Solubilization group for poorly water-soluble proteins

Abstract

[Problem] To provide a new means that can be used to solubilize poorly water-soluble proteins.　[Solution] Provided is a hydrophilic solubilization group for proteins or peptides, comprising: a sugar chain portion; and a functional group portion that is bonded to a carboxy group or an amino group of the sugar chain, and that binds to an amino acid side-chain and can be released from the protein or amino acid side-chain in the presence of an acid.　

Description

Solubilizing groups for poorly water-soluble proteins

[0001] The present disclosure relates to a solubilizing group for solubilizing a poorly water-soluble protein, and a method for producing a solubilized protein or the like using the solubilizing group.

[0002] There has long been a need for protein solubilization. For example, when using a protein as a pharmaceutical active ingredient, it is preferable that the protein be sufficiently soluble in water in order to improve absorption upon systemic administration (e.g., oral administration), to increase the concentration of the active ingredient in the formulation, and to achieve an appropriate viscosity in the formulation. Furthermore, even at the drug discovery stage, protein solubilization is still required for analyzing protein folding and complexes between folded proteins and disease-causing target molecules. Meanwhile, with the recent advancement and complexity of drug discovery technology, the number of poorly water-soluble candidate molecules is increasing, further increasing the need for solubilization technology.

[0003] One known method for solubilizing poorly water-soluble proteins is to use a hydrophilic tag. For example, Patent Document 1 describes a hydrophilic polylysine tag for solubilizing poorly water-soluble proteins.

[0004] Tsuda et al. , Angew. Chem. Int. Ed. 2018, 57, 2105-2109

[0005] The present disclosure aims to provide novel means that can be used to solubilize poorly water-soluble proteins.

[0006] Glycosylation is known to bind to membrane proteins, secretory proteins, and other proteins in vivo, exerting various effects on their biological activity. However, despite their usefulness, the use of glycosylation has been limited due to the difficulty of obtaining them and their high cost. The present inventors conducted extensive research to solve the above-mentioned problems and discovered that poorly water-soluble proteins can be solubilized by attaching glycosylation-solubilizing groups to them. Furthermore, they discovered that by incorporating specific functional groups into the glycosylation-solubilizing groups, the glycosylation-solubilizing groups used for solubilization can be separated, recovered, and reused after obtaining the desired solubilized protein, thereby completing the present invention.

[0007] That is, the present disclosure includes the following features: (1) A hydrophilic solubilizing group for a protein or peptide, 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, the functional group being bound to an amino group or a carboxy group of the sugar chain.

[0008] (2) The solubilizing group according to (1), which comprises a sugar chain portion and a triphenylmethanol portion bonded to an amino group or a carboxy group of the sugar chain.

[0009] (3) The solubilizing group according to (2), wherein the triphenylmethanol moiety is bonded to a carboxy group of the sugar chain.

[0010] (4) The solubilizing group according to (1), wherein the sugar chain is composed of four or more sugars.

[0011] (5) The solubilizing group according to (1), characterized in that the sugar chain is composed of seven or more sugars.

[0012] (6) The solubilizing group according to (1), characterized in that the sugar chain is composed of 9 or more sugars.

[0013] (7) The solubilizing group according to (1), wherein the sugar chain is a two-antennary complex sugar chain, a three-antennary complex sugar chain, or a four-antennary complex sugar chain.

[0014] (8) The solubilizing group according to (1), wherein the sugar chain is a double-stranded complex-type sugar chain.

[0015] (9) The solubilizing group according to (8), wherein the double-stranded complex-type sugar chain is a sugar chain selected from the group consisting of a disialo sugar chain, a monosialo sugar chain, an asialo sugar chain, a diglucan sugar chain, and a dimannose sugar chain.

[0016] (10) The solubilizing group according to (1), wherein the sugar chain is represented by the following formula: [In the formula, R １ and R ２ are the same or different, wherein Ac represents an acetyl group.

[0017] (11) The solubilizing group according to (1), having the following structure: Solubilizing group.

[0018] (12) A peptide or protein having one or more solubilizing groups according to (1).

[0019] (13) The peptide or protein according to (12), which is a poorly water-soluble peptide or protein.

[0020] (14) The peptide or protein according to (12), which is selected from the group consisting of interleukins and transmembrane proteins, and fragments thereof.

