WO2014084110A1 - 糖鎖付加リンカー、糖鎖付加リンカーと生理活性物質とを含む化合物またはその塩、及びそれらの製造方法 - Google Patents
糖鎖付加リンカー、糖鎖付加リンカーと生理活性物質とを含む化合物またはその塩、及びそれらの製造方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- 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
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
Definitions
- the present invention relates to a sugar chain addition linker, a compound containing a sugar chain addition linker and a physiologically active substance or a salt thereof, and a method for producing them.
- physiologically active substances are, for example, unable to perform (sufficient) filter sterilization due to low water solubility.
- physiologically active substances are, for example, unable to perform (sufficient) filter sterilization due to low water solubility.
- carrier-drug conjugates such as physiologically active substances
- drug derivatives a highly water-soluble carrier (carrier) is artificially added directly to a drug.
- carrier a hydrophilic amino acid sequence or polyethylene glycol (PEG) is known.
- the three-dimensional structure is different from the original drug.
- the drug derivative exhibits different pharmacokinetic, immunogenic, toxicological or pharmacological properties compared to the original drug molecule.
- the antigenicity of the drug derivative is usually reduced as compared to the original drug molecule.
- a drug to which PEG is added as a carrier has biodegradation resistance. Therefore, if the PEGylated drug is continuously administered in vivo, it accumulates in the living body and there is a risk of causing phytotoxicity to the living body, and it is difficult to say that biocompatibility is sufficiently established (patent document). 1). Furthermore, PEG has a molecular weight distribution (polydisperse nature). When a drug is PEGylated, since the attachment position or molecular weight of the added PEG is different, many monomeric isoforms (proteins having different structures but the same function) are generated. These generated isoforms may compete with each other for binding of a drug to a receptor molecule (Non-patent Document 1).
- a carrier linker-drug conjugate in which a drug and a carrier are bonded via a linker moiety has also been developed. They can be designed such that when acting at a target location (such as in the blood), the bond between the carrier linker moiety and the drug is cleaved and the drug itself is released.
- a carrier linker-drug conjugate When such a carrier linker-drug conjugate is used, light or enzymatic cleavage has been used as a trigger for cleaving the bond between the carrier linker moiety and the drug.
- light it is difficult to irradiate the target site with light, and there is a concern about damage to the living body.
- enzymatic cleavage it is known that the amount of enzyme varies greatly not only between individuals but also at the administration site. Therefore, there arises a problem that variation in the effect of drug treatment occurs between patients.
- Patent Document 2 a carrier linker-drug conjugate in which a carrier linker moiety is bonded to a physiologically active substance moiety via an amide group has been reported (Patent Document 2).
- the technique disclosed in Patent Document 2 utilizes autohydrolysis by intramolecular catalysis in the carrier linker moiety so that the bond breakage between the carrier linker moiety and the drug can be controlled.
- the mechanism of cleavage of the bond between the carrier linker moiety and the bioactive substance moiety is based on cyclization-activation by cyclic imide formation for amide bond cleavage.
- the present invention is to provide a carrier linker that can improve the water solubility of a physiologically active substance and that can release the physiologically active substance faster under specific conditions. .
- Patent Document 2 merely confirms that a large number of carrier linker-drug conjugates having various structures release the drug itself by cleaving the amide bond. Since there are many examples using PEG as a carrier, Patent Document 1 does not pay attention to the biodegradability of the carrier. Furthermore, since there are many examples using higher fatty acids that are hardly soluble in water as the carrier, Patent Document 1 does not mention the solubility of the carrier linker-drug conjugate or the carrier linker itself.
- the present invention in one aspect thereof, is a glycosylated linker for binding to a physiologically active substance having at least one carboxy group
- the sugar chain addition linker is represented by the following formula (A): XR 1 -YR 2 (A) [In the formula (A), X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S); R 1 is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, substituted or unsubstituted C 2 -C 5 alkenyl, substituted or unsubstituted C 2 -
- R 5- means where R 3 and R 5 are substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl, R 4 is a substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, or a sulfur atom (S); Y may or may not be present.
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 means —R 6 -R 7 , where R 6 is a sugar chain, a sugar chain An added amino acid or a glycosylated polypeptide, wherein R 7 is a hydrogen atom (H), —NH 2 , substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, Substituted or unsubstituted C 5 -C 16 heteroaryl, nucleic acid, or PEG.
- the sugar chain addition linker can be bonded to the carboxy group of the physiologically active substance by elimination of a leaving group at the oxygen atom (O) or sulfur atom (S).
- the sugar chain addition linker is represented by the following formula (A): a sugar chain addition linker XR 1 -YR 2 (A) [In the formula (A), X means a sulfur atom (S) having a leaving group, R 1 is, -R 3 -R 4 -, - R 4 -R 5 -, or -R 3 -R 4 -R 5 - means, wherein, R 3 and R 5 is a substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl, wherein R 4 is substituted or unsubstituted C 5 -C 16 aryl Substituted or unsubstituted C 5 to C 16 heteroaryl, or a sulfur atom (S), Y may or may not be present.
- X means a sulfur atom (S) having a leaving group
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide. ] It is characterized by being.
- the sugar chain addition linker is represented by the following formula (A): a sugar chain addition linker XR 1 -YR 2 (A) [In the formula (A), X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S); R 1 means —R 3 —R 4 —, or —R 4 —R 5 —, wherein R 3 and R 5 are substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl, and R 4 is substituted or unsubstituted C 5 -C 16 aryl, or substituted or unsubstituted C 5 -C 16 heteroaryl, Y may or may not be present.
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide. ] It is characterized by being.
- the glycosylation linker is the R 2 or R 6 “glycosylated amino acid or glycosylated polypeptide”, and the sugar chain is an amino acid or It is characterized by binding to Asn or Cys in a polypeptide.
- the sugar chain addition linker is a sugar chain in the “sugar chain, glycosylated amino acid, or glycosylated polypeptide” of R 2 or R 6 . It consists of four or more sugar residues.
- the sugar chain addition linker is a sugar chain in the “sugar chain, glycosylated amino acid, or glycosylated polypeptide” in R 2 or R 6 . It is a double-chain complex sugar chain, a three-chain complex sugar chain, or a four-chain complex sugar chain.
- the sugar chain addition linker is a group in which the sugar chain is composed of a dicialo sugar chain, a monosialo sugar chain, an asialo sugar chain, a diglucnac sugar chain, and a dimannose sugar chain. It is a selected double-stranded complex type sugar chain.
- the sugar chain addition linker is a sugar chain in the “sugar chain, glycosylated amino acid, or glycosylated polypeptide” of R 2 or R 6 .
- R 10 and R 11 are the same or different, Indicates. Ac represents an acetyl group. ] It is the sugar chain represented by these.
- the glycosylated linker is the above-mentioned “glycosylated amino acid or glycosylated polypeptide”, wherein the sugar chain is an amino acid or polypeptide and a linker. It is characterized by being connected without intervening.
- any combination of one or more features of the present invention described above is also a glycosylated linker of the present invention.
- a compound comprising a sugar chain addition linker moiety derived from the sugar chain addition linker and a physiologically active substance moiety or a salt thereof,
- the physiologically active substance has at least one carboxy group;
- the glycosylated linker moiety forms an ester bond or a thioester bond with the carboxy group of the physiologically active substance moiety by elimination of the leaving group at the oxygen atom (O) or sulfur atom (S), Bound to the physiologically active substance moiety,
- O oxygen atom
- S sulfur atom
- the compound or salt thereof is characterized in that the physiologically active substance is a low molecular weight physiologically active substance or a biopolymer.
- the compound or salt thereof is characterized in that the biopolymer is selected from the group consisting of a protein, a polypeptide, a polynucleotide, and a peptide nucleic acid. To do.
- the compound or the salt thereof is characterized by having improved water solubility as compared with an unmodified physiologically active substance.
- the compound or a salt thereof has an improved water solubility in a molar concentration of 10 to 1 compared with the “unmodified physiologically active substance”. 1,000,000 times.
- the compound or a salt thereof includes the oxygen atom (O) or sulfur atom (S) in the sugar chain addition linker moiety and the carboxy group in the physiologically active substance moiety.
- an arbitrary combination of one or more features of the present invention described above is a compound or a salt thereof containing the glycosylated linker moiety of the present invention and a physiologically active substance moiety.
- composition comprising the compound or a salt thereof, wherein the sugar chains in the compound or the salt thereof are substantially uniform.
- a pharmaceutical composition comprising: A pharmaceutical composition comprising (I) the compound according to claim 10 or a salt thereof, and (II) a pharmacologically acceptable carrier is provided.
- the pharmaceutical composition is characterized in that the physiologically active substance exhibits activity after administration to a subject.
- the pharmaceutical composition is used for vaccination.
- any combination of one or more features of the present invention described above is also a pharmaceutical composition of the present invention.
- the sugar chain addition linker is represented by the following formula (A): XR 1 -YR 2 (A)
- X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S);
- R 1 is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, substituted or unsubstituted C 2 -C 5 alkenyl, substituted or unsubstituted C 2 -C 5 alkynyl, or R 1 is —R 3 —R 4 —, —R 4 —R 5 —, or —R 3 —R 4 —.
- R 5- means where R 3 and R 5 are substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl, R 4 is a substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, or a sulfur atom (S); Y may or may not be present.
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 means —R 6 -R 7 , where R 6 is a sugar chain, a sugar chain An added amino acid or glycosylated polypeptide, wherein R 7 is a hydrogen atom (H), substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted Of C 5 -C 16 heteroaryl, nucleic acid, or PEG.
- H hydrogen atom
- the physiologically active substance has at least one carboxy group;
- the method comprises the following steps: (A) An ester bond or a thioester bond is formed between the oxygen atom (O) or sulfur atom (S) having a leaving group in the sugar chain addition linker and the carboxy group of the physiologically active substance. Including conducting a condensation reaction, A manufacturing method is provided.
- the production method includes the step of the condensation reaction in which the sugar chain addition linker is fixed. It is characterized in that it is carried out in a state bound to a resin for phase synthesis (provided that the glycosylated linker has a glycosylated amino acid or a glycosylated polypeptide).
- the production method comprises (a ′) the following formula: Step of preparing a glycosylated linker represented by (A) XR 1 -YR 2 (A) [In the formula (A), X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S); R 1 is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, substituted or unsubstituted C 2 -C 5 alkenyl, substituted or unsubstituted C 2 -C 5 alkynyl, or R 1 is —R 3 —R 4 —, —R 4 —R 5 —, or —R 3
- R 5- means where R 3 and R 5 are substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl, R 4 is a substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, or a sulfur atom (S); Y may or may not be present.
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 means —R 6 -R 7 , where R 6 is a sugar chain, a sugar chain An added amino acid or glycosylated polypeptide, wherein R 7 is a hydrogen atom (H), substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted Of C 5 -C 16 heteroaryl, nucleic acid, or PEG. ] Is further included.
- the production method comprises: The step (a ′) and / or the step (a) is performed on a resin.
- the production method comprises:
- the physiologically active substance has at least one carboxy group;
- the method comprises the following steps: (A) a step of binding a linker represented by the following formula (B) to a resin,
- the linker is represented by the following formula (B): XR 1 -YR 2 (B) [In the formula (B) X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S);
- R 1 is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, substituted or unsubstituted C 2 -C 5 alkenyl, substituted or unsubstituted C 2 -C 5 alkynyl,
- R 5- means R 3 and R 5 are substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 alkenyl, or substituted or unsubstituted C 2 -C 5 Alkynyl, R 4 is a substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, or sulfur atom (S); Y may or may not be present.
- R 2 is an amino acid or a polypeptide
- R 2 means —R 6 —R 7 , wherein R 6 is an amino acid or a polypeptide, and R 7 is a hydrogen atom (H), —NH 2 , substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, nucleic acid, or PEG is there.
- O oxygen atom
- S sulfur atom
- a compound or a salt thereof obtainable by a method for producing a compound or a salt thereof comprising the above-mentioned sugar chain addition linker moiety and a physiologically active substance moiety.
- an arbitrary combination of one or more features of the present invention described above is also a method for producing the compound of the present invention or a salt thereof.
- the sugar chain-added linker according to the present invention has a sugar chain structure portion having many hydroxyl groups and high polarity, the water solubility of the physiologically active substance can be improved by binding to the physiologically active substance.
- the glycosylated linker according to the present invention can release a physiologically active substance bound to the glycosylated linker under specific conditions (for example, in vivo) without depending on light or enzymatic cleavage. it can.
- the compound or a salt thereof containing the sugar chain addition linker moiety and the physiologically active substance moiety according to the present invention is water-soluble.
- sugar chain in the sugar chain addition linker portion according to the present invention is advantageous because it has biodegradable properties.
- the sugar chain in the sugar chain addition linker portion according to the present invention is advantageous for reducing the antigenicity of the physiologically active substance.
- FIG. 1A and FIG. 1B are graphs showing the results of hydrolysis tests of a thioalkyl-type glycosylation linker-HER2 (8-16) conjugate (Compound 1) according to one embodiment of the present invention. This graph plots the relative concentration of starting material against incubation time.
- 2A and 2B are graphs showing the results of hydrolysis tests of a thioaryl-type glycosylation linker-HER2 (8-16) conjugate (Compound 2) according to one embodiment of the present invention. This graph plots the relative concentration of starting material against incubation time.
- FIG. 1A and FIG. 1B are graphs showing the results of hydrolysis tests of a thioalkyl-type glycosylation linker-HER2 (8-16) conjugate (Compound 1) according to one embodiment of the present invention. This graph plots the relative concentration of starting material against incubation time.
- Compound 2A and 2B are graphs showing the results of hydrolysis tests of a thioaryl-type glycosylation linker-
- FIG. 3 shows a thioalkyl type glycosylation linker-HER2 (8-16) conjugate (compound 1) having a thioester bond and a thioaryl type glycosylation linker-HER2 (8-16) conjugate having a thioester bond
- compound 2 is a graph showing the results of a hydrolysis test of 2
- a glycosylated linker-HER2 (8-16) conjugate compound 6) having an ester bond. This graph plots the relative concentration of starting material against incubation time.
- FIG. 4 is a graph showing the results of a hydrolysis test of a glycosylated (Cys (disialo) type) linker-chemerin 9 conjugate (Compound 8). This graph plots the relative concentration of starting material against incubation time.
- the “glycosylation linker” is a linker having, as a carrier, a sugar chain capable of improving the water solubility of the physiologically active substance by binding to the physiologically active substance having at least one carboxy group.
- the glycosylated linker bonded to the physiologically active substance is characterized by being hydrolyzed at a desired rate under specific conditions, for example, in vivo. By this hydrolysis, the glycosylated linker is detached from the physiologically active substance, and the physiologically active substance can be released. The released physiologically active substance returns to the state before addition of the glycosylated linker.
- the glycosylated linker is represented by the following formula (A). XR 1 -YR 2 (A)
- X in the above formula (A) means an oxygen atom (O) having a leaving group or a sulfur atom (S) having a leaving group.
- the oxygen atom having a leaving group (O) or the sulfur atom having a leaving group (S) means a sugar chain represented by the formula (A): X—R 1 —Y—R 2
- the leaving group may be any group that is eliminated when the carboxy group of the physiologically active substance and the oxygen atom (O) having a leaving group or the sulfur atom (S) having a leaving group are combined. It is not limited, For example, they are a hydrogen atom, lithium, sodium, potassium, rubidium, cesium, francium, silver etc. which become a monovalent cation.
- the sugar chain addition linker of this invention couple
- X in the above formula (A) is a sulfur atom (S) having a leaving group, it is bonded by forming a thioester with a physiologically active substance.
- R 1 is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, substituted Or unsubstituted C 2 -C 5 alkenyl, substituted or unsubstituted C 2 -C 5 alkynyl, or R 1 is —R 3 —R 4 —, —R 4 —R 5 —, Or, it means -R 3 -R 4 -R 5- .
- R 3 and R 5 mean substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl.
- R 4 represents a substituted or unsubstituted C 5 -C 16 aryl, a substituted or unsubstituted C 5 -C 16 heteroaryl, or a sulfur atom (S).
- substituted or unsubstituted C 1 -C 5 alkyl includes linear or branched alkyl.
- Examples of “C 1 -C 5 alkyl” include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl Tert-pentyl and the like.
- These “C 1 -C 5 alkyl” can be independently substituted with one or more “substituents”.
- substituteduents include C 1 -C 4 alkoxy groups (eg, methoxy, ethoxy, propoxy, butoxy, etc.), amino groups, hydroxy groups, thiol groups, carboxy groups, nitro groups, mesyl groups, tosyl groups, Or a halogen atom (for example, fluorine, chlorine, bromine, iodine) etc. can be mentioned.
