recovering the solubilizing group is not particularly limited as long as it is a method capable of separating substances based on molecular weight, charge, or other chemical properties, but it is preferable to use liquid column chromatography.
• first and second may be used to describe various elements, but it is understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another element, and for example, it is possible to refer to a first element as a second element and similarly, to refer to a second element as a first element without departing from the scope of this disclosure.
• the solid obtained was purified by reversed-phase HPLC (Proteonavi column; gradient ratio A:B, 90/10 to 10/90, 60 min, 2.5 ml/min, solvent A: 0.1% TFA in H 2 O, solvent B: 0.1% TFA in 90/10 acetonitrile/H 2 O) to obtain target product 1 as a colorless solid (3.5 mg, 75% yield) (FIG. 1).
• ESI-MS [M-OH+H]+2 calculated 1012.36 found 1012.32
• the SUMO tag protein that solubilizes hydrophobic peptides and the HisTag for affinity purification were removed using SUMO protease.
• Compound 3 (1 mg, 0.56 ⁇ mol) was dissolved in a Tris buffer solution (50 mM Tris-HCl (FUJIFILM Wako Pure Chemical Industries, Ltd.), 75 mM NaCl (FUJIFILM Wako Pure Chemical Industries, Ltd.), 1 mM dithiothreitol (DTT, FUJIFILM Wako Pure Chemical Industries, Ltd.), pH 8.0), and an appropriate amount of SUMO protease was added to proceed with the reaction. The reaction was monitored by LC/MS at 30° C. until completion.
• Example 3 Glycoprotection of interleukin-6 (IL-6) N-terminal fragment (1-141) using glycan-solubilizing groups
• Compound 1 (1 mg, 0.49 ⁇ mol, 16 equivalents relative to the peptide) was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 60 ⁇ L).
• Peptide 5 (0.5 mg, 0.03 ⁇ mol) prepared by E. coli expression was added to this yellow solution and dissolved to initiate the reaction. The reaction was monitored by LC/MS to confirm completion.
• Compound 6 (1.1 mg, 0.05 ⁇ mol) was dissolved in 200 mM phosphate buffer solution, and each mixture was adjusted to the following concentrations (compound 6 : 2.0 mM, 8 M Gu-HCl, 80 mM 4-mercaptophenylacetic acid: MPAA, Fujifilm Wako Pure Chemical Industries, Ltd., 20 mM tris(2-carboxyethyl)phosphine: TCEP, Tokyo Chemical Industry Co., Ltd., pH 6.9).
• Compound 7 (0.5 mg, 0.10 ⁇ mol), which was bound to a sugar chain by the E. coli expression method and the liquid phase method, was dissolved in this solution, and a peptide linking reaction was performed. The reaction was performed at room temperature for 2 days.
• Compound 8 obtained by purification by gel filtration was dissolved in a buffer solution (8 M Gu-HCl, 0.2 M phosphate, 200 mM TCEP, 40 mM glutathione (Sigma-Aldrich (registered trademark)), and 20 mM VA-044 (FUJIFILM Wako Pure Chemical Industries, Ltd.)) and allowed to stand at room temperature to perform desulfurization of thiols. The reaction was monitored by LC/MS.
• a buffer solution 8 M Gu-HCl, 0.2 M phosphate, 200 mM TCEP, 40 mM glutathione (Sigma-Aldrich (registered trademark)), and 20 mM VA-044 (FUJIFILM Wako Pure Chemical Industries, Ltd.)

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