[0021] (15) The peptide or protein according to (14), which is rhodopsin or IL-6, or a fragment thereof.

[0022] (16) A method for producing a solubilized protein or peptide, comprising the step of attaching the solubilizing group according to claim 1 to a sulfhydryl group, a carboxyl group, or an amino group of one or more amino acid side chains constituting any protein or peptide.

[0023] (17) A method for producing a target protein or peptide using a poorly water-soluble protein or peptide, comprising: solubilizing the poorly water-soluble protein or peptide by binding the solubilizing group according to claim 1 to a sulfhydryl group, a carboxy group, or an amino group in the side chain of one or more amino acids constituting 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 the target protein.

[0024] (18) The method according to (17), further comprising the steps of: removing the solubilizing group from the target protein in the presence of an acid; and recovering the solubilizing group.

[0025] According to the present disclosure, a novel means is provided that can be used to solubilize poorly water-soluble proteins and the like.

[0026] FIG. 1 shows the results of HPLC purification of the produced glycan-solubilizing group 1. FIG. 2 shows the reaction progress of glycan-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 glycan-solubilizing group 1 from compound 3 and the results of HPLC purification of the reaction product, peptide 2. FIG. 4 shows the progress of the removal reaction of SUMO-tagged protein from compound 3 and the results of HPLC purification of the reaction products, compound 4 and the SUMO-tagged protein. FIG. 5 shows the reaction progress of glycan-solubilizing group 1 with 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 glycan-solubilizing group 1 from compound 6 and the results of HPLC purification of the reaction products, compound 5 and glycan-solubilizing group 1. Figure 7 shows the progress of the peptide ligation reaction between peptide 6 having glycan-solubilizing group 1 and solubilized peptide 7, and the results of HPLC purification of the reaction product, compound 8. Figure 8 shows the progress of the desulfurization reaction of compound 8, and the results of HPLC purification of the reaction product, compound 9. Figure 9 shows an HPLC chart of compound 10, which is the product of the deprotection reaction of glycan-solubilizing group 1 from compound 9. Figure 10 shows the reaction progress of the folding reaction of compound 10, and the HPLC chart of the reaction product, compound 11.

[0027] The present disclosure relates to a hydrophilic solubilizing group for solubilizing a protein (also referred to herein simply as a "solubilizing group according to the present disclosure").

[0028] In 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. Protein derivatives refer to derivatives of natural proteins, and include, but are not limited to, for example, proteins in which some of the amino acids constituting natural proteins are replaced with non-natural amino acids, natural proteins that have been subjected to chemical or genetic processing, combinations of natural protein fragments, etc.

[0029] In the present disclosure, proteins include any poorly water-soluble proteins that need 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.

[0030] 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 that is bound to an amino group or a carboxy group of the sugar chain.

[0031] In the present disclosure, the functional group moiety may include, but is not limited to, triphenylmethanol, methoxybenzyl, phenacyl, diphenylmethyl, etc. The functional group moiety may further include a carboxyl group, an amino group, a hydroxyl group, etc. for bonding to a sugar chain moiety.

[0032] In the present disclosure, "in the presence of an acid" refers to acidic conditions commonly used for deprotecting a protecting group. Examples of acids that can be used in the present disclosure include, but are not limited to, inorganic acids such as hydrochloric acid and sulfuric acid; Lewis acids such as boron trifluoride diethyl etherate (BF3.OEt2), dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST), trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tripropylsilyl trifluoromethanesulfonate, dimethylethylsilyl trifluoromethanesulfonate, tribenzylsilyl trifluoromethanesulfonate, trinaphthylsilyl trifluoromethanesulfonate, tribenzylmethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate, cyclopentadienyl hafnium chloride, cyclopentadienyl zirconium chloride, and tin chloride; and organic acids such as formic acid, acetic acid, trifluoroacetic acid, trifluoroacetic anhydride, trifluoromethanesulfonic acid, and tetrafluoromethanesulfonic acid. These acids can be used alone or in combination of two or more. The amount of acid used can be adjusted appropriately by those skilled in the art, and it is preferable to use an excess amount of acid relative to the solubilized protein.