- substituted or unsubstituted C 5 -C 16 aryl is not limited to the following, but includes, for example, phenyl, biphenyl, naphthyl, anthranyl, phenanthryl, anthryl, o-tolyl, m-tolyl, p- Examples include tolyl, xylyl, ethylphenyl and benzyl.
- “Substituted or unsubstituted C 5 -C 16 aryl” is not limited to the above, and one or more hydrogen atoms in “C 5 -C 16 aryl” are each independently substituted with “substituent”. Also included.
- substituteduents include C 1 -C 4 alkyl groups, C 1 -C 4 alkoxy groups (eg, methoxy, ethoxy, propoxy, butoxy, etc.), amino groups, hydroxy groups, thiol groups, carboxy groups, nitro groups, Group, mesyl group, tosyl group, halogen atom (eg, fluorine, chlorine, bromine, iodine), C 1 -C 4 halogenated alkyl group (eg, methyl chloride group), phenyl group, o-tolyl group, m-tolyl group , P-tolyl group, xylyl group, ethylphenyl group or benzyl group.
- C 1 -C 4 alkyl groups eg, methoxy, ethoxy, propoxy, butoxy, etc.
- amino groups eg, methoxy, ethoxy, propoxy, butoxy, etc.
- amino groups hydroxy groups
- substituted or unsubstituted C 5 -C 16 heteroaryl is not limited to the following, but includes those in which the carbon atoms forming the ring structure are substituted with nitrogen atoms or oxygen atoms. More specifically, indole, quinoline, chromene and the like can be mentioned. “Substituted or unsubstituted C 5 -C 16 heteroaryl” is not limited to the above, but one or more hydrogen atoms bonded to the carbon atom forming the ring structure of “C 5 -C 16 heteroaryl” Including those in which atoms are each independently substituted by “substituents”.
- substituted examples include an alkyl group, an alkoxy group (eg, methoxy, ethoxy, propoxy, butoxy, etc.), a hydroxy group, a carboxy group, a nitro group, a mesyl group, a halogen atom (eg, fluorine, chlorine, bromine, Iodine), an alkyl halide group (for example, a methyl chloride group) and the like.
- an alkyl group an alkoxy group (eg, methoxy, ethoxy, propoxy, butoxy, etc.), a hydroxy group, a carboxy group, a nitro group, a mesyl group, a halogen atom (eg, fluorine, chlorine, bromine, Iodine), an alkyl halide group (for example, a methyl chloride group) and the like.
- an alkyl group an alkoxy group (eg, methoxy, ethoxy, propoxy, butoxy, etc.
- substituted or unsubstituted C 2 -C 5 alkenyl includes linear or branched alkenyl.
- substituted or unsubstituted C 2 -C 5 alkenyl include ethenyl, peropenyl, butenyl and the like.
- Substituted or unsubstituted C 2 -C 5 alkenyl is not limited to the above, and “C 2 -C 5 alkenyl” is independently substituted with one or more “substituents”. Including those.
- substituteduents include C 1 -C 4 alkoxy groups (eg, methoxy, ethoxy, propoxy, butoxy, etc.), amino groups, hydroxy groups, thiol groups, carboxy groups, nitro groups, mesyl groups, tosyl groups, Or a halogen atom (for example, fluorine, chlorine, bromine, iodine) etc. can be mentioned.
- substituted or unsubstituted C 2 -C 5 alkynyl includes linear or branched alkynyl.
- substituted or unsubstituted C 2 -C 5 alkynyl include ethynyl, propynyl, butynyl and the like.
- substituted or unsubstituted C 2 -C 5 alkynyl is not limited to the above, and “C 2 -C 5 alkynyl” is independently substituted with one or more “substituents”. Including things.
- substituteduents include C 1 -C 4 alkoxy groups (eg, methoxy, ethoxy, propoxy, butoxy, etc.), amino groups, hydroxy groups, thiol groups, carboxy groups, nitro groups, mesyl groups, tosyl groups, Or a halogen atom (for example, fluorine, chlorine, bromine, iodine) etc. can be mentioned.
- Y in the above formula (A) may or may not be present in the formula (A).
- Y is —CO— or —CONH— (wherein C is bonded to R 1 in the formula (A) and N is in the formula (A). R 2 is bound).
- R 2 is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 represents —R 6 -R 7 .
- R 6 means a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide.
- R 7 is a hydrogen atom (H), —NH 2 , substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 Means heteroaryl, nucleic acid, or PEG.
- sugar chain refers to a compound in which one or more unit sugars (monosaccharides and / or derivatives thereof) are linked. When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond.
- Such sugar chains include, for example, monosaccharides and polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes thereof contained in the living body.
- sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, etc., but are not limited thereto.
- the sugar chain may be linear or branched.
- sugar chain also includes sugar chain derivatives.
- sugar chain derivatives include sugars having a carboxy group (for example, C-1 Oxidized aldonic acid converted to carboxylic acid (for example, D-gluconic acid oxidized D-glucose), uronic acid whose terminal C atom became carboxylic acid (D-glucose oxidized D-glucose) Glucuronic acid)), sugars having amino groups or derivatives of amino groups (eg acetylated amino groups) (eg N-acetyl-D-glucosamine, N-acetyl-D-galactosamine etc.), amino groups and carboxy Sugars having both groups (eg, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.), deoxygenated sugars (eg, 2-deoxy-D-ribo Scan), sulfated sugar including a sulfuric acid group, including but sugar chains are such
- a preferable sugar chain is a sugar chain that improves the water solubility of the physiologically active substance when added to the physiologically active substance as a sugar chain addition linker.
- preferred sugar chains are sugar chains that reduce the antigenicity of the physiologically active substance when added to the physiologically active substance as a sugar chain addition linker.
- the sugar chain in the glycosylated linker of the present invention is not particularly limited, and may be a sugar chain that exists as a complex carbohydrate (glycopeptide (or glycoprotein), proteoglycan, glycolipid, etc.) in vivo. It may be a sugar chain that does not exist as a complex carbohydrate in vivo.
- a sugar chain that exists as a complex carbohydrate in vivo is preferable from the viewpoint that the glycosylated linker of the present invention is administered to the living body.
- sugar chains include N-linked sugar chains and O-linked sugar chains that are sugar chains bound to peptides (or proteins) as glycopeptides (or glycoproteins) in vivo.
- an N-linked sugar chain is used.
- the N-linked sugar chain include a high mannose type, a complex type, and a hybrid type, and a complex type is particularly preferable.
- the sugar chain in the sugar chain addition linker of the present invention is a complex type sugar chain.
- the complex-type sugar chain includes two or more types of monosaccharides and has a basic structure shown below and a lactosamine structure represented by Gal ⁇ 1-4GlcNAc.
- the complex type sugar chain includes a double chain complex type sugar chain.
- the double-stranded complex type sugar chain refers to one in which a single-chain sugar chain composed of 0 to 3 sugars is bonded to two mannoses at the ends of the basic structure.
- double-stranded complex type sugar chain examples include the following diasial sugar chain, Monosialo sugar chain, Asialo sugar chain, Digul Knack sugar chain, Dimannose sugar chain, Etc. are preferred.
- the double-stranded complex sugar chain is more preferably a disialo sugar chain or an asialo sugar chain, and most preferably a disialo sugar chain.
- the complex sugar chain of the present invention includes the above-described double-chain complex sugar chain (two-branch complex sugar chain) and a three-chain complex sugar chain (three-branch complex sugar chain). Also included are four-chain complex sugar chains (four-branch complex sugar chains).
- a trisialo sugar chain represented by the following structural formula Tetrasialo sugar chain represented by the following structural formula can be mentioned.
- examples of the three-chain complex sugar chain and the four-chain complex sugar chain include sugar chains that have lost one or more sugar residues from the non-reducing ends of these trisialo sugar chains and tetrasialo sugar chains. it can.
- the complex type sugar chain of the present invention includes those with fucose.
- a fucose-containing complex type sugar chain represented by the following structural formula: Can be mentioned.
- sugar chains in which one or more sugars have been lost from the non-reducing end of these fucose-containing complex sugar chains can also be mentioned.
- double-stranded complex sugar chain include those shown in the above chemical formula, and those having different binding modes from the examples shown in the chemical formula.
- Such a sugar chain is also preferably used as the sugar chain of the present invention.
- sugar chains include those in which sialic acid and galactose are bonded by an ( ⁇ 2 ⁇ 3) bond in a dicialosaccharide chain or a monosialosugar chain.
- the complex type sugar chain of the present invention includes a sugar chain having a polylactosamine structure or a sialylpolylactosamine structure represented by the following formula. (In the formula, n is an integer of 2 to 3.) (In the formula, n is an integer of 2 to 3.)
- the high mannose type sugar chain used in the present invention is a sugar chain in which two or more mannoses are further bonded to the basic structure of the complex type sugar chain described above. Since the high mannose type sugar chain is bulky, the stability in blood can be further increased by binding the high mannose type sugar chain to the peptide.
- a sugar chain containing 5 to 9 mannose is preferable like a mammalian high mannose sugar chain, but may be a sugar chain containing more mannose like a high mannose sugar chain of yeast.
- the high mannose type sugar chain preferably used in the present invention, for example, Highman North-5 (M-5) Highman North-9 (M-9) Etc.
- preferable sugar chains include, for example, sugar chains existing as glycoproteins bound to proteins in the human body (for example, sugar chains described in “FEBS LETTERS Vol. 50, No. 3, Feb. 1975”). ) And a sugar chain having the same structure (a sugar chain having the same kind of constituent sugars and their coupling mode) or a sugar chain from which one or more sugars have been lost from the non-reducing end thereof. Specific examples include the sugar chains listed below.
- a preferable sugar chain is a sugar chain having a linear structure.
- sugar chains include oligohyaluronic acid.
- oligohyaluronic acid refers to a sugar chain in which N-acetylglucosamine and glucuronic acid are alternately linked to 2 to 32 sugars, preferably 2 to 16 sugars, more preferably 4 to 8 sugars, and linearly linked.
- oligohyaluronic acids used in the present invention particularly preferred are sugars having 2 units (4 sugars) or more and 8 units (16 sugars) or less when the unit consisting of N-acetylglucosamine and glucuronic acid is 1 unit.
- a chain more preferably 2 units (4 sugars) to 4 units (8 sugars), most preferably 2 units (4 sugars).
- hyaluronic acid preferably used in the present invention, for example, Tetrasaccharide oligohyaluronic acid, Octasaccharide oligohyaluronic acid Etc.
- the hydroxy group and / or carboxy group of each sugar residue constituting it may be protected by a protecting group.
- the protecting group is, for example, a protecting group known to those skilled in the art introduced for the purpose of protecting the hydroxy group and / or carboxy group of the sugar residue from a chemical reaction.
- an alkyl group methyl group, ethyl group, etc.
- benzyl group acyl group (acetyl group, benzoyl group, pivaloyl group, etc.), tert-butyldimethylsilyl group, tert -Butyldiphenylsilyl group, phenacyl group, allyl group and the like
- tert-butyldimethylsilyl group tert -Butyldiphenylsilyl group
- phenacyl group allyl group and the like
- amino acid is used in its broadest sense, and is a natural amino acid such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile).
- amino acids in the present specification, for example, L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and amino acid derivatives; norleucine, ⁇ -alanine, It should be understood that ornithine and other amino acids that are not constituents of proteins in vivo; and chemically synthesized compounds having amino acid properties known to those skilled in the art.
- non-natural amino acids include ⁇ -methylamino acids ( ⁇ -methylalanine etc.), D-amino acids (D-aspartic acid, D-glutamic acid etc.), histidine-like amino acids (2-amino-histidine, ⁇ -hydroxy-histidine) , Homohistidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, etc.), amino acids with extra methylene in the side chain (“homo” amino acids), and carboxylic acid functional amino acids in the side chain are replaced with sulfonic acid groups Amino acids such as cysteic acid.
- ⁇ -methylamino acids ⁇ -methylalanine etc.
- D-amino acids D-aspartic acid, D-glutamic acid etc.
- histidine-like amino acids (2-amino-histidine, ⁇ -hydroxy-histidine)
- the “glycosylated amino acid” is not particularly limited in the binding site between the sugar chain and the amino acid, but an amino acid is preferably bonded to the reducing end of the sugar chain.
- an amino acid is preferably bonded to the reducing end of the sugar chain.
- the type of amino acid to which the sugar chain is bound there are no particular limitations on the type of amino acid to which the sugar chain is bound, and any of natural amino acids, unnatural amino acids, and D-amino acids can be used.
- the glycosylated amino acid has the same or similar structure as that present as a glycopeptide (glycoprotein) in the living body
- the glycosylated amino acid is a glycosylated Asn such as an N-linked sugar chain.
- Glycosylated Ser such as O-linked sugar chain and glycosylated Thr are preferable.
- the sugar chain and the amino acid may be bonded without using a linker, or may be bonded through a linker.
- the amino acid of the glycosylated amino acid has two or more carboxy groups in the molecule such as aspartic acid or glutamic acid.
- Amino acids having two or more amino groups in the molecule such as lysine, arginine, asparagine, histidine and tryptophan; amino acids having a hydroxy group in the molecule such as serine, threonine and tyrosine; thiols in the molecule such as cysteine Amino acids having a group; amino acids having an amide group in the molecule such as asparagine and glutamine are preferred.
- the amino acid of the glycosylated amino acid is preferably aspartic acid, glutamic acid, lysine, arginine, serine, threonine, cysteine, asparagine, or glutamine, and more preferably cysteine or asparagine.
- linkers used in the art can be used.
- a is an integer and is not limited as long as the desired linker function is not inhibited, but preferably represents an integer of 0 to 4
- C1-10 polymethylene —CH 2 —R—;
- R is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, carbocyclic group, substituted carbocyclic group, heterocyclic group and This is a group formed by elimination of one hydrogen atom from a group selected from the group consisting of substituted heterocyclic groups.
- a is an integer and is not limited as long as the desired linker function is
- the hydrogen atom on the amino group of the side chain of asparagine is at the reducing end portion of the sugar chain. May be substituted.
- the leaving group present at the reducing end of the sugar chain is not limited, and may be, for example, chlorine, bromine or fluorine.
- the hydrogen atom on the thiol group of the side chain of cysteine is reduced in the sugar chain via the linker.
- the linker is —CH 2 —CONH—
- the reducing end of the sugar chain is bonded to the nitrogen atom in the linker.
- the leaving group of the linker bonded to the reducing end of the sugar chain is not limited, and may be, for example, chlorine, bromine or fluorine.
- glycosylated polypeptide is not particularly limited as long as it is a compound in which at least one sugar chain is added to a protein (or polypeptide or peptide).
- Glycosylated polypeptides may be used interchangeably herein with “glycoprotein” or “glycopeptide”.
- the glycosylated polypeptide may be a polypeptide containing the aforementioned glycosylated amino acid.
- the binding mode between the sugar chain and the amino acid in the glycosylated polypeptide and the type of amino acid constituting the polypeptide may be defined in the same manner as in the glycosylated amino acid in the present invention.
- the amino acid (residue) in the polypeptide that binds to the sugar chain is not limited to the N-terminus or C-terminus of the polypeptide, and if appropriate, any of the amino acids (residues) that constitute the polypeptide. Also good.
- the amino acid residue in the glycosylated polypeptide of the present invention may preferably be 2 to 100 amino acid residues, more preferably 2 to 10 amino acid residues.
- amino acids other than the amino acid at the portion where the sugar chain and the polypeptide are bonded may be selected relatively freely.
- the amino acid at the portion where the sugar chain and the polypeptide are bonded is, for example, asparagine, cysteine, lysine or glutamine, while the amino acid other than the amino acid at the portion where the sugar chain and the polypeptide are bonded (for example, (sugar chain).
- the amino acids bound to the linker moiety is not particularly limited.
- the glycosylated amino acid or the amino acid constituting the glycosylated polypeptide in the present invention is preferably composed of amino acids present in the living body.
- the sugar chain addition linker of the present invention can be a thioalkyl type sugar chain addition linker.
- the thioalkyl-type glycosylation linker refers to a glycosylation linker that can be bound to a physiologically active substance via a thioester bond and has an alkyl structure in the structure.
- the thioalkyl-type glycosyl linker is a glycosyl linker that can be bound to a physiologically active substance via a thioester bond, and has a alkynyl or alkenyl structure in its structure.
- a chain addition linker is also included.