[0033] In one aspect of the present disclosure, the solubilizing group comprises a sugar chain moiety and a triphenylmethanol moiety bound to an amino group or a carboxy group of the sugar chain. In a more specific embodiment of the present disclosure, the solubilizing group comprises a triphenylmethanol moiety bound to a carboxy group of the sugar chain.

[0034] In the present disclosure, the term "glycan" refers to a compound formed by linking one or more sugar units (monosaccharides and / or derivatives thereof). When two or more sugar units are linked, the sugar units are bonded to each other by dehydration condensation via a glycosidic bond. Examples of such sugar chains include, but are not limited to, a wide range of sugar chains, 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 sugar chains decomposed or derived from complex biomolecules such as decomposed polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids. The sugar chain may be linear or branched.

[0035] In the present disclosure, the term "sugar chain" also includes derivatives of sugar chains. Examples of sugar chain derivatives include, but are not limited to, sugars constituting the sugar chain having a carboxy group (for example, an aldonic acid oxidized at the C-1 position to become a carboxylic acid (for example, D-gluconic acid obtained by oxidizing D-glucose), and an uronic acid having a carboxylic acid at the terminal C atom (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.), deoxygenated sugars (for example, 2-deoxy-D-ribose), sulfated sugars containing a sulfate group, and phosphorylated sugars containing a phosphate group.

[0036] In the present disclosure, the type of unit sugar constituting the sugar chain 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. In one embodiment, the sugar chain according to the present disclosure contains at least two mannose units.

[0037] The sugar chain in the present disclosure may also be a sugar chain that exists in vivo as a complex carbohydrate (glycopeptide (or glycoprotein), proteoglycan, glycolipid, etc.), or a sugar chain that does not exist in vivo as a complex carbohydrate.

[0038] Glycochains that exist as complex carbohydrates in vivo are preferred, for example, when the solubilizing group according to the present disclosure is attached to a poorly water-soluble protein, solubilizing the protein, and administering it to a living body. Examples of such glycochains include N-linked glycochains and O-linked glycochains, which are glycochains that are bound to peptides (or proteins) in vivo as glycopeptides (or glycoproteins). N-linked glycochains are preferably used. Examples of N-linked glycochains include high-mannose, complex, and hybrid types, with complex types being preferred.

[0039] In one embodiment, the complex sugar chain used in the present disclosure may be, for example, a sugar chain represented by the following general formula: [In the formula, R １ and R ２ are the same or different, where Ac represents an acetyl group.]

[0040] In one aspect of the present disclosure, the number of sugar chains is not limited as long as they contain enough sugars to impart sufficient solubility to the poorly water-soluble protein, but it is preferable that the sugar chain is composed of 4 or more sugars, for example, 5 or more, 7 or more, 9 or more, or 11 or more sugars.

[0041] In another embodiment of the present disclosure, the sugar chain is a sugar chain consisting of 4 to 11, 7 to 11, for example, 9 sugars.

[0042] In one embodiment of the present disclosure, the sugar chain is a biantennary complex sugar chain. A complex sugar chain is characterized by containing two or more types of monosaccharides and having the basic structure shown below and a lactosamine structure represented by Galβ1-4GlcNAc.

[0043] A double-antennary complex-type sugar chain is one in which a single sugar chain consisting of 0 to 3 sugars is bound to each of the two terminal mannoses of the basic structure. Examples of double-antennary complex-type sugar chains include the disialo sugar chain shown below: Monosialoglycan, Asialoglycan, Digluconate sugar chain, Dimannose sugar chain, In one embodiment of the present disclosure, the biantennary complex-type sugar chain is an asialo sugar chain.

[0044] Furthermore, the complex-type sugar chains of the present disclosure include not only biantennary complex-type sugar chains but also triantennary complex-type sugar chains (triantennary complex-type sugar chains) and quadrantennary complex-type sugar chains (tetraantennary complex-type sugar chains). For example, triantennary and quadrantennary complex-type sugar chains include trisialo-sugar chains represented by the following structural formula: Tetrasialoglycan represented by the following structural formula Furthermore, the triple-antennary complex-type glycans and quadruple-antennary complex-type glycans also include glycans in which one or more sugars have been lost from the non-reducing end of these trisialo- or tetrasialo-glycans.