- the thioalkyl-type glycosyl linker is X in the above formula (A) is a sulfur atom (S) having a leaving group, Whether R 1 in the above formula (A) is substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 2 -C 5 alkynyl; or, R 1 is, -R 3 -R 4 -, - R 4 -R 5 -, or -R 3 -R 4 -R 5 - means, wherein, R 3 and R 5 are substituted or Unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 2 -C 5 alkenyl, or substituted or unsubstituted C 1 -C 5 alkynyl, wherein R 4 is substituted or unsubstituted C 5- C 16 aryl, substituted
- R 2 in the above formula (A) is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 means —R 6 -R 7 , where R 6 is , A sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, wherein R 7 is a hydrogen atom (H), —NH 2 , substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, nucleic acid, or PEG, Glycosylation linker.
- the sugar chain addition linker of the present invention can be a thioaryl-type sugar chain addition linker.
- the thioaryl-type glycosylation linker refers to a glycosylation linker that can be bound to a physiologically active substance via a thioester bond, and has an aryl structure in the structure. .
- the thioalkyl-type glycosyl linker is X in the above formula (A) is a sulfur atom (S) having a leaving group, R 1 in the above formula (A) is substituted or unsubstituted C 1 -C 5 aryl, or substituted or unsubstituted C 5 -C 16 heteroaryl, Y in the formula (A) may or may not be present in the formula (A).
- R 2 in the above formula (A) is a sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, or R 2 means —R 6 -R 7 , where R 6 is , A sugar chain, a glycosylated amino acid, or a glycosylated polypeptide, wherein R 7 is a hydrogen atom (H), —NH 2 , substituted or unsubstituted C 1 -C 5 alkyl, substituted or unsubstituted C 5 -C 16 aryl, substituted or unsubstituted C 5 -C 16 heteroaryl, nucleic acid, or PEG, Glycosylation linker.
- the sugar chain contained in the compound or salt thereof containing the glycosylated linker moiety and the physiologically active substance moiety of the present invention is uniform.
- the sugar chain is uniform when the sugar chains are compared between the sugar chain addition linker parts, the sugar chain addition site, the type of each sugar constituting the sugar chain, the binding order, and the binding between the sugars.
- the mode is the same between the glycosylated linker moieties.
- the sugar chain is uniform means that the structure of the plurality of sugar chains added in the sugar chain addition linker part.
- the types of sugars constituting the sugar chain, the binding order, and the binding mode between sugars are the same.
- the structure of the sugar chain is at least 90% or more, preferably 95% or more, more preferably 99% or more.
- the ratio of the uniform sugar chain and the ratio of the uniform sugar chain-added linker can be measured by a method using, for example, HPLC, capillary electrophoresis, NMR, mass spectrometry or the like.
- the glycosylated amino acid or glycosylated polypeptide having substantially uniform amino acid sequence and / or sugar chain used in the present invention is a solid phase synthesis, liquid phase synthesis, cell synthesis, naturally occurring one. It can be produced by incorporating a glycosylation step into a peptide production method known to those skilled in the art, such as a separation and extraction method.
- a glycosylation step into a peptide production method known to those skilled in the art, such as a separation and extraction method.
- International Publication No. 2010/021126, International Publication No. 2004/005330, etc. may be referred to.
- WO 03/008431 pamphlet for example, WO 2004/058984 pamphlet, WO 2004/008431 pamphlet, WO 2004/0084 / Reference may be made to the pamphlet No. 058824, the pamphlet of International Publication No. 2004/070046, the pamphlet of International Publication No. 2007/011055, and the like.
- the glycosylated polypeptide used in the present invention includes a sugar chain in which an amino acid-free sugar chain is bound to an amino acid or an amino acid on the polypeptide directly or via a linker.
- Addition polypeptide one or more (for example, 2 to 30, preferably 2 to 10) amino acids bonded to an amino group and / or carboxy group of a glycosylated amino acid, Glycosylated polypeptide linked to amino acid or polypeptide; glycan linked to amino acid is linked to amino acid on polypeptide via linker Is not a glycosylated polypeptide, it may also be included such as.
- glycosylated amino acid or glycosylated polypeptide of the present invention by transferring various sugars (for example, fucose and the like) to the glycosylated amino acid or glycosylated polypeptide of the present invention by glycosyltransferase, the glycosylated amino acid or sugar chain having a desired sugar chain structure is transferred. Additional polypeptides may be obtained efficiently.
- a glycosylated amino acid or a glycosylated polypeptide having a desired sugar chain structure containing fucose can be obtained by transferring fucose by glycosyltransferase (fucose transferase).
- fucose transferase Depending on the glycosyltransferase used, a glycosylated amino acid or glycosylated polypeptide having a desired glycosylated structure with a different binding mode can be obtained.
- fucose commercially available fucose or chemically synthesized can be used.
- fucose transferase commercially available ones, naturally-derived ones, and ones produced by gene recombination can be used, and can be appropriately selected depending on the type of fucose to be transferred. Specific examples include Fucoyltransferase® V (Human, Recombinant, plasma-derived, serum-derived, milk-derived, liver-derived) which is an enzyme that transfers fucose to N-acetylglucosamine on the non-reducing end side of the sugar chain asparagine. it can. Further, fucose may be transferred by using fucose hydrolase and shifting the equilibrium by adjusting pH or the like.
- Fucoyltransferase® V Human, Recombinant, plasma-derived, serum-derived, milk-derived, liver-derived
- fucose may be transferred by using fucose hydrolase and shifting the equilibrium by adjusting pH or the like.
- nucleic acid refers to DNA or RNA in which nucleotides consisting of a base (adenine, guanine, thymine, cytosine, uracil), a sugar residue, and a phosphate are linked by a phosphate ester bond.
- base adenine, guanine, thymine, cytosine, uracil
- a sugar residue and a phosphate are linked by a phosphate ester bond.
- PEG is a polymer of ethylene glycol, and can be represented by, for example, “(—CH 2 —CH 2 —O—) n” (n is an integer of 2 to 10,000).
- a preferred glycosylated linker is In the glycosylated linker represented by the formula (A): X—R 1 —Y—R 2 , X represents an oxygen atom having a leaving group (O) or a sulfur atom having a leaving group (S); R 1 is benzyl, aryl represented by tolyl or the like, or R 1 means —R 3 —R 4 —R 5 —, R 3 is —CH 2 CH 2 —, and R 4 Is a sulfur atom (S), R 5 is —CH 2 — (that is, R 1 is a thioether represented by —CH 2 CH 2 SCH 2 —), Y means —CO—, R 2 is NH-sugar chain, glycosylated Asn, glycosylated Cys, or one or more amino acids at the C-terminus of glycosylated Asn or glycosylated Cys (eg, 2, 3, 4 5) A glycosyl
- the glycosylated linker of the present invention can be produced by solid phase synthesis, liquid phase synthesis or the like.
- a glycosylated linker represented by the formula (A): X—R 1 —Y—R 2 is produced by solid phase synthesis, R 2 , Y (if present), R In the order of 1 and X, an appropriate compound is bonded onto the resin.
- R 2 , Y, R 1 and X may be bonded on the resin in order for each component, or compounds corresponding to a plurality of consecutive components may be bonded on the resin.
- R 2 having a leaving group is first bonded on the resin.
- a glycosylated linker represented by the formula (A): X—R 1 —Y—R 2 can be prepared.
- a compound corresponding to a plurality of consecutive structures includes not only XR 1 -Y but also other combinations in which two or more of four structures of X, R 1 , Y, and R 2 are selected.
- the production method by the solid phase synthesis method is A step of binding an R 2 compound having a leaving group (sugar chain, glycosylated amino acid, glycosylated polypeptide, etc.) on a resin; A step of bonding Y having at least two leaving groups to R 2 on the resin, wherein the leaving group of R 2 and Y is removed, whereby R 2 and Y are bonded; Process, A step of bonding a compound corresponding to a portion of R 1 having at least two leaving groups to Y—R 2 on the resin, wherein the leaving group of Y and R 1 is eliminated Y and R 1 are bonded together; A step of bonding a compound corresponding to a moiety of X having at least two leaving groups to R 1 —Y—R 2 on the resin, wherein the leaving groups of R 1 and X are eliminated A step of bonding R 1 and X; Cleaving XR 1 -YR 2 synthe
- a glycosylated linker can be produced by a production method including a step of cleaving XR 1 -YR 2 synthesized on a resin from the resin.
- an amino acid can be further linked without cleaving the glycosylated linker formed on the resin from the resin (more specifically, X
- An amino acid can be further linked to an oxygen atom having a leaving group represented by the formula (1) or a sulfur atom having a leaving group. This makes it possible to produce a compound containing a physiologically active substance moiety and a sugar chain-added linker moiety on the resin without having to separate the sugar chain-added linker from the resin.
- the resin used in the solid phase synthesis may be any resin (resin) that is usually used in solid phase synthesis.
- resin for example, 2-chlorotrityl chloride resin (Merck) functionalized with chlorine.
- a linker may be present between the amino-PEGA resin and the amino acid. Examples of such a linker include 4-hydroxymethylphenoxyacetic acid (HMPA), 4- (4-hydroxymethyl-3-methoxyphenoxy).
- H-Cys (Trt) -Trity NovaPEG resin manufactured by Merck or the like in which the C-terminal amino acid is bonded to the resin in advance can also be used.
- a Rink-Amide-PEGA resin functionalized with an amino group manufactured by Merck & Co., Inc.
- the 2-chlorotrityl chloride resin is preferable because it can prevent racemization of Cys at the terminal when the peptide chain is extended in solid phase synthesis.
- a compound corresponding to the R 2 moiety sucgar chain, glycosylated amino acid, glycosylated polypeptide, etc.
- the glycosylated amino acid when bound on the resin, it is fat-soluble.
- a glycosylated amino acid whose amino acid is protected by a protecting group is bound.
- a glycopolypeptide is bound on a resin, a glycosylated polypeptide can be synthesized on the resin by sequentially binding a desired amino acid and a glycosylated amino acid onto the resin.
- the fat-soluble protective group examples include carbonate-based or amide groups such as 9-fluorenylmethoxycarbonyl (Fmoc) group, t-butyloxycarbonyl (Boc) group, benzyl group, allyl group, allyloxycarbonyl group, and acetyl group.
- Protecting groups of the system can be mentioned.
- a fat-soluble protecting group into an amino acid, for example, when introducing an Fmoc group, it can be introduced by adding 9-fluorenylmethyl-N-succinimidyl carbonate and sodium hydrogen carbonate to carry out the reaction. The reaction is carried out at 0 to 50 ° C., preferably at room temperature, for about 1 to 5 hours.
- amino acids protected with a fat-soluble protecting group can also be used as amino acids protected with a fat-soluble protecting group.
- amino acids protected with a fat-soluble protecting group and having a protecting group introduced in the side chain include, for example, Fmoc-Arg (Pbf) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asp (OtBu ) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Cys (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc -Gln (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Trp (Boc) -
- glycosylated amino acid in which the amino acid is protected with a fat-soluble protecting group examples include Fmoc-glycosylated Asn and Boc-glycosylated Asn.
- sugar chain-added amino acids are the above-described sugar chains and amino acids to which sugar chains having the same sugar chain structure are added.
- Such a sugar chain can be obtained by any known method. Specific techniques include, but are not limited to, for example, chemical synthesis of sugar chains (see, for example, J. Seifert et al. Angew Chem Int. Ed. 2000, 39, p531-534), natural or Those separated from an artificial sugar chain source or commercially available can be used. In this method, the glycosylated amino acids having the same structure are not limited.
- separation of a sugar chain having the same structure from a natural or artificial sugar chain source is described in, for example, WO2004 / 058789. It can be done by a method. Specifically, from a natural sugar chain source such as hen's egg, Seko et al. Biochim Biophys Acta. 1997; 1335 (1-2): 23-32 and the like, and a mixture containing glycan asparagine (sialylglycopeptide (SGP)) is isolated, and a lipophilic protecting group (for example, Fmoc) is contained in the glycan asparagine.
- a natural sugar chain source such as hen's egg, Seko et al. Biochim Biophys Acta. 1997; 1335 (1-2): 23-32 and the like
- a mixture containing glycan asparagine sialylglycopeptide (SGP)
- a lipophilic protecting group for example, Fmoc
- sugar chain asparagine derivatives which is subjected to chromatography, whereby sugar chains of various structures contained in the mixture can be separated according to the structure.
- sugar chain asparagine having a specific structure with or without various protecting groups can be obtained from, for example, Sugar Chain Engineering Laboratory.
- the reaction for binding the resin to the amino acid or glycosylated amino acid is preferably performed, for example, by placing the resin in a solid phase column, washing the resin with a solvent, and then adding the amino acid solution.
- the cleaning solvent include dimethylformamide (DMF), 2-propanol, dichloromethane, and the like.
- solvents that dissolve amino acids include dimethyl sulfoxide (DMSO), DMF, dichloromethane, and the like.
- the coupling reaction between the resin and the amino acid or glycosylated amino acid is carried out at 0 to 50 ° C., preferably at room temperature, for about 10 minutes to 30 hours, preferably about 15 minutes to 24 hours.
- dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC / HCl), diphenylphosphoryl azide (DPPA), carbonyldiimidazole (CDI), Diethyl cyanophosphonate (DEPC), 1,3-diisopropylcarbodiimide (DIC), benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP), 3-diethoxyphosphoryloxy-1,2,3-benzotriazine -4 (3H) -one (DEPBT), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP), 1-hydroxy-7-azabenzo Riazole (HOAt), 3-hydroxy-4-oxo-3,4-dihydro
- DCC dicyclohexylcar
- sialic acid may be removed from the resin by acid treatment in the cutting step. Therefore, when a sugar chain having sialic acid is introduced into the linker portion before the cleavage step using an acid, a sugar chain in which the carboxy group of sialic acid on the sugar chain to be introduced is protected by a protecting group is used. It is preferable.
- a carboxy group of sialic acid is converted to —COOBn, —COOEt, —COOMe, —COOCH (Ph) 2 , —COOCH 2 COPh, —COOCH 2 PhOMe, —COOCH 2 Ph (OMe) 2 ,
- a protecting group protecting as represented by —COOCH 2 PhNO 2 or —COOCH 2 Ph (NO 2 ) 2 is preferable. In this way, by protecting the carboxy group of sialic acid with a benzyl group or the like, it is possible to prevent the elimination of acid-labile sialic acid.
- the protection reaction of the carboxy group of sialic acid on the sugar chain can be performed by methods well known to those skilled in the art.
- the deprotection of the protective group of the carboxy group of sialic acid protected by a benzyl group, diphenylmethyl group, or phenacyl group can be performed by methods well known to those skilled in the art.
- the deprotection reaction is not limited to the following, but can be performed by hydrolysis under basic conditions.
- the deprotection reaction is usually carried out at 0 to 50 ° C., preferably 0 to 40 ° C., more preferably 0 to 30 ° C.
- the reaction time is preferably about 5 minutes to 5 hours.
- the reaction solution is neutralized with a weak acid such as phosphoric acid or acetic acid and then appropriately purified by a known method (for example, high performance liquid column chromatography (HPLC)).
- HPLC high performance liquid column chromatography
- R 2 is a nucleic acid or PEG
- those skilled in the art can appropriately bind the corresponding compound to the resin by a known method.
- R 2 is a sugar chain
- it is produced by synthesizing a linker structure without a sugar chain on the resin and adding the sugar chain to the end of the linker after being cleaved and isolated from the resin.
- it is designed to have a thiol group at the end of the linker to which a sugar chain is to be added.
- the thiol group present at the end of the linker after isolation from the resin is linked to the haloacetylated complex type sugar chain derivative (or haloacetamido complex type sugar chain derivative), and the sugar chain is introduced to the end of the linker.
- Can do
- R 2 is a nucleic acid or PEG
- a linker structure to which no nucleic acid or PEG is added is synthesized on the resin, and the nucleic acid or PEG is added to the linker end after being cleaved and isolated from the resin.
- the terminal of the linker to which nucleic acid or PEG is to be added is designed to have, for example, a thiol group.
- the thiol group present at the end of the linker after isolation from the resin and the nucleic acid or PEG made into a haloacetylated product (or a haloacetamidized product) or the like can be bound to introduce the nucleic acid or PEG.
- R 2 is a glycosylated amino acid or a glycosylated polypeptide
- the sugar chain is later moved to the R 2 site. It can also be combined.
- the reaction for binding the sugar chain to R 2 may be performed on the resin continuously with the solid-phase synthesis, or may be performed after separation from the resin.
- the sugar chain addition step may be performed after the synthesis of the physiologically active substance portion, or the sugar chain addition step before the synthesis of the physiologically active substance portion. May be performed.
- the haloacetylated complex type sugar chain derivative (or haloacetamido complex type sugar chain derivative) is By reacting a sugar chain with a thiol group of unprotected Cys by reacting with a linker (including unprotected Cys) or a compound (including unprotected Cys) having the linker moiety and a physiologically active substance moiety; Can be conjugated to a peptide.
- the above reaction is usually performed at 0 to 80 ° C., preferably 10 to 60 ° C., more preferably 15 in a phosphate buffer, Tris-HCl buffer, citrate buffer, acetonitrile, DMSO, or a mixed solution thereof. It is better to perform at ⁇ 35 ° C.
- the reaction time is usually about 10 minutes to 24 hours, preferably about 30 minutes to 5 hours. After completion of the reaction, it may be appropriately purified by a known method (for example, HPLC).
- the haloacetylated complex type sugar chain derivative (or haloacetamido complex type sugar chain derivative), for example, represents a hydroxyl group bonded to the 1st-position carbon of the reducing end of a complex type asparagine-linked sugar chain by —NH— (CH 2 ) a— (CO) —CH 2 X
- X is a halogen atom, a is an integer, and is not limited as long as the desired linker function is not inhibited, but preferably represents an integer of 0 to 4. ).
- the reaction can be performed in a phosphate buffer at room temperature. After completion of the reaction, a glycosylated polypeptide substituted with glycosylated Cys can be obtained by purification with HPLC.
- the reaction can also be performed in a mixed solution of an organic solvent such as DMSO, DMF, methanol, and acetonitrile and the above buffer solution. At this time, the ratio of the organic solvent can be added to the buffer solution in the range of 0 to 99% (v / v).
- a peptide containing unprotected Cys having low solubility in a buffer solution is preferable because it can improve solubility in a reaction solution by adding such an organic solvent.
- the reaction can also be carried out in an organic solvent such as DMSO, DMF, methanol, acetonitrile, or a mixed solution thereof. In that case, it is preferable to carry out in the presence of a base.
- the base include DIPEA, triethylamine, pyridine, 2,4,6-collidine and the like.
- the reaction can also be performed in a mixed solution in which guanidine hydrochloride or urea is added to the buffer solution.
- Guanidine hydrochloride and urea can be added to the buffer so that the final concentration is 1M to 8M. Addition of guanidine hydrochloride or urea is preferable because it can improve the solubility of a peptide having low solubility in a buffer solution. Further, those skilled in the art can appropriately carry out the reaction of nucleic acid or PEG converted to haloacetylated form (or haloacetamidolated form) or the like with a Cys-containing peptide by a known method.
- Respect R 2 on the resin the step of coupling a compound corresponding to the portion of the Y, with respect to Y-R 2 on the resin, the step of coupling a compound corresponding to the portion of R 1, on the resin R 1
- the step of bonding a compound corresponding to the X moiety to —YR 2 those skilled in the art appropriately design and select a compound corresponding to each component and condense it to R 2 on the resin. Can do.
- Those skilled in the art can also design and select the compounds and reaction conditions as appropriate even when compounds corresponding to a plurality of consecutive components are bound onto the resin.
- the part corresponding to X of the sugar chain addition linker may require a protective group for synthesis.
- the protecting group for oxygen atom include trityl group, methoxytrityl group, t-butyl group, and benzyl group.
- the protecting group for sulfur atom include trityl group, methoxytrityl group, t-butyl group, t -A butylthio group, an Acm group, etc. can be mentioned.
- the protecting group can be introduced by a conventionally known method.
- the step of separating the glycosylated linker (formula (A): X—R 1 —Y—R 2 ) synthesized on the resin from the resin is preferably treated with an acid.
- the acid include a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane, ethanedithiol, and water (90: 5: 2.5: 2.5), and a mixed solution of acetic acid and trifluoroethanol (50 : 50), HCl and the like.
- TFA trifluoroacetic acid
- ethanedithiol ethanedithiol
- water 90: 5: 2.5: 2.5
- a mixed solution of acetic acid and trifluoroethanol 50 : 50
- the step of separating the compound from the resin is preferably treated with an acid.
- the acid used and the reaction conditions can be the same as the conditions for separating the glycosylated linker from the resin.
- the glycosylated linker thus produced binds to a physiologically active substance at an oxygen atom having a leaving group or a sulfur atom having a leaving group.
- the glycosylated linker can enhance the water solubility of the physiologically active substance by binding to the physiologically active substance.
- the glycosylated linker can preferably reduce the antigenicity of the physiologically active substance.
- the glycosylated linker bonded to the physiologically active substance can release the physiologically active substance within a certain period of time under specific temperature and pH conditions depending on the structure.
- the released physiologically active substance has an original function.
- the physiologically active substance released from the sugar chain-added linker in vivo exhibits the original function.
- the rate of hydrolysis can be increased when the linkage between the sugar chain-added linker moiety and the physiologically active substance moiety is a thioester bond rather than an ester bond.
- the sugar chain addition linker having a thioaryl structure is hydrolyzed faster than the sugar chain addition linker having a thioalkyl structure.
- a person skilled in the art can design a glycosylated linker having a desired physiologically active substance release time by appropriately changing the structure of the glycosylated linker moiety.
- the physiologically active substance can be bonded to the glycosylated linker moiety by changing (modifying) a part of its structure. However, once the glycosylated linker moiety is cleaved, the physiologically active substance is released.
- the structure of the released physiologically active substance is preferably the same as the compound structure before binding (before modification) to the glycosylated linker moiety.
- a physiologically active substance that is not bonded to a glycosylated linker is referred to as an “unmodified physiologically active substance”.
- the unmodified physiologically active substance preferably has the pharmacokinetic, immunogenic, toxicological or pharmacological characteristics inherent to the physiologically active substance itself, but the characteristics are altered, modified, etc. Also good.
- the “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” of the present invention releases an unmodified physiologically active substance by cleaving the glycosylated linker moiety under a predetermined condition. It is preferable to do.
- glycosylated linker of the present invention preferably does not adversely affect the pharmacokinetic, immunogenic, toxicological or pharmacological properties of the physiologically active substance that becomes a binding partner.
- the “physiologically active substance” means a substance that brings about some action / influence directly or indirectly on the physiological activity of a living body, although not limited thereto.
- the physiologically active substance may be intended for use in vitro and in vivo.
- the physiologically active substance may be one that does not exhibit its function in vivo.
- the physiologically active substance may be used synonymously with a drug.
- the physiologically active substance may include not only those useful as vaccines or pharmaceuticals, but also substances that do not directly act on or influence physiological activities of living bodies, such as diagnostic agents.
- the physiologically active substance may include not only naturally derived substances but also those obtained by deleting, modifying or substituting a part thereof (also referred to as derivatives).
- the physiologically active substance in the present invention includes, for example, a substance fused with a reporter protein such as GFP (green fluorescent protein) or a fluorescent dye such as fluorescein.
- a reporter protein such as GFP (green fluorescent protein) or a fluorescent dye such as fluorescein.
- the physiologically active substance in the present invention has at least one carboxy group.
- the physiologically active substance in the present invention binds to a glycosyl linker at at least one carboxy group of the physiologically active substance.
- the physiologically active substance in the present invention is preferably a low molecular weight physiologically active substance or a biopolymer having at least one carboxy group.
- biopolymer may mean a high molecular organic compound among physiologically active substances.
- the “low molecular biologically active substance” may mean a low molecular organic compound among the physiologically active substances.
- the biopolymer may be, for example, a polymer compound such as protein, nucleic acid or polysaccharide or a part thereof, but may be artificially synthesized.
- the low molecular weight physiologically active substance may be, for example, a substance capable of interacting with a biopolymer in a living body or may be artificially synthesized.
- the biopolymer and the low-molecular physiologically active substance may refer to the same thing depending on the case.
- the biopolymer in the present invention is a protein, polypeptide, polynucleotide, or peptide nucleic acid having at least one carboxy group, or a part of the structure thereof includes the above-mentioned “protein, polypeptide , Polynucleotide or peptide nucleic acid ".
- a portion derived from a protein or polypeptide is also referred to as a “peptide portion”.
- the “protein” is not particularly limited as long as a plurality of amino acids are bonded by an amide bond, and includes known proteins, novel proteins, or modifications thereof.
- the “variant” is a compound obtained by partially modifying a protein naturally or artificially. Such modifications include, for example, alkylation, acylation (eg, acetylation), amidation (eg, C-terminal amidation of a protein), carboxylation, ester formation of one or more amino acid residues of a protein. , Disulfide bond formation, glycosylation, lipidation, phosphorylation, hydroxylation, dehydration condensation or label component conjugation.
- examples of the variant include those obtained by deleting, substituting, or fusing a part of the structure of a known protein or a novel protein.
- the biopolymer as a physiologically active substance is a protein
- the protein is not limited.
- those skilled in the art such as solid phase synthesis, liquid phase synthesis, cell synthesis, and methods for separating and extracting naturally occurring substances can be used. May be synthesized using known methods.
- polypeptide and peptide are used in the same meaning as protein in principle.
- a polypeptide and a peptide may be used to indicate a relatively short amino acid chain that does not have a higher-order structure when it is a part of the structure of a protein (protein fragment).
- the polypeptide or peptide in the present invention includes, for example, a dipeptide in which two amino acids are bonded, a tripeptide in which three amino acids are bonded, a tetrapeptide in which four amino acids are bonded, and an oligo in which the number of amino acids is usually 10 or less.
- Peptides may also be included.
- polynucleotide includes, but is not limited to, single-stranded or double-stranded DNA or RNA having 2 to 2000 nucleotide residues; single-stranded or double-stranded siRNA, miRNA or nucleic acid. (DNA or RNA) aptamers; or compounds in which they are chemically modified. Such modifications further include, but are not limited to, charge, polarizability, hydrogen bonding, electrostatic interaction, or fluxionality to all or part of the polynucleotide. Modifications with other chemical groups are mentioned.
- a polynucleotide may be an oligonucleotide having a size of 20 base pairs or less.
- the “peptide nucleic acid” means a modified nucleic acid obtained by converting a sugar phosphate skeleton of a nucleic acid (DNA or RNA) into an N- (2-aminoethyl) glycine skeleton, although not limited thereto.
- the peptide nucleic acid may be further modified by methods known to those skilled in the art.
- the biopolymer in the present invention is not limited to the following, but in one embodiment, for example, adrenocorticotropic hormone (ACTH), oxytocin, adenosine deaminase, agarsidase, ⁇ 1 antitrypsin, ⁇ 1 protease inhibitor, alteplase, amylin, simulin , Anistreplase, ancrodose serine protease, antithrombin III, antitrypsin, aprotinin, asparaginase, atosiban, biphalin, bivalirudin, bone morphogenetic protein, pancreatic trypsin inhibitor, cadherin fragment, calcitonin (eg salmon derived), collagenase, Complement C1 esterase inhibitor, conotoxin, cytokine receptor fragment, DNase, dynorphin A, endorphin, et Nfuvirtide, enkephalin, erythropoiet
- HBs antigen, etc. influenza vaccine, Lyme disease vaccine, etc.
- VEGF vascular endothelial growth factor
- chemerin vascular endothelial growth factor
- HER2 protein human epidermal growth factor receptor
- epidermal growth factor EGF
- vasoactive Intestinal peptides vasopressin, ziconotide, lectin, cholinesterase, amylase, or pepsin, or variants or fragments thereof are included.
- the low molecular weight biologically active substance in the present invention includes, for example, a central nervous system active agent, anti-infective agent, anti-allergic agent, immunomodulator, anti-obesity agent, anti-coagulant having at least one carboxy group.
- a central nervous system active agent for example, a central nervous system active agent, anti-infective agent, anti-allergic agent, immunomodulator, anti-obesity agent, anti-coagulant having at least one carboxy group.
- Blood, antidiabetic, anticancer, anti-neoplastic, antibacterial, antifungal, analgesic, contraceptive, anti-inflammatory, steroid, vasodilator, vasoconstrictor or cardiovascular agonist Can be mentioned.
- the low molecular weight biologically active substance in the present invention is not limited to the following.
- Amoxapine Amoxicillin, Amphetamine, Amphotericin B, Ampicillin, Amprenavir, Amrinone, Anileridine, Apraclonidine, Apramycin, Articaine, Atenolol, Atomoxetine, Abizaphone, Baclofen, Benazepril, Benserazolol, Bentaxolbromine , Casin, Cathinone, Carbutamide, Cephalexin, Clinafloxacin, Ciprofloxy Syn, deferoxamine, delavirdine, desipramine, daunorubicin, dexmethylphenidate, dexmethylphenidate, diaphenylsulfone, dizocilpine, dopamine, dobutamine, dorzolamide, doxorubicin, duloxetine, eflornithine, enalapril, e
- the compound in which the glycosylated linker of the present invention is bound to a physiologically active substance can be produced by coupling the glycosylated linker synthesized and isolated by the above method to the physiologically active substance.
- the bond between the glycosylated linker and the physiologically active substance is an oxygen atom (O) having a leaving group of the glycosylated linker or a sulfur atom (S) having a leaving group and at least one carboxy group of the physiologically active substance.
- O oxygen atom
- S sulfur atom
- the condensing agent for the condensation reaction can be used as the condensing agent for the condensation reaction.
- a solvent for the condensation reaction for example, DMF, DMSO, dichloromethane or the like can be used.
- the condensation reaction can be performed, for example, by dissolving a peptide having a protected amino acid side chain and a sugar chain addition linker having a thiol group in DMF and adding PyBOP and DIPEA.
- the physiologically active substance is a peptide
- the isomerization of the C-terminal amino acid of the peptide can be suppressed, and therefore, the reaction is preferably performed at a low temperature ( ⁇ 15 ° C. to ⁇ 30 ° C.).
- the side chain of the peptide is preferably protected with a protecting group.
- the protecting group protecting the side chain of the peptide can be deprotected after the glycosylated linker is bound to the peptide.
- a protecting group for protecting the side chain of the peptide a protecting group well known to those skilled in the art can be used.
- the protecting group for the amino acid used in the solid phase synthesis described above can be used.
- those skilled in the art can also implement the introduction and deprotection of a protecting group to a peptide as appropriate.
- the physiologically active substance is a polypeptide or the like
- the amino acid constituting the physiologically active substance is sequentially directly bonded directly to the glycosylated linker bonded to the resin during solid phase synthesis.
- a compound containing a glycosylated linker moiety and a physiologically active substance moiety can be produced.
- the reaction conditions for synthesizing the physiologically active substance moiety on the resin by solid phase synthesis can be appropriately set by those skilled in the art.
- the present invention preferably provides a compound or a salt thereof that can be obtained by any of the above-described production methods.
- the compound or salt thereof that can be obtained is not limited to those produced by any of the production methods described above, and those produced by other production methods are also targeted.
- the present invention preferably provides a compound or a salt thereof obtained by any of the above-described production methods (obtained).
- the glycosylated linker of the present invention regardless of whether or not the physiologically active substance is sparingly soluble, “a compound comprising a glycosylated linker moiety and a physiologically active substance moiety or a compound thereof As a salt, the physiologically active substance can be easily dissolved in an aqueous solution or an emulsion prepared from the aqueous solution. After the dissolution, the glycosylated linker moiety is cleaved, whereby an unmodified physiologically active substance can be released.
- the glycosylated linker moiety in the present invention is cleaved from a “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” by a hydrolysis reaction.
- the glycosylated linker moiety can be cleaved from the “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” by self-hydrolysis by its intramolecular catalysis. is there.
- the cleavage is not intended to exclude biological cleavage such as cleavage by an enzyme existing in a living body (for example, esterase which is an enzyme that cleaves an ester bond).
- the compound of the present invention or a salt thereof has a feature that, after being dissolved in an aqueous solution or emulsion, the cleavage of the glycosylated linker moiety is accelerated depending on pH and / or temperature ( pH and / or temperature dependent cleavage).
- the compound of the present invention or a salt thereof and a glycosylated linker may be stored, for example, at a low temperature (eg, ⁇ 80 ° C. to 4 ° C.) and a low pH (eg, pH 1 to pH 4).
- the step of preparing a “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” by binding a physiologically active substance to the glycosylated linker moiety is performed at, for example, a low temperature (eg, 0 ° C. to 25 ° C. ), Low pH (eg, pH 1 to pH 7).
- a low temperature eg, 0 ° C. to 25 ° C.
- Low pH eg, pH 1 to pH 7
- glycosylated linker By protecting the N-terminal amino group of the glycosylated amino acid with a C 1 -C 16 acyl group, Fmoc group, Alloc group or the like, a “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” And the glycosylated linker may be stabilized.