[0045] Furthermore, the complex-type sugar chains of the present disclosure also include those to which fucose is attached. The complex-type sugar chains to which fucose is attached include the fucose-containing complex-type sugar chains represented by the following structural formula: Further examples include sugar chains that have lost one or more sugars from the non-reducing end of these fucose-containing complex sugar chains.

[0046] Furthermore, in the present disclosure, "disialoglycan," "monosialoglycan," "asialoglycan," "diglucunacglycan," "dimannoseglycan," "bi-antennary complex-type glycan," "tri-antennary complex-type glycan," "quadruple-antennary complex-type glycan," and "fucose-containing complex-type glycan" include those represented by the above chemical formulas as well as those with a different bond type from the examples represented by the chemical formulas, and such glycans are also included in the glycans of the present disclosure. Examples of such glycans include disialoglycan or monosialoglycan in which sialic acid and galactose are linked by an (α2→3) bond.

[0047] Furthermore, in the case of a sugar chain having sialic acid at the non-reducing end of the sugar chain, 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 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 thereof include alkylamino groups having 1 to 30 carbon atoms, benzyl groups, amino groups, aminoethylamino groups, etc. By introducing these modifying groups, the negative charge of the carboxy group of sialic acid can be eliminated (benzyl group, amino group, etc.) or converted to a positive charge (aminoethylamino group, etc.), thereby making it possible to intend to control the blood clearance or biodistribution of the glycosylated polypeptide.

[0048] Furthermore, 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. Because high-mannose glycans are bulky, binding a high-mannose glycan to a peptide can increase stability in the blood. While a glycan containing 5 to 9 mannoses, like mammalian high-mannose glycans, may also be a glycan containing more mannoses, like yeast high-mannose glycans. Examples of high-mannose glycans preferably used in the present disclosure include high mannose-5 (M-5) High mannose-9 (M-9) The following can be mentioned:

[0049] In the present disclosure, preferred sugar chains include, for example, sugar chains that have the same structure (sugar chains with the same types of constituent sugars and their bonding mode) as sugar chains that exist in the human body as glycoproteins bound to proteins (for example, sugar chains described in "FEBS LETTERS Vol. 50, No. 3, February 1975"), or sugar chains that have lost one or more sugars from the non-reducing end thereof, such as the sugar chains described below.

[0050]

[0051]

[0052]

[0053]

[0054] In one embodiment of the present disclosure, the solubilizing group according to the present disclosure has the following structure:

[0055] The solubilizing group according to the present disclosure can be produced according to methods well known to those skilled in the art. For example, it can be produced by dehydration condensation between a reactive group (e.g., an amino group) of a glycosylated amino acid having a desired sugar chain (e.g., glycosylated asparagine, glycosylated cysteine) and a reactive group (e.g., a carboxy group) of a functional group moiety according to the present disclosure. In an exemplary embodiment, for example, 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 the functional group moiety. The reaction time varies depending on the reagents and solvent used, but can be 1 minute to 48 hours, preferably 10 minutes to 24 hours, for example, 6 hours. The reaction temperature varies depending on the reagents and solvent used, but can be −78°C to 300°C, preferably −78°C to 150°C, for example, room temperature.

[0056] Reagents capable of activating the carboxy group of the functional group moiety include, but are not limited to, carbodiimide condensing agents such as 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD), triazine 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. One type of the above-mentioned reagent may be used, or multiple types may be used in combination.

[0057] 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-hexafluoroisopropanol (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, hydrochloric acid, sulfuric acid, ethyl acetate, acetone, methyl ethyl ketone, guanidine, and acetonitrile mixed with an aqueous urea solution. One of the above solvents may be used alone, or multiple solvents may be used in combination.

[0058] In another aspect, the present disclosure relates to a protein or peptide comprising one or more of the solubilizing groups, which typically have increased aqueous solubility due to the attached solubilizing groups.

[0059] In the present disclosure, the site at which the solubilizing group is attached to a protein or peptide is not particularly limited, but typically, the solubilizing group can be attached to a sulfhydryl group, carboxyl group, or amino group in the side chain of an amino acid that constitutes the protein or peptide. In one embodiment, the solubilizing group according to the present disclosure is attached to a sulfhydryl group in the side chain of an amino acid that constitutes the protein or peptide.