- the compound of the present invention or a salt thereof has a temperature and pH close to physiological conditions (for example, a physiological environment in a mammal's living body or an environment close thereto, such as 35 ° C to 43 ° C, pH 6.8 to 7. 8 etc.).
- physiological conditions for example, a physiological environment in a mammal's living body or an environment close thereto, such as 35 ° C to 43 ° C, pH 6.8 to 7. 8 etc.
- the physiologically active substance can be efficiently dissolved in an aqueous solution or an emulsion prepared from an aqueous solution by using the compound of the present invention or a salt thereof. Therefore, in a preferred embodiment, filter sterilization can be performed even with a physiologically active substance having low water solubility (less soluble) by using the compound of the present invention or a salt thereof. Furthermore, in another preferred embodiment, even a physiologically active substance having low water solubility can be administered to a living body by using the compound of the present invention or a salt thereof.
- the present invention advantageously reduces the “loss” that can arise from the insolubility of the substance in the preparation or administration process of the preparation containing an expensive physiologically active substance.
- the release time and timing of the active substance can be controlled. For example, it is advantageous for delivery of a physiologically active substance that is desired to exert its effect quickly at a desired site after administration in vivo.
- the “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” of the present invention can provide improved water solubility as compared with an unmodified physiologically active substance.
- the improved water solubility is preferably 2 to 1,000,000 times in molar concentration, more preferably 10 to 1,000,000 times, and more preferably 100 to 1,000,000 times. It is more preferable that the ratio is 500 times to 1,000,000 times or more.
- a person skilled in the art appropriately selects a “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” or a glycosylated linker depending on the use and purpose of the physiologically active substance. Can do.
- the molar extinction coefficient (specific absorbance) necessary for determining the solubility of the “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” or the unmodified physiologically active substance of the present invention is determined by those skilled in the art. Determined by UV-visible spectrophotometry (for example, wavelength in the UV-visible region such as 280 nm) using a known protein concentration solution measured by a known method such as amino acid composition analysis or nitrogen quantification method as a sample. It's okay.
- composition comprising a compound or a salt thereof comprising a glycosylated linker moiety and a physiologically active substance moiety.
- composition includes any one or more other components (active ingredient or inactive ingredient) in addition to one or more compounds of the present invention or a salt thereof.
- the use of the composition of the present invention is not particularly limited.
- the composition may be used in an assay system (for example, an in vitro assay system).
- the sugar chain structure of the sugar chain addition linker moiety can be made uniform.
- the sugar chain addition linker part contained in the composition containing the compound having a sugar chain addition linker part and a physiologically active substance part or a salt thereof is not limited to the sugar chain structure, but the whole sugar chain addition linker part.
- the structure is also preferably uniform.
- the structure of the glycosylated linker moiety is uniform when the glycosylated linker moiety contained in the composition is compared with each other when the sugar chain and the linker moiety are compared.
- the site, the type of each saccharide constituting the glycan, the linking order of the glycans, the bonding mode between saccharides, and the structure constituting the linker moiety are the same.
- the structure of the sugar chain and the linker part are uniform in at least 90% or more, preferably 95% or more, more preferably 99% or more, between the sugar chain-added linker parts contained in the composition.
- a composition containing a glycosylated linker moiety having a uniform sugar chain has a constant quality, and is particularly preferable in the fields of pharmaceutical production and assay.
- the ratio of the uniform sugar chain and the ratio of the uniform sugar chain-added linker can be measured by a method using, for example, HPLC, capillary electrophoresis, NMR, mass spectrometry or the like.
- the “pharmaceutical composition” of the present invention is a composition suitable for pharmaceutical use, and usually used fillers, extenders, binders, moistening agents, disintegrating agents, surfactants, lubricants, etc.
- a pharmaceutical composition suitable for pharmaceutical use, and usually used fillers, extenders, binders, moistening agents, disintegrating agents, surfactants, lubricants, etc.
- examples of such a pharmaceutical composition include, but are not limited to, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections, and the like.
- the pharmaceutical use targeted by the pharmaceutical composition may be intended for a disease or disease involving a physiologically active substance contained in the composition as a physiologically active substance moiety.
- the physiologically active substance is GLP-1 or a derivative thereof
- the intended pharmaceutical use may be diabetes or the like.
- Other medical uses can be similarly understood by those skilled in the art by considering the diseases and types of diseases in which each physiologically
- the “pharmacologically acceptable carrier” is not particularly limited. By blending a pharmacologically acceptable carrier, the absorbability and blood concentration of the “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof” of the present invention are affected, and the pharmacokinetics May bring about changes.
- the compound of the present invention or a salt thereof and the pharmaceutical composition of the present invention containing the same can also be used as a vaccine.
- a poorly soluble antigen can be dissolved in an aqueous solution or emulsion as the compound of the present invention or a salt thereof, and after cleavage of the glycosylated linker moiety in vivo. Unmodified antigen can be released.
- the compound of the present invention or a salt thereof and a glycosylated linker can be used for development of various vaccines such as peptide vaccines.
- vaccine means a substance capable of producing an immune response when inoculated into an animal.
- a vaccine contains an antigen or is capable of expressing an antigen, which can induce an immune response against the antigen.
- the pharmaceutical composition of the present invention can be used not only for the prevention or treatment of viral infections, bacterial infections (sepsis, etc.), infectious diseases, but also any disease that can be related to the immune response, such as cancer, autoimmune diseases It can also be used for the treatment of (for example, type I diabetes, multiple sclerosis, rheumatoid arthritis, etc.).
- an “antigen” is a molecule containing one or more epitopes, and may be any one that can induce an antigen-specific immune response by stimulating the host immune system.
- the immune response may be a humoral immune response and / or a cellular immune response.
- As few as 3 to several (eg, 5 or 6) amino acids can be an epitope, but usually one epitope in a protein is 7 to 15 amino acids, eg 8, 9, 10, 12, or 14 Contains amino acids.
- the antigen is preferably a peptide or epitope in one embodiment. When an antigen is used for the treatment of cancer, such a peptide is also referred to as a cancer peptide.
- the pharmaceutical composition of the present invention may be administered to a living body.
- the administration method It administers by the method according to various formulation forms, a patient's age, sex, a disease state, and other conditions.
- the administration method in the case of tablets, pills, liquids, suspensions, emulsions, granules and capsules include oral administration.
- it can be administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally alone or mixed with a normal fluid such as glucose or amino acid.
- a suppository it is administered intrarectally.
- the pharmaceutical composition of the present invention may be subcutaneous, intramuscular, oral, stamped, intradermal and the like.
- the dosage of the pharmaceutical composition of the present invention may be appropriately selected according to the usage, the patient's age, sex, degree of disease, and other conditions.
- the number of administrations may be appropriately selected according to usage, patient age, sex, disease severity, and other conditions, such as 3 times / 1 day, 2 times / 1 day, 1 time / 1 day, Depending on the blood stability, a less frequent administration frequency (for example, once / week, once / month, etc.) may be selected.
- the pharmaceutical composition of the present invention may provide sustained release properties to the physiologically active substance by gradual cleavage of the sugar chain linker moiety.
- the pharmaceutical composition of the present invention may provide rapid action to a physiologically active substance by rapid cleavage of the sugar chain linker moiety.
- the present invention provides a glycosylation linker or a disease or disease targeted by a physiologically active substance, such as a compound or a salt thereof containing a glycosyl linker moiety and a physiologically active substance moiety. It also relates to the use for the manufacture of a medicament for treatment or prevention.
- the present invention provides a glycosylation linker, or a disease or disease targeted by a physiologically active substance, such as a “compound containing a glycosylated linker moiety and a physiologically active substance moiety or a salt thereof”. It also relates to the use for the treatment or prevention of.
- the physiologically active substance is HER2 or a derivative thereof
- the target disease may be cancer (for example, breast cancer) or the like.
- the sugar chain addition linker of the present invention employs a structure for adding a sugar chain having biodegradable properties, so that adverse effects on the living body are reduced compared to a structure for adding PEG. As a result, long-term administration is expected when it is administered to a living body as a pharmaceutical composition.
- aqueous solution in the present specification may be any liquid as long as a substance (for example, acetic acid) is dissolved in water as a solvent, and any aqueous solution known or new to those skilled in the art is targeted.
- a substance for example, acetic acid
- the emulsion in this specification is not limited, but any emulsion prepared from an aqueous solution may be used.
- the emulsion may be, but is not limited to, an oil-in-water (O / W type) emulsion or a water-in-oil (W / O type) emulsion.
- O / W type oil-in-water
- W / O type water-in-oil
- As a method of dispersing and emulsifying in an aqueous solution a method known to those skilled in the art may be used.
- the “subject” to which the compound of the present invention or a salt thereof, or the pharmaceutical composition of the present invention is administered (applied) includes, but is not limited to, an animal (human, non-human mammal (eg, mouse, rat, dog, cat). , Rabbits, cows, horses, sheep, goats, pigs, etc.) or non-mammals (eg fish, reptiles, amphibians or birds)), plants, insects, bacteria, or cells derived from them (including cultured cells), tissues Or an organ etc. are included.
- the “target” may be an artificial environment (for example, an in vitro reaction system).
- the “subject” in the present invention is a human.
- a conjugate in which a glycosylated linker and a physiologically active substance are bound is referred to as a conjugate.
- a conjugate in which a glycan-added linker having an asialog sugar chain and a part of HER2 which is a physiologically active substance (part containing the 8th to 16th amino acids in the amino acid sequence of HER) are bound to cysteine in the linker Is denoted as a glycosylation (Cys (asialo) type) linker-HER2 (8-16) conjugate.
- HER2 (8-16) corresponds to 8 to 16 amino acid residues in the amino acid sequence of HER2 / neu protein, which is one of HER (Human Epidermal Growth Factor Receptor) family. It is a peptide.
- This HER2 (8-16) has a binding ability to HLA-A24, which is one of HLA (Human Leukocyte Antigen), and cytotoxic T cells (CTL) by antigen presentation via HLA. It is a peptide fragment identified as a tumor vaccine candidate peptide showing inducibility (Tanaka, H., et al., Brit. J. Cancer, 84 (1), 94-99, 2001).
- Rink-Amide-PEGA resin 100 ⁇ mol was taken in a solid phase synthesis column and washed with dichloromethane and DMF. After washing, DMF containing Fmoc-Cys (Trt) -OH (234 mg, 0.399 mmol), HCTU (157 mg, 0.380 mmol), and 2,4,6-trimethylpyridine (79.6 ⁇ L, 0.600 mmol) ( 2.5 mL) solution was added and shaken at room temperature for 10 minutes. After 10 minutes, after washing with DMF, this condensation operation was repeated once more. After completion of the second condensation operation, the resin was washed with DMF and dichloromethane.
- DMF containing Fmoc-Cys (Trt) -OH 234 mg, 0.399 mmol
- HCTU 157 mg, 0.380 mmol
- 2,4,6-trimethylpyridine 79.6 ⁇ L, 0.600 mmol
- the compound 5 Fmoc-Arg (Pbf) -Trp (Boc) -Gly-Leu-Leu on the resin was synthesized on the resin by a peptide solid phase synthesis method by the Fmoc method using a Prelude (trademark) peptide synthesizer.
- -Leu-Ala-Leu-Leu-HMBA-Cys (Trt) (SEQ ID NO: 1) was synthesized.
- the condensation reaction in the solid phase synthesis method was performed in DMF using HCTU as the condensing agent and N-methylmorpholine as the base.
- the Fmoc protecting group on compound 5 was removed by treatment with 20% piperidine in DMF.
- the obtained crude peptide 6 (15.5 mg) was dissolved in a DMSO-0.1M phosphate buffer (pH 7.4) mixed solution (9/1, v / v, 240 ⁇ L) containing 50 mM DTT, and 30 mM asialo- A mixed solution (9/1, v / v, 946 ⁇ L) of DMSO-0.1 M phosphate buffer (pH 7.4) in which BrAc7 was dissolved was added, and the mixture was shaken at room temperature for 2 hours.
- a DMSO-0.1M phosphate buffer (pH 7.4) mixed solution (9/1, v / v, 240 ⁇ L) containing 50 mM DTT, and 30 mM asialo- A mixed solution (9/1, v / v, 946 ⁇ L) of DMSO-0.1 M phosphate buffer (pH 7.4) in which BrAc7 was dissolved was added, and the mixture was shaken at room temperature for 2 hours.
- Example 1-1 The crude peptide 6 (15.5 mg) obtained in Example 1-1 was added to a DMSO-0.1M phosphate buffer (pH 7.4) mixed solution (9/1, v / v, 240 ⁇ L) containing 50 mM DTT. Dissolved. To this mixed solution, a DMSO-0.1M phosphate buffer (pH 7.4) mixed solution (9/1, v / v, 3.8 mL) containing 7.5 mM disialo-BrAc9 was added, and at room temperature. Shake for 5 hours.
- a DMSO-0.1M phosphate buffer (pH 7.4) mixed solution 9/1, v / v, 3.8 mL
- Example 1-3 Solubility measurement
- SEQ ID NO: 5 The solubility of Leu-Leu-Ala-Leu-Leu
- the target glycosylation (Cys (asialo) type) linker-HER2 (8-16) conjugate (compound 1), glycosylation (Cys (disialo) type) linker -HER2 (8- 16) was taken in a microtube, and 30 ⁇ L of water was added. The mixture was shaken at 25 ° C. for 15 minutes and then centrifuged at 25 ° C. and 16100 ⁇ g for 10 minutes.
- the molar extinction coefficient at 280 nm is the glycosylation (Cys (asialo) type) linker-HER2 (8-16) conjugate (Compound 1) or glycosylation (Cys (disialo) type) linker-HER2 (8-16) It calculated
- N Trp represents the number of tryptophan residues
- n Tyr represents the number of tyrosine residues
- n SS represents the number of disulfide bonds.
- the solubility in water of the HER2 (8-16) peptide to which no glycosylated linker was bound was 0.22 mg / mL (2.1 ⁇ 10 2 ⁇ M)). At this time, precipitation of the HER2 (8-16) peptide could be visually confirmed in the microtube.
- the solubility in water of the glycosylated (Cys (asialo) type) linker-HER2 (8-16) conjugate (Compound 1) was confirmed to be 144 mg / mL or more.
- glycosylation (Cys (asialo) type) linker-HER2 (8-16) conjugate (compound 1) and glycosylation (Cys (disialo) type) linker-HER2 (8-16) conjugate It was found that the solubility of (Compound 8) in an aqueous solution improved by 190 times or more in terms of molar concentration compared to the unmodified HER2 (8-16) peptide (Compound 10) (Table 1A). ).
- the solubility of the HER2 (8-16) peptide to which the glycosylated linker was not bound in an aqueous acetic acid solution was 0.52 mg / mL (4.9 ⁇ 10 2 ⁇ M)). At this time, precipitation of the HER2 (8-16) peptide could be visually confirmed in the microtube.
- the solubility of the glycosylated (Cys (asialo) type) linker-HER2 (8-16) conjugate (Compound 1) in an aqueous acetic acid solution was confirmed to be 110 mg / mL or more.
- glycosylation (Cys (asialo) type) linker-HER2 (8-16) conjugate (compound 1) and glycosylation (Cys (disialo) type) linker-HER2 (8-16) conjugate It was found that the solubility of (Compound 8) in an aqueous solution was improved by 69 times or more in terms of molar concentration compared to the unmodified HER2 (8-16) peptide (Compound 10) (Table 1B). ).
- Example 1-4 Tracking hydrolysis behavior in aqueous solution
- the hydrolysis behavior of the linker-HER2 (8-16) conjugate (compound 8) was followed.
- the hydrolysis reaction was started by adding a buffer solution (acetic acid buffer (pH 4.0) or PBS (pH 7.4)) that had been previously set to the reaction temperature (25 ° C. or 37 ° C.) to 8). The temperature during the reaction was maintained at a constant temperature (25 ° C. or 37 ° C.) using a block incubator. The hydrolysis reaction was followed by injecting a certain amount of solution into the HPLC at appropriate time intervals.
- the relative starting material concentration was determined from the HPLC peak area corresponding to the starting material.
- both Compound 1 and Compound 8 are very stable, and the hydrolyzate 10 produced after 48 hours of follow-up is almost 1% or less of the starting material, almost hydrolyzed. It was found that it was not decomposed.
- the peptide having the disialo-glycosylation linker was faster in hydrolysis than the peptide having the asialo-glycosylation linker.
- the peptide having the disialo-glycosylation linker resulted in a slower hydrolysis rate than the peptide having the asialo-glycosylation linker.
- the preferable hydrolysis rate under specific conditions can also be adjusted by selecting the kind of sugar chain to be added.
- Rink-Amide-PEGA resin 100 ⁇ mol was taken in a solid phase synthesis column and washed with dichloromethane and DMF. After washing, contains Fmoc-Cys (tButio) -OH (173.4 mg, 0.402 mmol), HCTU (157 mg, 0.380 mmol), and 2,4,6-trimethylpyridine (79.6 ⁇ L, 0.600 mmol) DMF (2.5 mL) solution was added and shaken at room temperature for 10 minutes. After 10 minutes, the resin was washed with DMF. After washing, the Fmoc protecting group was removed by treatment with 20% piperidine in DMF to obtain Compound 11 in which Cys (StBu (tButio)) was bound on the resin.
- a peptide with a protected amino acid side chain synthesized on a resin with a Prelude (trademark) peptide synthesizer was cleaved from the resin by treatment with an AcOH-TFE (1/1, v / v) solution.
- Peptide in which amino acid side chain is protected by concentrating the filtrate under reduced pressure (Compound 18): Boc-Arg (Pbf) -Trp (Boc) -Gly-Leu-Leu-Leu-Ala-Leu-Leu-Leu (SEQ ID NO: 9) was obtained.
- Compound 23 (2.8 g) was obtained by allowing trityl chloride (2.0 g, 7.1 mmol) to act on 4-mercaptophenylacetic acid (compound 22) (1.0 g, 6.1 mmol) in dichloromethane.
- Compound 24 (62 ⁇ mol), compound 23 (320 ⁇ mol), HOBt (50.7 mg, Gly), a glycopeptide consisting of two amino acids on Rink-Amide-PEGA resin (Asn (asialo) -Gly), 375 ⁇ mol) and a DMF (2.0 mL) solution containing DIC (54 ⁇ L, 522 ⁇ mol) were added, and the mixture was shaken at room temperature for 1 hour to obtain Compound 25 on the resin.
- the solubility of the unmodified HER2- (8-16) peptide was measured.
- the solubility in water of the HER2 (8-16) peptide to which no glycosylated linker was bound was 0.22 mg / mL (2.1 ⁇ 10 2 ⁇ M)).
- precipitation of the HER2 (8-16) peptide could be visually confirmed in the microtube.
- the solubility in water of the thioalkyl-type glycosylated linker-HER2 (8-16) conjugate was 77.4 mg / mL or more.
- Example 3-4 Tracking of hydrolysis behavior in aqueous solution
- the behavior of (Compound 21) during hydrolysis was followed.
- FIGS. 1A and 1B graphs plotting the relative concentration of the starting material against the incubation time for the thioalkyl-type glycosylation linker-HER2 (8-16) conjugate (Compound 20) are shown in FIGS. 1A and 1B. Show. In addition, Table 5 shows the half-life of the thioalkyl-type glycosylated linker-HER2 (8-16) conjugate (Compound 20) under each condition.
- the thioalkyl type glycosylation linker-HER2 (8-16) conjugate having a thioester bond is similar to the glycosylation linker-HER2 (8-16) conjugate having an ester linkage (compounds 1 and 8).
- Compound 21 was also confirmed to have a higher hydrolysis rate as the temperature and / or pH was higher.
- the thioaryl-type glycosylation linker-HER2 (8-16) conjugate (Compound 21) was hydrolyzed and subjected to HPLC analysis. As a result, the thioalkyl-type glycosylation linker-HER2 (8-16) conjugate (compound) As in 20), it was confirmed that an unmodified HER2- (8-16) peptide (Compound 10) was produced.
- the following chemical formula shows the hydrolysis reaction of the thioaryl-type glycosylation linker-HER2 (8-16) conjugate (compound 21), and compound 28 shows the glycosylation linker generated by the hydrolysis reaction of compound 21.
- R represents the following chemical formula.
- glycosylation linker-HER2- (8-16) conjugate having a thioester linkage has a high temperature and / or pH as well as the glycosylation linker-HER2- (8-16) conjugate having an ester linkage. It was confirmed that the hydrolysis rate was faster.
- the hydrolysis rate of compounds 20, 21, and 1 as an asialo-glycosylation linker-HER2 (8-16) conjugate in 37 ° C. in PBS (pH 7.4) is determined by the thioaryl-type sugar having a thioester bond.
- Example 4-1 Synthesis of Glycosylation (Cys (disialo) Type) Linker-Chemerin 9 Conjugate (Compound 29) Since this chemerin 9 has ChemR23 agonist activity which is a G protein-coupled receptor, it has potential as a therapeutic and / or prophylactic agent for immune diseases, inflammatory diseases and diabetes. However, chemerin 9 is known to be very unstable due to degradation by proteolytic enzymes in vivo (Patent Document JP2010-229093). Therefore, a glycosylated linker according to one embodiment of the present invention was introduced into this chemerin 9, and the hydrolysis half-life of the produced conjugate was evaluated.
- Rink-Amide-PEGA resin (100 ⁇ mol) was taken in a solid phase synthesis column and washed with dichloromethane and DMF. After washing, DMF containing Fmoc-Cys (Trt) -OH (234 mg, 0.399 mmol), HCTU (157 mg, 0.380 mmol), and 2,4,6-trimethylpyridine (79.6 ⁇ L, 0.600 mmol) 2.5 mL) solution was added and shaken at room temperature for 10 minutes. After 10 minutes, after washing with DMF, this condensation operation was repeated once more. After completion of the second condensation operation, the resin was washed with DMF and dichloromethane.
- Example 4-2 Tracking of hydrolysis behavior in aqueous solution
- the hydrolysis behavior of the glycosylated (Cys (disialo) type) linker-chemerin 9 conjugate (Compound 29) obtained in Example 4-1 was followed.
- FIG. 4 shows a graph plotting the relative concentration of the starting material against the incubation time for the glycosylated (Cys (disialo) type) linker-chemerin 9 conjugate (Compound 29) in the same manner as in Example 1-4.
- Table 7 shows the half-life of the glycosylated (Cys (disialo) type) linker-chemerin 9 conjugate (Compound 29) under each condition.
- the hydrolyzate (compound 33) produced after 49 hours of follow-up in the acetate buffer (0.1 M, pH 4.0) at 37 ° C. was 1 of the starting material (compound 29). % was found to be hardly hydrolyzed (in addition, the elimination of sialic acid present at the non-reducing end in the sugar chain structure was observed. The content of the desialic acid was determined by the hydrolysis behavior test. Before and after the start, it increased from 4.7% to 8.7%). On the other hand, hydrolysis reaction proceeds in PBS (pH 7.4) and borate buffer (pH 9.0), and an unmodified peptide (compound 33) having the amino acid sequence of chemerin 9 is obtained. It was.
- the half-life determined from the obtained curve was 45.0 hours (1.9 days) in PBS at 37 ° C. Further, as a result of tracing the hydrolysis behavior in a borate buffer solution (0.1 M, pH 9.0) at 37 ° C., the half-life was 0.83 hours (50 minutes). Further, when a glycosylated (Cys (disialo) type) linker-chemerin 9 conjugate (compound 29) was dissolved in a 50 mM aqueous sodium hydroxide solution, it disappeared completely in 2 minutes, and the amino acid sequence having chemerin 9 amino acid sequence was lost. A modified peptide (compound 33) was produced. As reported in the literature, it was confirmed that hydrolysis was very rapid under basic conditions.
- the conjugate to which the glycosylated linker of the present invention was bound was prepared in a low pH solution at room temperature before administration, and the compound did not hydrolyze. It can be expected that the peptide will be decomposed and exhibit the original activity of the peptide.
Abstract
Description
すなわち、本発明は、その一態様において、少なくとも1つのカルボキシ基を有する生理活性物質との結合用の、糖鎖付加リンカーであって、
前記糖鎖付加リンカーは、下記式(A)で表わされ、
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記糖鎖付加リンカーは、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質のカルボキシ基と結合することができることを特徴とする。
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する硫黄原子(S)を意味し、
R1は、-R3-R4-、-R4-R5-、または-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドである。]
であることを特徴とする。
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、-R3-R4-、または-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、または置換もしくは非置換のC5~C16ヘテロアリールであり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドである。]
であることを特徴とする。
で表される糖鎖であることを特徴とする。
前記生理活性物質は、少なくとも1つのカルボキシ基を有しており、
前記糖鎖付加リンカー部分は、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質部分のカルボキシ基とエステル結合またはチオエステル結合を形成し、前記生理活性物質部分と結合している、
化合物またはその塩を提供する。
(I)請求項10に記載の化合物またはその塩、および
(II)薬理学的に許容される担体
を含む、医薬組成物を提供する。
ここで、糖鎖付加リンカーは、下記式(A)で表わされ、
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記生理活性物質は、少なくとも1つのカルボキシ基を有しており、
前記方法は、以下のステップ、
(a)前記糖鎖付加リンカーにおける脱離基を有する酸素原子(O)または硫黄原子(S)と、前記生理活性物質のカルボキシ基との間で、エステル結合またはチオエステル結合が形成されるように縮合反応を行うステップを含む、
製造方法を提供する。
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
をさらに含むことを特徴とする。
前記ステップ(a’)および/または前記ステップ(a)が、樹脂上で行われることを特徴とする。
前記生理活性物質が、少なくとも1つのカルボキシ基を有しており、
前記方法は、以下のステップ、
(a)樹脂に、下記式(B)で表わされるリンカーを結合するステップであって、
前記リンカーは、下記式(B)で表わされ、
X-R1-Y-R2 (B)
[式(B)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、アミノ酸、もしくはポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、アミノ酸、またはポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記リンカーにおけるR2のアミノ酸またはポリペプチドのカルボキシ基と前記樹脂とを結合するステップと、
(b)前記樹脂に結合した前記リンカーと前記生理活性物質とを結合するステップであって、前記リンカーは、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質のカルボキシ基とエステル結合またはチオエステル結合を形成し、前記生理活性物質と結合するステップと、
(c)前記リンカーにおけるR2のアミノ酸またはポリペプチドの側鎖に、糖鎖を付加するステップと
を含む、製造方法であることを特徴とする。
また、本発明に係る、糖鎖付加リンカーは、光や酵素的切断に依らずに、特定の条件下(例えば、生体内)において当該糖鎖付加リンカーと結合した生理活性物質を放出することができる。
X-R1-Y-R2 (A)
本明細書において、「脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)」とは、式(A):X-R1-Y-R2で表わされる糖鎖付加リンカーのXの位置存在する原子であって、当該原子に結合する脱離基が脱離することにより生理活性物質と結合可能な原子をいう。当該原子と生理活性物質との結合は、生理活性物質中のカルボキシ基を介して行われる。
ここで、脱離基としては、生理活性物質のカルボキシ基と脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)とが結合する際に脱離するものであれば限定されず、例えば、水素原子や、一価の陽イオンとなるリチウム、ナトリウム、カリウム、ルビジウム、セシウム、フランシウム、銀等である。
また、「置換もしくは非置換のC5~C16アリール」は、上記に限定されず、「C5~C16アリール」における1つまたは複数の水素原子は、それぞれ独立に「置換基」により置換されたものも含む。「置換基」の例としては、C1~C4アルキル基、C1~C4アルコキシ基(例えば、メトキシ、エトキシ、プロポキシ、ブトキシ等)、アミノ基、ヒドロキシ基、チオール基、カルボキシ基、ニトロ基、メシル基、トシル基、ハロゲン原子(例えば、フッ素、塩素、臭素、ヨウ素)、C1~C4ハロゲン化アルキル基(例えば塩化メチル基)、フェニル基、o-トリル基、m-トリル基、p-トリル基、キシリル基、エチルフェニル基またはベンジル基等を挙げることができる。