[0060] Furthermore, the number of solubilizing groups that a protein or peptide according to the present disclosure has per molecule can be appropriately changed 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 it is sufficient to use one solubilizing group per 20 to 30 amino acid residues.

[0061] In the present disclosure, 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. For example, 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.

[0062] In the above reaction, a catalyst may be used as needed, and although not limited thereto, for example, trifluoroethanol or the like can be used.

[0063] In another aspect, the present disclosure relates to a method for producing a solubilized protein or peptide (also referred to in the present disclosure as the "method for producing a solubilized protein, etc. according to the present disclosure"). The method for producing a solubilized protein, etc. according to the present disclosure comprises a step of binding a solubilizing group according to the present disclosure to a sulfhydryl group, a carboxy group, or an amino group in the side chain of one or more amino acids constituting any protein or peptide (also referred to in the present disclosure as the "binding step").

[0064] In the method for producing a solubilized protein or the like according to the present disclosure, the binding step can be carried out in an appropriate solvent, for example, but not limited to, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), trifluoroethanol, etc., by mixing the target protein with the solubilizing group according to the present disclosure. This reaction may be carried out in the presence of a catalyst, if necessary, for example, but not limited to, trifluoroethanol.

[0065] The reaction time in the binding step may vary depending on the reagents and solvent 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 solvent used, but may be −10° C. to 50° C., preferably −10° C. to 40° C., for example, room temperature.

[0066] In the method for producing a solubilized protein or the like according to the present disclosure, the target to which the solubilizing group according to the present disclosure is introduced does not necessarily have to be a poorly water-soluble protein. For example, a method in which a solubilizing 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 solubilized protein is also within the scope of the present disclosure.

[0067] In another aspect, the present disclosure relates to a method for producing a protein of interest using a poorly water-soluble protein or peptide (also referred to in the present disclosure as a "protein production method according to the present disclosure"). Specifically, the protein production method according to the present disclosure includes a step of solubilizing a 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 the side chain of one or more amino acids constituting the protein or peptide (also referred to in the present disclosure as a "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 a protein of interest (also referred to in the present disclosure as a "linking step").

[0068] 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 protein of interest. In addition, the poorly water-soluble protein or peptide to be solubilized in the solubilization step may be one type or multiple types.

[0069] The solubilization step can be carried out in an appropriate solvent, such as, but not limited to, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), trifluoroethanol, etc., by mixing the target protein with the solubilizing group of the present disclosure. The reaction may be carried out in the presence of a catalyst, if necessary, such as, but not limited to, trifluoroethanol.

[0070] The reaction time in the solubilization step may vary depending on the reagents and solvent 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 solvent used, but may be −10° C. to 50° C., for example, room temperature.

[0071] 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 the present disclosure) or another poorly water-soluble protein or peptide solubilized by the method of the present disclosure or a method other than the method of the present disclosure. Solubilization by a method other than the method of the present disclosure includes, but is not limited to, chemical or genetic preparation of a fusion protein with a water-soluble peptide.

[0072] Linking of a soluble protein or peptide to a soluble protein or peptide can be performed using any ligation method known to those skilled in the art. In one embodiment of the present disclosure, the linking step is performed using native chemical ligation (NCL). The NCL method is a chemoselective reaction between a first peptide having an α-carboxythioester moiety at its C-terminus and a second peptide having a cysteine ​​residue at its N-terminus (e.g., International Publication No. 96 / 34878). The reaction conditions and methods for preparing the first and second peptides are also known to those skilled in the art (e.g., International Publication No. 2013 / 047372). Alternatively, linking of a soluble protein or peptide to a soluble protein or peptide may be performed via a peptide linker or the like.

[0073] In one embodiment of the present disclosure, 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 structure of the target protein, solubility may be maintained by linking it to a water-soluble peptide or protein even without the solubilizing group according to the present disclosure. In such cases, it would be extremely beneficial to be able to remove and recover the solubilizing group from the target protein and repeatedly use the sugar chain-containing solubilizing group, which is difficult and expensive to obtain.

[0074] Specifically, in one embodiment, the method for producing a protein according to the present disclosure further comprises the steps of removing the solubilizing group from the target protein in the presence of an acid, and recovering the solubilizing group.

[0075] In the protein production method according to the present disclosure, "in the presence of an acid" refers to acidic conditions that are generally used for deprotection of a protecting group. Acids that can be used in the protein production methods of the present disclosure include, but are not limited to, inorganic acids such as hydrochloric acid and sulfuric acid; Lewis acids such as boron trifluoride diethyl etherate (BF3.OEt2), dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST), trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tripropylsilyl trifluoromethanesulfonate, dimethylethylsilyl trifluoromethanesulfonate, tribenzylsilyl trifluoromethanesulfonate, trinaphthylsilyl trifluoromethanesulfonate, tribenzylmethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate, cyclopentadienyl hafnium chloride, cyclopentadienyl zirconium chloride, and tin chloride; and organic acids such as formic acid, acetic acid, trifluoroacetic acid, trifluoroacetic anhydride, trifluoromethanesulfonic acid, and tetrafluoromethanesulfonic acid. These acids can be used alone or in combination of two or more. Furthermore, those skilled in the art can determine the appropriate amount of acid to be used, and it is preferable to use an excess amount of acid relative to the solubilized protein.

[0076] In the protein production method according to the present disclosure, the removed solubilizing group can be recovered by a method known to those skilled in the art. For example, the recovery of the solubilizing group is not particularly limited as long as it is a method that can separate substances based on molecular weight, charge, or other chemical properties, but it is preferable to use liquid column chromatography.

[0077] It should be noted that the terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0078] Furthermore, the term "comprise" used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0079] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0080] Although terms such as "first" and "second" may be used to describe various elements, it is understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another, and for example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the present disclosure.

[0081] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure can be embodied in various forms and should not be construed as being limited to the examples set forth herein.

[0082] Example 1: Preparation of sugar chain solubilizing groups

[0083] 4-(Diphenylhydroxymethyl)benzoic acid (4.5 mg, 15 μmol, 6 equivalents; Sigma-Aldrich®) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (3.8 mg, 10 μmol, 4 equivalents; Novabiochem®) were dissolved in dry DMF (150 μL; Kanto Chemical Co., Inc.), followed by the addition of triethylamine (1.4 μL, 10 μmol, 4 equivalents; Nacalai Tesque®). This mixture was stirred at room temperature for 10 minutes to activate 4-(diphenylhydroxymethyl)benzoic acid. H ２N-Asn(asialo-N-glycan)-COOH (4.4 mg, 2.5 μmol, 1 equivalent) dissolved in dry DMF:DMSO (7:3, 250 μL) was added and reacted at room temperature. The reaction status was monitored by LC / MS to confirm completion. After the reaction was completed, chilled diethyl ether was added to the solution to precipitate the product, and the residue, the target product, was isolated by centrifugation. The obtained solid was analyzed by reversed-phase HPLC (Proteonavi column; gradient ratio A:B, 90 / 10 to 10 / 90, 60 minutes, 2.5 ml / min, solvent A: 0.1% TFA (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0 mL) to give the target compound 1 as a colorless solid (3.5 mg, 75% yield) (Figure 1). ESI-MS [M-OH+H]+2 calculated 1012.36 found 1012.32

[0084] Example 2: Glycoprotection using model peptides

[0085] Compound 1 (1.4 mg, 0.686 μmol, 8 equivalents relative to the peptide) was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 180 μL; Tokyo Chemical Industry Co., Ltd.). Peptide 2 (1.2 mg, 0.09 μmol), which had been prepared by E. coli expression, was added to this yellow solution to initiate the reaction. The reaction status was monitored by LC / MS, and completion was confirmed. After completion of the reaction, chilled diethyl ether was added to this solution to precipitate the product. The residue, the target product, was isolated by centrifugation and then subjected to reverse-phase HPLC (Proteonavi column; gradient ratio A:B, 90 / 10 to 30 / 70, 60 minutes, 2.5 ml / min, solvent A: 0.1% TFA (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0 mL) to give the target compound 3 as a colorless solid (1.0 mg, 80% yield) (Figure 2). ESI-MS [M+1H]+1 calculated 18051.50 found 18051.73

[0086] Deprotection of sugar chain solubilizing groups-1

[0087] Compound 3 (1 mg, 0.56 μmol) was dissolved in 5% tetraisopropylsilane (TIPS; Tokyo Chemical Industry Co., Ltd.) / TFA (500 μL; Fujifilm Wako Pure Chemical Industries, Ltd.) and reacted at room temperature for 10 minutes. After the reaction was completed, the TFA was evaporated, and cooled diethyl ether was added to the remaining TFA solution to precipitate the product. The residue, which was the target product, was isolated by centrifugation and then analyzed by reverse-phase HPLC (Proteonavi column; gradient ratio A:B, 90 / 10 to 30 / 70, 60 minutes, 2.5 ml / min, solvent A: 0.1% TFA (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0 mL) to give the target compound 2 as a colorless solid (0.7 mg, 90% yield) (Figure 3).