また、「置換もしくは非置換のC5~C16ヘテロアリール」は、上記に限定されず、「C5~C16ヘテロアリール」の環構造を形成する炭素原子に結合する1つまたは複数の水素原子が、それぞれ独立に「置換基」により置換されたものを含む。「置換基」の例としては、アルキル基、アルコキシ基(例えば、メトキシ、エトキシ、プロポキシ、ブトキシ等)、ヒドロキシ基、カルボキシ基、ニトロ基、メシル基、ハロゲン原子(例えば、フッ素、塩素、臭素、ヨウ素)、ハロゲン化アルキル基(例えば塩化メチル基)等を挙げることができる。
また、本発明において、好ましい糖鎖は、糖鎖付加リンカーとして生理活性物質に付加された場合に、当該生理活性物質の抗原性を低減する糖鎖である。
このような、本発明の糖鎖付加リンカーにおける糖鎖は特に限定されず、生体内で複合糖質(糖ペプチド(または糖タンパク質)、プロテオグリカン、糖脂質等)として存在する糖鎖であってもよいし、生体内では複合糖質として存在しない糖鎖であってもよい。
さらに、本発明の複合型糖鎖には、フコースが付いたものも含む。フコースが付いた複合型糖鎖としては、以下の構造式で表わされるフコース含有複合型糖鎖
また、本明細書中において「2本鎖複合型糖鎖」、「ジシアロ糖鎖」、「モノシアロ糖鎖」、「アシアロ糖鎖」、「ジグルクナック糖鎖」、「ジマンノース糖鎖」、「3本鎖複合型糖鎖」、「4本鎖複合型糖鎖」、「フコース含有複合型糖鎖」には、上記化学式で示したもののほか、化学式で示した例と結合様式の異なるものも含まれ、かかる糖鎖も本発明の糖鎖として好ましく用いられる。かかる糖鎖としては、例えば、ジシアロ糖鎖またはモノシアロ糖鎖においてシアル酸とガラクトースが(α2→3)結合で結合しているもの等が挙げられる。
ハイマンノース-5(M-5)
本発明に用いられるオリゴヒアルロン酸のうち、特に好ましいものとして、N-アセチルグルコサミンとグルクロン酸とからなる単位を1単位とした場合、2単位(4糖)以上8単位(16糖)以下の糖鎖が挙げられ、さらに好ましくは、2単位(4糖)~4単位(8糖)、最も好ましくは2単位(4糖)である。
本発明に好ましく用いられるヒアルロン酸としては、例えば、
4糖のオリゴヒアルロン酸、
-NH-(CH2)a-(CO)-CH2-
(式中、aは整数であり、目的とするリンカー機能を阻害しない限り限定されるものではないが、好ましくは0~4の整数を示す。);
C1-10ポリメチレン;
-CH2-R-;
(ここで、Rは、アルキル、置換されたアルキル、アルケニル、置換されたアルケニル、アルキニル、置換されたアルキニル、アリール、置換されたアリール、炭素環基、置換された炭素環基、複素環基及び置換された複素環基からなる群より選択される基から水素原子が1つ脱離して生ずる基である。)
-(CO)-(CH2)a-(CO)-
(式中、aは整数であり、目的とするリンカー機能を阻害しない限り限定されるものではないが、好ましくは0~4の整数を示す。)
等を挙げることができる。
本明細書において、チオアルキル型の糖鎖付加リンカーとは、生理活性物質とチオエステル結合を介して結合可能な糖鎖付加リンカーであって、その構造内にアルキルの構造を有する糖鎖付加リンカーをいう。
また、本明細書において、チオアルキル型の糖鎖付加リンカーには、生理活性物質とチオエステル結合を介して結合可能な糖鎖付加リンカーであって、その構造内にアルキニル、またはアルケニルの構造を有する糖鎖付加リンカーも含まれる。
より具体的には、チオアルキル型の糖鎖付加リンカーは、
上記式(A)におけるXが脱離基を有する硫黄原子(S)であり、
上記式(A)におけるR1が、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC1~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
上記式(A)におけるYは、式(A)中に存在していても、存在していなくてもよく、Yが式(A)中に存在する場合には、Yは、-CO-、または-CONH-(ただし、Cが式(A)中のR1と結合し、Nが式(A)中のR2と結合する)であり、
上記式(A)におけるR2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである、
糖鎖付加リンカーである。
本明細書において、チオアリール型の糖鎖付加リンカーとは、生理活性物質とチオエステル結合を介して結合可能な糖鎖付加リンカーであって、その構造内にアリールの構造を有する糖鎖付加リンカーをいう。
より具体的には、チオアルキル型の糖鎖付加リンカーは、
上記式(A)におけるXが脱離基を有する硫黄原子(S)であり、
上記式(A)におけるR1が、置換もしくは非置換のC1~C5アリール、または置換もしくは非置換のC5~C16ヘテロアリールであり、
上記式(A)におけるYは、式(A)中に存在していても、存在していなくてもよく、Yが式(A)中に存在する場合には、Yは、-CO-、または-CONH-(ただし、Cが式(A)中のR1と結合し、Nが式(A)中のR2と結合する)であり、
上記式(A)におけるR2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである、
糖鎖付加リンカーである。
均一な糖鎖の割合や均一な糖鎖付加リンカーの割合は、例えば、HPLC、キャピラリー電気泳動、NMR、質量分析等を用いた方法によって測定することが可能である。
また、糖鎖付加工程で用いる糖鎖の製造方法に関しては、例えば、国際公開第03/008431号パンフレット、国際公開第2004/058984号パンフレット、国際公開第2004/008431号パンフレット、国際公開第2004/058824号パンフレット、国際公開第2004/070046号パンフレット、国際公開第2007/011055号パンフレット等を参照してよい。
また、本明細書において「PEG」とは、エチレングリコールの重合体であり、例えば、「(‐CH2‐CH2‐O‐)n」(nは、2~10000の整数)で表わすことができる。
式(A):X-R1-Y-R2で表わされる糖鎖付加リンカーにおいて、
Xが、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1が、ベンジル、またはトリル等で表わされるアリールであるか、またはR1が、-R3-R4-R5-を意味し、R3が-CH2CH2-であり、R4が硫黄原子(S)であり、R5が-CH2-であり(すなわち、R1が、-CH2CH2SCH2-で表わされるチオエーテルであり)、
Yが、-CO-を意味し、
R2が、NH-糖鎖、糖鎖付加Asn、糖鎖付加Cys、または糖鎖付加Asnもしくは糖鎖付加CysのC末端にアミノ酸が1つ又は複数(例えば、2つ、3つ、4つ、5つ)付加したものを意味する、糖鎖付加リンカーである。
例えば、固相合成により式(A):X-R1-Y-R2で表わされる糖鎖付加リンカーを製造する場合には、樹脂上に、R2、Y(存在する場合のみ)、R1、Xの順で、適当な化合物を樹脂上に結合させていく。このとき、R2、Y、R1、Xは、各構成ごとに順に樹脂上に結合させてもよいし、連続する複数の構成に相当する化合物を樹脂上に結合させることもできる。連続する複数の構成に相当する化合物を樹脂上に結合させる例としては、例えば、まず、樹脂上に、脱離基を有するR2を結合させる。次に、Yの末端に脱離基を有するX-R1-Yの1つの化合物を、樹脂上のR2と縮合させることにより、R2およびYの脱離基が脱離し、樹脂上に、式(A):X-R1-Y-R2で表わされる糖鎖付加リンカーを作製することができる。なお、連続する複数の構成に相当する化合物は、X-R1-Yに限らず、X、R1、Y、R2の4つの構成から2つ以上が選択されるその他の組み合わせも含む。
すなわち、固相合成法による製造方法は、
樹脂上に、脱離基を有するR2の化合物(糖鎖、糖鎖付加アミノ酸、糖鎖付加ポリペプチド等)を結合させる工程と、
樹脂上のR2に対して、少なくとも2つの脱離基を有するYを結合させる工程であって、R2とYとの脱離基が脱離することにより、R2とYとが結合する工程と、
樹脂上のY-R2に対して、少なくとも2つの脱離基を有するR1の部分に相当する化合物を結合させる工程であって、YとR1との脱離基が脱離することにより、YとR1とが結合する工程と、
樹脂上のR1-Y-R2に対して、少なくとも2つの脱離基を有するXの部分に相当する化合物を結合させる工程であって、R1とXとの脱離基が脱離することにより、R1とXとが結合する工程と、
樹脂上に合成されたX-R1-Y-R2を樹脂から切り離す工程と
を含む。
樹脂上に、脱離基を有するR2の化合物(糖鎖、糖鎖付加アミノ酸、糖鎖付加ポリペプチド等)を結合させる工程と
樹脂上のR2に対して、Yの末端に脱離基を有するX-R1-Yの化合物を結合させる工程であって、R2とYとの脱離基が脱離することにより、R2とYとが縮合し樹脂上にX-R1-Y-R2を形成させる工程と、
樹脂上に合成されたX-R1-Y-R2を樹脂から切り離す工程と
を含む製造方法により、糖鎖付加リンカーを作製することができる。
なお、固相合成法における糖鎖付加リンカーの製造方法において、樹脂上に形成された糖鎖付加リンカーを樹脂より切断することなく、さらにアミノ酸を連結させることができる(より具体的には、Xで表わされる脱離基を有する酸素原子または脱離基を有する硫黄原子へさらにアミノ酸を連結させることができる)。これにより、樹脂より糖鎖付加リンカーを切り離す必要なく、樹脂上で生理活性物質部分と糖鎖付加リンカー部分とを含む化合物を製造することができる。
また、固相合成により製造された糖鎖付加リンカー内に存在するアミノ酸またはペプチドのC末端をアミド化する場合には、例えば、アミノ基で官能化されたRink-Amide-PEGAレジン(メルク社製)を用いることができる。このレジンとペプチドを酸で切断することにより、糖鎖付加リンカーにおけるアミノ酸またはペプチドのC末端アミノ酸をアミド化することができる。
なお、2-クロロトリチルクロリド樹脂は、固相合成においてペプチド鎖を伸長する際、末端にあるCysのラセミ化を防止することができる点において、好ましい。
また、脂溶性保護基で保護したアミノ酸であって、側鎖に保護基を導入したものとして、例えば、Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Cys(StBu)-OH、Fmoc-Cys(tBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-His(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Tyr(tBu)-OH、Boc-Arg(di-Z)-OH、Fmoc-Asp(OBzl)-OH、Boc-Cys(Bzl)-OH、Boc-Glu(OBzl)-OH、Boc-His(Dnp)-OH、Boc-Lys(2-Cl-Z)-OH、Boc-Ser(Bzl)-OH、Boc-Thr(Bzl)-OH、Boc-Trp(For)-OH、Boc-Tyr(Bzl)-OHを挙げることができる。
なお、これらの糖鎖付加アミノ酸は、上述した糖鎖であって、同一の糖鎖構造を有する糖鎖が付加されたアミノ酸を用いる。このような糖鎖としては、任意の公知の方法により得ることができる。具体的な手法としては、限定されることなく、例えば、糖鎖を化学合成すること(例えば、J.Seifert et al. Angew Chem Int.Ed. 2000, 39, p531-534参照)や、天然または人工の糖鎖給源から分離したものや市販されているものを利用することができる。当該手法において、同一の構造を有する糖鎖付加アミノ酸としては、限定されることなく、例えば、天然または人工の糖鎖給源からの同一構造の糖鎖の分離は、例えば、WO2004/058789に記載の方法により行うことができる。具体的には、鶏卵などの天然の糖鎖給源から、Seko et al., Biochim Biophys Acta. 1997;1335(1-2):23-32などに記載の方法で糖鎖アスパラギンを含む混合物(シアリルグリコペプチド(SGP))を単離し、該糖鎖アスパラギンに脂溶性の保護基(例えば、Fmoc)を導入して糖鎖アスパラギン誘導体混合物を得、これをクロマトグラフィーに供することにより、該混合物に含まれる種々の構造の糖鎖を、その構造に応じて分離することができる。また、種々の保護基を有するまたは有しない特定の構造の糖鎖アスパラギンは、例えば、株式会社糖鎖工学研究所より入手可能である。
その際、縮合剤として、ジシクロヘキシルカルボジイミド(DCC)、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド・塩酸塩(WSC/HCl)、ジフェニルホスホリルアジド(DPPA)、カルボニルジイミダゾール(CDI)、ジエチルシアノホスホネート(DEPC)、1,3-ジイソプロピルカルボジイミド(DIC)、ベンゾトリアゾール-1-イルオキシ-トリスピロリジノホスホニウムヘキサフルオロホスフェート(PyBOP)、3-ジエトキシホスホリルオキシ-1,2,3-ベンゾトリアジン-4(3H)-オン(DEPBT)、1-ヒドロキシベンゾトリアゾール(HOBt)、ヒドロキシスクシンイミド(HOSu)、ジメチルアミノピリジン(DMAP)、1-ヒドロキシ-7-アザベンゾトリアゾール(HOAt)、3-ヒドロキシ-4-オキソ-3,4-ジヒドロ-5-アザベンゾ-1,2,3-トリアジン(HODhbt)、ヒドロキシフタルイミド(HOPht)、ペンタフルオロフェノール(Pfp-OH)、2-(1H-ベンゾトリアゾール-1-イル)-1,1,3,3-テトラメチルウロニウム ヘキサフルオロホスフェート(HBTU)、O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate(HCTU)、O-(7-アザベンゾトリアゾール-1-イル)-1,1,3,3-テトラメチルウロニウム ヘキサフルオロホスホネート(HATU)、O-ベンゾトリアゾール-1-イル-1,1,3,3-テトラメチルウロニウム テトラフルオロボレート(TBTU)等を用いることができる。アミノ酸または糖鎖付加アミノ酸と脱水縮合剤との使用割合は、前者1重量部に対して、後者が、通常1~10重量部、好ましくは2~5重量部である。
また、DMSO、DMF、メタノール、アセトニトリルといった有機溶媒と、上記の緩衝液との混合溶液中で反応を行うこともできる。このとき、有機溶媒の比率は、0~99%(v/v)の範囲で、上記緩衝液に添加することができる。緩衝液への溶解性が低い無保護のCysを含むペプチドは、このような有機溶媒を添加することにより反応溶液への溶解性を向上させることができ、好ましい。
また、DMSO、DMF、メタノール、アセトニトリルといった有機溶媒や、それらの混合溶液中で反応を行うこともできる。その際、塩基の存在下で行うのが好ましい。塩基としては、例えばDIPEA、トリエチルアミン、ピリジン、2,4,6-コリジン等を挙げることができる。
また、グアニジン塩酸塩や尿素を緩衝溶液に加えた混合溶液中においても反応を行うことができる。なお、グアニジン塩酸塩や尿素は、最終濃度が1M~8Mとなるように上記緩衝液に加えることができる。グアニジン塩酸塩や尿素の添加によっても、緩衝液への溶解性の低いペプチドの溶解性を向上させることができ、好ましい。
また、ハロアセチル化体(またはハロアセトアミド化体)等にした核酸またはPEGとCys含有ペプチドとの反応についても、当業者は公知の方法により適宜実施することができる。
また、当業者は、連続する複数の構成に相当する化合物を樹脂上へ結合する場合も、適宜、化合物および反応条件を設計・選択することができる。
なお、糖鎖付加リンカーのXに相当する部分(脱離基を有する酸素原子または脱離基を有する硫黄原子)は合成上保護基を必要とする場合がある。酸素原子の保護基としては、トリチル基、メトキシトリチル基、t-ブチル基、ベンジル基等を挙げることができ、硫黄原子の保護基としては、トリチル基、メトキシトリチル基、t-ブチル基、t-ブチルチオ基、Acm基等を挙げる事ができる。保護基の導入は、従来周知の方法により行うことができる。
また、樹脂上に、糖鎖付加リンカーと生理活性物質とが結合した化合物を合成した際にも、樹脂から当該化合物を切り離す工程は、酸で処理するのが好ましい。用いる酸や反応条件は、糖鎖付加リンカーを樹脂から切り離す条件と同様に行うことができる。
また、生理活性物質と結合した糖鎖付加リンカーは、その構造に依存して、特定の温度およびpHの条件下、当該生理活性物質を一定の時間内に遊離させることができる。なお、この遊離した生理活性物質は、本来の機能を有しており、例えば、生体内で糖鎖付加リンカーから遊離した生理活性物質は、本来の機能を発揮する。
また、糖鎖付加リンカー部分と生理活性物質部分との結合が、エステル結合よりもチオエステル結合である方が、加水分解速度を速くすることが可能である。また、チオエステル結合のうち、チオアルキル構造を有する糖鎖付加リンカーよりもチオアリール構造を有する糖鎖付加リンカーの方がより速く加水分解される。
当業者は、糖鎖付加リンカー部分の構造を適宜変更することで所望の生理活性物質放出時間を有する糖鎖付加リンカーを設計することができる。
糖鎖付加リンカーと生理活性物質との結合は、糖鎖付加リンカーの脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)と生理活性物質の少なくとも1つのカルボキシ基とが縮合反応し、エステル結合またはチオエステル結合を介して結合する。
この縮合反応の条件は、当業者が適宜設定することができる。例えば、縮合反応の縮合剤には、PyBOP、DMAP、HCTU等を使用することができる。また、縮合反応の溶媒として、例えば、DMF、DMSO、ジクロロメタン等を使用することができる。縮合反応の反応は、例えば、アミノ酸側鎖が保護されたペプチドとチオール基を有する糖鎖付加リンカーをDMFに溶解させ、PyBOPおよびDIPEAを添加することで行うことができる。このとき、生理活性物質がペプチドである場合、ペプチドのC末端アミノ酸の異性化を抑制できることから、低温(-15℃~-30℃)で反応を行うことが好ましい。
また、生理活性物質がペプチドである場合には、当該ペプチドの側鎖は保護基により保護されていることが好ましい。ペプチドの側鎖を保護している保護基は、糖鎖付加リンカーと当該ペプチドとの結合後に脱保護することができる。ペプチドの側鎖を保護する保護基は、当業者に周知の保護基を使用することができ、例えば、上述した固相合成に用いるアミノ酸の保護基を使用することができる。また、ペプチドへの保護基の導入や脱保護も、当業者は適宜実施することができる。
本発明の「組成物」は、1種以上の本発明の化合物またはその塩に加えて、任意の1種以上の他の成分(活性成分または不活性成分)を含む。本発明の組成物の使用は、特に限定されないが、例えば、アッセイ系(例えばin vitroアッセイ系等)に用いてよい。
特に、糖鎖が均一である糖鎖付加リンカー部分を含む組成物等は、品質が一定であり、特に医薬品の製造や、アッセイなどの分野において好ましい。均一な糖鎖の割合や均一な糖鎖付加リンカーの割合は、例えば、HPLC、キャピラリー電気泳動、NMR、質量分析等を用いた方法によって測定することが可能である。
本発明の実施態様は模式図を参照しつつ説明される場合があるが、模式図である場合、説明を明確にするために、誇張されて表現されている場合がある。
第一の、第二のなどの用語が種々の要素を表現するために用いられるが、これらの要素はそれらの用語によって限定されるべきではないことが理解される。これらの用語は一つの要素を他の要素と区別するためのみに用いられているのであり、例えば、第一の要素を第二の要素と記し、同様に、第二の要素は第一の要素と記すことは、本発明の範囲を逸脱することなく可能である。
Ac:アセチル(基)
AcOH:酢酸
Asn:アスパラギン
Boc:tert-ブチルオキシカルボニル基
BrAc:ブロモアセトアミド
Cys:システイン
DIC:ジイソプロピルカルボジイミド
DIPEA:N,N-ジイソプロピルエチルアミン
DMAP:4-ジメチルアミノピリジン
DMF:N,N-ジメチルホルムアミド
DMSO:ジメチルスルホキシド
DTT:ジチオトレイトール
ESI-MS:エレクトロスプレーイオン化(ElectroSpray Ionization)質量分析
Fmoc(基):9-フルオレニルメチルオキシカルボニル(基)
HCL:塩酸
HCTU:O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
HMPB:4-ヒドロキシメチル-3-メトキシフェノキシ酪酸
HMBA:4-ヒドロキシメチル安息香酸
HOBt:1-ヒドロキシベンゾトリアゾール
HPLC:高速液体クロマトグラフィー
H2O:水
MSNT:1-(メシチレン-2-スルホニル)-3-ニトロ-1,2,4-トリアゾール(1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole)
PBS:リン酸緩衝生理食塩水
Pbf:2,2,4,6,7-ペンタメチルジヒドロベンゾフラン-5-スルホニル
SPPS:ペプチド固相合成
tBu:tert-ブチル基
TFA:トリフルオロ酢酸
Trt:トリチル基
なお、HER2(8-16)は、HER(ヒト上皮増殖因子受容体:Human Epidermal Growth Factor Receptor)ファミリーの1つであるHER2/neuタンパク質のアミノ酸配列のうち、8~16アミノ酸残基に相当するペプチドである。このHER2(8-16)は、HLA(ヒト白血球抗原分子:Human Leukocyte Antigen)の一つであるHLA-A24に結合能を有し、HLAを介した抗原提示により細胞傷害性T細胞(CTL)誘導能を示す、腫瘍ワクチン候補ペプチドとして同定されたペプチドフラグメントである(Tanaka,H.,et al.,Brit.J.Cancer,84(1),94-99,2001)。
化合物5上のFmoc保護基を、DMF中の20%のピペリジンで処理することにより除去した。DMFおよびジクロロメタンで洗浄後、TFA:トリイソプロピルシラン:エタンジチオール:水(=90:5:2.5:2.5)を加え、室温で3時間振盪した。ろ液に冷却したエーテルを加え、粗ペプチド6:Arg-Trp-Gly-Leu-Leu-Leu-Ala-Leu-Leu-HMBA-Cys(配列番号2)を沈殿として得た。