[0088] Enzymatic removal of SUMO-tagged proteins

[0089] SUMO protease was used to remove the SUMO-tagged protein that solubilizes the hydrophobic peptide and the HisTag used for affinity purification. Compound 3 (1 mg, 0.56 μmol) was dissolved in 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 allow the reaction to proceed. The reaction was monitored by LC / MS at 30°C until completion. After completion of the reaction, the solution was directly subjected to reverse-phase HPLC (Proteonavi column; gradient ratio A:B, 90 / 10 to 30 / 70, 60 minutes, 2.5 ml / min, solvent A: 0.1% TFA (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0) to give the target compound 2 as a colorless solid (0.3 mg, 90% yield) (FIG. 4). ESI-MS [M+H]+1 calculated 5311.04, found 5311.70

[0090] Example 3: Glycoprotection of interleukin-6 (IL-6) N-terminal fragment (1-141) using sugar chain solubilizing groups Compound 1 (1 mg, 0.49 μmol, 16 equivalents relative to the peptide) was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 60 μL). Peptide 5 (0.5 mg, 0.03 μmol), which had been prepared by E. coli expression, was added to this yellow solution and dissolved, initiating the reaction. The reaction status was monitored by LC / MS, and completion was confirmed. After completion of the reaction, chilled diethyl ether was added to this solution to precipitate the product, and the residue, the target product, was isolated by centrifugation and then analyzed by reverse-phase HPLC (Proteonavi column; gradient ratio A:B, 70 / 30 to 20 / 80, 30 minutes, 1.0 ml / min, solvent A: 0.1% TFA (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0) to give the target compound 6 as a colorless solid (0.4 mg, 80% yield) (FIG. 5). ESI-MS [M+H]+1 calculated 24088.8770, found 24087.0751

[0091] Deprotection of sugar chain solubilizing groups-2

[0092] Compound 6 (0.4 mg, 0.02 μmol, 1 equivalent) was dissolved in 0.1 M HCl / HFIP solution (25 μL) to remove the sugar chain solubilizing groups. The reaction was monitored by LC / MS. After the reaction was completed, Tris buffer solution (6 M Gn-HCl (Fujifilm Wako Pure Chemical Industries, Ltd.), 50 mM Tris-HCl, pH 8.0, 500 μL) was added, and the resulting solution was directly analyzed 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 (H ２ in 0), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The sugar chain-solubilizing group recovered was confirmed to have the structure of 1.

[0093] Example 4: Synthesis of hydrophobic glycoprotein IL6 using native chemical ligation and sugar chain solubilizing groups

[0094] Compound 6 (1.1 mg, 0.05 μmol) was dissolved in 200 mM phosphate buffer solution, and the 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 μmol), which had been glycosylated using E. coli expression and liquid-phase methods, was dissolved in this solution, and a peptide ligation reaction was carried out. The reaction was carried out at room temperature for two days. TCEP was added to the reaction solution and the progress was monitored by LC / MS. After completion of the reaction, the product was purified using an HPLC gel filtration column (column: TSKgel SuperSW3000, 4.6 mm x 30 cm, 4 μm). The mobile phase was 0.1 M phosphoric acid solution containing 6 M Gu-HCl (pH 6.7, 0.2 ml / min, 35 min), and 8 was obtained (Figure 7). ESI-MS [M+H]+1 calculated 30596.0850, found 30594.6350. The product was directly used in the subsequent desulfurization experiment.

[0095] Desulfurization of thiols in the presence of sugar chain solubilizing groups

[0096] 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 left at room temperature to carry out desulfurization of the thiol. The reaction was monitored by LC / MS, and after completion of the reaction, the compound was directly analyzed by reversed-phase HPLC (Proteonavi column; gradient ratio A:B, 70 / 30 to 10 / 90, 60 minutes, 1.0 ml / min, solvent A: 0.1% TFA (H ２ O), Solvent B: 0.1% TFA (90 / 10 acetonitrile / H ２ The product was purified by HPLC (in 0) to give the target compound 9 as a colorless solid (0.9 mg, 60% yield over two steps) (FIG. 8). ESI-MS [M+H]+1 calculated 30531.9650, found 30529.8548

[0097] Deprotection of sugar chain solubilizing groups using IL-6

[0098] Compound 9 (0.9 mg, 0.03 μmol) was dissolved in HFIP, and HCl solution was added to make the solution 100 mM. This solution was left at room temperature, and it was confirmed by LC / MS that the sugar chain solubilizing groups had been removed ( FIG. 9 ), and the protein folding procedure was then carried out.

[0099] Removal of sugar chain solubilizing groups and folding of IL-6 To the HFIP solution containing compound 10, 50 mM Tris buffer solution (final pH 7.3-7.5) containing 6 M Gu-HCl and 1 mM cysteamine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and left at room temperature. The folding reaction was monitored by LC / MS. After folding, the mixture was dialyzed against 50 mM Tris buffer solution (pH = 8) to obtain folded IL-6 11 (Figure 10). ESI-MS [M+H]+1 calculated 22432.2730, found 22430.8209

Claims

1. A hydrophilic solubilizing group for a protein or peptide, comprising a glycan portion and a functional portion bound to an amino or carboxy group of the glycan, the functional portion being bound to an amino acid side chain and capable of being detached from the protein or amino acid side chain in the presence of an acid.

2. The solubilizing group according to claim 1, comprising a sugar chain portion and a triphenylmethanol portion bonded to an amino group or a carboxy group of the sugar chain.

3. The solubilizing group according to claim 2, wherein the triphenylmethanol moiety is bonded to a carboxy group of the sugar chain.

4. The solubilizing group according to claim 1, characterized in that the sugar chain is composed of four or more sugars.

5. The solubilizing group according to claim 1, characterized in that the sugar chain is composed of 7 or more sugar units.

6. The solubilizing group according to claim 1, characterized in that the sugar chain is composed of 9 or more sugar units.

7. The solubilizing group according to claim 1, characterized in that the glycan is a two-antennary complex-type glycan, a three-antennary complex-type glycan, or a four-antennary complex-type glycan.

8. The solubilizing group according to claim 1, characterized in that the sugar chain is a double-stranded complex-type sugar chain.

9. The solubilizing group according to claim 8, characterized in that the double-stranded complex-type glycan is a glycan selected from the group consisting of a disialo-glycan, a monosialo-glycan, an asialo-glycan, a diglucunac-glycan, and a dimannose-glycan.

10. The solubilizing group according to claim 1, wherein the sugar chain is represented by the following formula: [In the formula, R １ and R ２ are the same or different, and Ac represents an acetyl group.

11. The solubilizing group of claim 1, having the following structure: Solubilizing group.

12. A peptide or protein having one or more solubilizing groups according to claim 1.

13. The peptide or protein according to claim 12, which is a poorly water-soluble peptide or protein.

14. A peptide or protein according to claim 12, selected from the group consisting of interleukins and transmembrane proteins, and fragments thereof.

15. A peptide or protein according to claim 14, which is rhodopsin or IL-6, or a fragment thereof.

16. A method for producing a solubilized protein or peptide, comprising the step of attaching the solubilizing group according to claim 1 to a sulfhydryl group, a carboxyl group or an amino group of one or more amino acid side chains constituting any protein or peptide.

17. A method for producing a target protein or peptide using a poorly water-soluble protein or peptide, comprising the steps of: binding the solubilizing group described in claim 1 to a sulfhydryl group, a carboxy group or an amino group in one or more amino acid side chains constituting the poorly water-soluble protein or peptide to solubilize it; and linking the solubilized protein or peptide to another soluble protein or peptide, or a solubilized protein or peptide, to obtain the target protein.

18. The method according to claim 17, further comprising the steps of: removing the solubilizing group from the target protein in the presence of an acid; and recovering the solubilizing group.