得られた粗ペプチド6(15.5mg)を50mM DTTを含むDMSO-0.1Mリン酸緩衝液(pH7.4)混合溶液(9/1、v/v、240μL)に溶解し、30mMアシアロ-BrAc7が溶解したDMSO-0.1Mリン酸緩衝液(pH7.4)混合溶液(9/1、v/v、946μL)を添加し、室温で2時間振盪した。
この画分を、HPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ20x250mm、流速:7.0mL/分、溶離液 A:0.1%AcOH水 B:0.09%AcOH/10%水/90%アセトニトリル、グラジエント A:B=75:25→60:40(30分)直線濃度勾配溶出]を用いてさらに精製し、糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)(17.2mg、5.79μmol)を得た。
ESI-MS calcd for C127H204N20O58S [M+2H]2+ 1485.7、[M+3H]3+ 990.8、[M+4H]4+ 743.3、found 1485.7、990.8、743.3。
この画分を、HPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ20x250mm、流速:7.0mL/分、溶離液 A:0.1%AcOH水 B:0.09%AcOH/10%水/90%アセトニトリル、グラジエント A:B=70:30→50:50(30分)直線濃度勾配溶出]を用いてさらに精製し、糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)(21.8mg、6.13μmol)を得た。
ESI-MS calcd for C149H238N22O74S [M+2H]2+ 1776.8、[M+3H]3+ 1184.8、[M+4H]4+ 888.9、found 1776.8、1184.8、888.9。
上記の実施例1-1および実施例1-2で得られた糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)と、糖鎖付加リンカーを有さない無修飾のHER2-(8-16)ペプチド(化合物10)(Arg-Trp-Gly-Leu-Leu-Leu-Ala-Leu-Leu)(配列番号5)とについて、水溶液への溶解度を測定した。
(参考文献:C. N. Pace et al., Prot. Sci., 1995, 4, 2411-2423)。
実施例1-1および実施例1-2で得られた糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)の加水分解時の挙動を追跡した。凍結乾燥した糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)に対し、あらかじめ反応温度(25℃または37℃)にした緩衝溶液(酢酸緩衝液(pH4.0)またはPBS(pH7.4))を加えることで、加水分解反応を開始した。また、反応中の温度は、ブロックインキュベーターを用いて一定の温度(25℃または37℃)となるように維持した。適当な時間間隔で、一定の量の溶液をHPLCにインジェクトすることで加水分解反応を追跡した。出発物質に相当するHPLCのピーク面積から相対出発物質濃度をもとめた。インキュベーション時間に対して、出発物質の相対濃度をプロットした。その結果、直線状のプロットが得られたことから、加水分解反応が一次反応であることが示された。また、出発物質の消失半減期t1/2を、式t1/2=ln(2)/k(kは直線プロットの傾き)から算出した。各条件下における糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)の半減期を、表2及び表3に示す。
<チオール基を有する糖鎖付加リンカー(化合物17)の合成>
ESI-MS calcd for C66H111N5O46S2: [M+2H]2+ 888.36、found 888.34。
得られたペプチド(化合物18)(63.5mg、41.8μmol)、チオール基を有する糖鎖付加リンカー(化合物17)(41.5mg、23.4μmol)、PyBOP(121.8mg、234μmol)をDMF(1mL)に溶解させ、窒素雰囲気下、-15℃に冷却した。この溶液に、DIPEA(40.0μL、40.7μmol)を加え-15℃で撹拌した。3時間後、TFA(100μL)を加え、減圧下で濃縮乾固した。得られた残渣に、TFA-H2O(95/5、v/v)溶液(1mL)を加え、3時間撹拌した。溶液にエーテルを加え、粗ペプチドを沈殿として得た。得られた粗ペプチドをHPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ20x250mm、流速:7.0mL/分、溶離液 A:0.1%TFA水 B:0.09%TFA/10%水/90%アセトニトリル、グラジエント A:B=65:35→35:55(30分)直線濃度勾配溶出]を用いて精製し、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20):Arg-Trp-Gly-Leu-Leu-Leu-Ala-Leu-Leu-(sugar chain added-linker:SCH2CH2SCH2CONH(asialo))(配列番号10)(6.9mg、収率10.5%)を得た。
ESI-MS calcd for C118H196N18O55S2: [M+2H]2+ 1406.52、[M+3H]3+ 938.01、found 1406.10、937.73。
ESI-MS calcd for C76H120N8O49S: [M+2H]2+ 981.34、found 981.37。
得られた粗ペプチドをHPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ20x250mm、流速:7.0mL/分、溶離液 A:0.1%TFA水 B:0.09%TFA/10%水/90%アセトニトリル、グラジエント A:B=65:35→35:55(30分)直線濃度勾配溶出]を用いて精製し、チオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21):Arg-Trp-Gly-Leu-Leu-Leu-Ala-Leu-Leu-(sugar chain added-linker:4-thiobenzoic acid-Asn(asialo)-Gly)(配列番号11)(2.3mg、収率17.5%)を得た。
ESI-MS calcd for C128H205N21O58S1: [M+2H]2+ 1500.09、[M+3H]3+ 1000.39、found 1499.67、1000.08。
糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物7)の代わりに、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)およびチオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)を用いた以外は、実施例1-3と同様にして、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)およびチオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)の水への溶解度を測定した。なお、比較として、無修飾のHER2-(8-16)ペプチド(化合物10)の溶解度を測定した。その結果、糖鎖付加リンカーが結合していないHER2(8-16)ペプチドの、水への溶解度は、0.22mg/mL(2.1×102μM))であった。この時、マイクロチューブ中、HER2(8-16)ペプチドの沈殿を目視で確認することができた。一方、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)の、水への溶解度は、77.4mg/mL以上であることを確認した。驚くべきことに、77.4mg/mLの濃度でさえも、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)の沈殿は確認できなかった。また、チオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)の、水への溶解度は、76.7mg/mL以上であることを確認した。驚くべきことに、76.7mg/mLの濃度でさえも、チオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物8)の沈殿は確認できなかった。これらの結果より、チオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)およびチオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)の水溶液への溶解度は、無修飾のHER2-(8-16)ペプチド(化合物10)と比較して、モル濃度にしていずれも100倍以上溶解度が向上することが分かった。
実施例3-1および実施例3-2で得られたチオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)およびチオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)の加水分解時の挙動を追跡した。具体的には、凍結乾燥した糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)の代わりに、凍結乾燥したチオアルキル型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物20)およびチオアリール型糖鎖付加リンカー-HER2(8-16)コンジュゲート(化合物21)を用い、反応温度を4℃、25℃、または37℃とした以外は、実施例1-4と同様にして、加水分解の挙動を追跡した。
このケメリン9は、Gタンパク質共役型レセプターであるChemR23アゴニスト活性を有することから、免疫疾患、炎症性疾患、糖尿病の治療及び/又は予防剤としての可能性を有している。しかしながら、ケメリン9は、生体内においてタンパク分解酵素による分解を受け、非常に不安定であることが知られている(特許文献JP2010-229093)。そこで、このケメリン9に、本発明の一実施の態様である糖鎖付加リンカーを導入し、製造されたコンジュゲートの加水分解半減期の評価を行った。まず、エステル結合を介して、糖鎖付加(Cys(disialo)型)リンカーとケメリン9(配列番号12:YFPGQFAFS、特許文献US2003096299に開示される、配列番号31-36のアミノ酸配列に相当)が結合したコンジュゲートを合成した。
得られた化合物3が結合している樹脂の一部(50μmol)に、Fmoc-Ser(tBu)-OH(96.9mg、0.253mmol)、MSNT(74.1mg、0.250mmol)、およびN-メチルイミダゾール(14.0μL、0.177mmol)を含むジクロロメタン(2.5mL)溶液を加え、室温で1時間振盪した。1時間振盪後、ジクロロメタンおよびDMFで洗浄した。洗浄後、Fmoc保護基をDMF中の20%のピペリジンで処理することにより除去することで、樹脂上にSer(tBu)-HMBA-Cys(Trt)が結合した化合物30を得た。DMFで洗浄後、Prelude(商標)ペプチド合成機を用いて、Fmoc法によるペプチド固相合成法にて樹脂上にアミノ酸側鎖が保護されたペプチドが結合した化合物31:Fmoc-Tyr(tBu)-Phe-Pro-Gly-Gln(Trt)-Phe-Ala-Phe-Ser(tBu)-HMBA-Cys(Trt)(配列番号13)を合成した。固相合成法における縮合反応は、縮合剤としてHCTU、塩基としてN-メチルモルホリンを使用してDMF中で行った。
化合物31上のFmoc保護基を、DMF中の20%のピペリジンで処理することにより除去した。DMFおよびジクロロメタンで洗浄後、TFA:トリイソプロピルシラン:エタンジチオール:水(=90:5:2.5:2.5)を加え、室温で3時間振盪した。ろ液に冷却したエーテルを加え、粗ペプチド32:Tyr-Phe-Pro-Gly-Gln-Phe-Ala-Phe-Ser-HMBA-Cys(配列番号14)を沈殿として得た。
得られた粗ペプチド32(14.2mg)、ジシアロ-BrAc9(41.6mg、17.7μmol)、およびTCEP(16.0mg、55.8μmol)を7Mグアニジン塩酸塩を含む0.2Mリン酸緩衝液(pH6.8、1.15mL)に溶解し、室温で反応させた。3時間後、反応溶液をHPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ20x250mm、流速:7.0mL/分、溶離液 A:0.1%AcOH水 B:0.09%AcOH/10%水/90%アセトニトリル、グラジエント A:B=70:30→55:45(10分)直線濃度勾配溶出]を用いて精製し、糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29):Tyr-Phe-Pro-Gly-Gln-Phe-Ala-Phe-Ser-(sugar chain added-linker:HMBA-Cys(disialo))(配列番号15)(19.0mg、5.33μmol)を得た。
ESI-MS calcd for C151H217N19O77S [M+3H]3+ 1187.8、[M+4H]4+ 891.1、[M+5H]5+ 713.1、found 1187.8、891.1、713.1。
(実施例4-2:水溶液中での加水分解挙動の追跡)
実施例4-1で得られた糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)の加水分解時の挙動を追跡した。具体的には、凍結乾燥した糖鎖付加(Cys(asialo)型)リンカー-HER2(8-16)コンジュゲート(化合物1)および糖鎖付加(Cys(disialo)型)リンカー-HER2(8-16)コンジュゲート(化合物8)の代わりに、凍結乾燥した糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)を用い、緩衝溶液として酢酸緩衝液(pH4.0)、PBS(pH7.4)、ホウ酸緩衝液(pH9.0)を用いた以外は、実施例1-4と同様にして、加水分解の挙動を追跡した。
糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)を加水分解し、HPLC分析を行ったところ、ケメリン9のアミノ酸配列を有する無修飾のケメリン9のぺプチド:Tyr-Phe-Pro-Gly-Gln-Phe-Ala-Phe-Ser(化合物33)が生成したことを確認した。下記化学式は、糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)の加水分解反応を示す。
また、50mM水酸化ナトリウム水溶液に糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)を溶解させたところ、2分で完全に消失し、ケメリン9のアミノ酸配列を有する無修飾のペプチド(化合物33)が生成した。文献での報告の通り、塩基性条件においては、加水分解が非常に速やかであることが確認された。
また、糖鎖付加(Cys(disialo)型)リンカー-ケメリン9コンジュゲート(化合物29)は他の化合物と同様に、pHをあげることで加水分解速度が向上することが確認された(Entry1、2、および3)。また、同じpHの場合、温度が高いほど加水分解が促進することが確認された(Entry3および4)。
加水分解の挙動試験後、反応液のHPLC[カラム:SHISEIDO CAPCELL PAK C18 UG-120(5μm)、φ4.6x250mm、流速:0.7mL/分、溶離液A:0.1%TFA水 溶離液B:0.09%TFA/10%水/90%アセトニトリル、グラジエントA:B=95:5→50:50(20分) 直線濃度勾配溶出]分析を行った。その結果、保持時間10.2分で糖鎖付加リンカー部分(化合物34)のピークが確認された。
化合物34のESI-MS:(m/z)calcd for C97H153N9O65S[M+2H]2+ 1258.93、[M+3H]3+ 839.62、found 1258.98、839.65。
Claims (23)
- 少なくとも1つのカルボキシ基を有する生理活性物質との結合用の、糖鎖付加リンカーであって、
前記糖鎖付加リンカーは、下記式(A)で表わされ、
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記糖鎖付加リンカーは、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質のカルボキシ基と結合することができる、
糖鎖付加リンカー。 - 請求項1に記載の糖鎖付加リンカーであって、
前記糖鎖付加リンカーは、下記式(A)で表わされる、糖鎖付加リンカー。
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する硫黄原子(S)を意味し、
R1は、-R3-R4-、-R4-R5-、または-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドである。] - 請求項1に記載の糖鎖付加リンカーであって、
前記糖鎖付加リンカーは、下記式(A)で表わされる、糖鎖付加リンカー。
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、-R3-R4-、または-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、または置換もしくは非置換のC5~C16ヘテロアリールであり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドである。] - 請求項1に記載の糖鎖付加リンカーであって、
前記R2またはR6の「糖鎖付加アミノ酸、または糖鎖付加ポリペプチド」において、糖鎖は、アミノ酸またはポリペプチドにおける、AsnまたはCysに結合している、
糖鎖付加リンカー。 - 請求項1に記載の糖鎖付加リンカーであって、
前記R2またはR6の「糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチド」における糖鎖は、4個以上の糖残基からなる、
糖鎖付加リンカー。 - 請求項1に記載の糖鎖付加リンカーであって、
前記R2またはR6における「糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチド」における糖鎖は、2本鎖複合型糖鎖、3本鎖複合型糖鎖、または4本鎖複合型糖鎖である、
糖鎖付加リンカー。 - 請求項6に記載の糖鎖付加リンカーであって、
前記糖鎖が、ジシアロ糖鎖、モノシアロ糖鎖、アシアロ糖鎖、ジグルクナック糖鎖およびジマンノース糖鎖からなる群から選択される2本鎖複合型糖鎖である、
糖鎖付加リンカー。 - 請求項1に記載の糖鎖付加リンカーであって、
前記「糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチド」において、糖鎖は、アミノ酸またはポリペプチドと、リンカーを介することなく結合している、
糖鎖付加リンカー。 - 請求項1~9のいずれか1項に記載の糖鎖付加リンカーに由来する糖鎖付加リンカー部分と生理活性物質部分とを含む化合物またはその塩であって、
前記生理活性物質は、少なくとも1つのカルボキシ基を有しており、
前記糖鎖付加リンカー部分は、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質部分のカルボキシ基とエステル結合またはチオエステル結合を形成し、前記生理活性物質部分と結合している、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩であって、
前記生理活性物質は、低分子生理活性物質または生体高分子である、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩であって、
前記生体高分子は、タンパク質、ポリペプチド、ポリヌクレオチド、およびペプチド核酸からなる群より選択される、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩であって、
無修飾の生理活性物質と比較して、向上した水溶性を有する、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩であって、
前記向上した水溶性が、モル濃度において、前記「無修飾の生理活性物質」と比較して、10~1,000,000倍である、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩であって、
前記糖鎖付加リンカー部分における前記酸素原子(O)または硫黄原子(S)と前記生理活性物質部分におけるカルボキシ基との間に形成されたエステル結合またはチオエステル結合が、pHおよび/または温度に依存して切断される、
化合物またはその塩。 - 請求項10に記載の化合物またはその塩を含む組成物であって、
前記化合物又はその塩における糖鎖は、実質的に均一である、
組成物。 - 医薬組成物であって、
(I)請求項10に記載の化合物またはその塩、および
(II)薬理学的に許容される担体
を含む、
医薬組成物。 - 請求項17に記載の医薬組成物であって、
前記生理活性物質は、対象に投与後に、活性を発揮する、
医薬組成物。 - 請求項17に記載の医薬組成物であって、
ワクチン接種に用いられる、
医薬組成物。 - 糖鎖付加リンカー部分と生理活性物質部分とを含む化合物またはその塩の製造方法であって、
ここで、糖鎖付加リンカーは、下記式(A)で表わされ、
X-R1-Y-R2 (A)
[式(A)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC2~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、糖鎖、糖鎖付加アミノ酸、もしくは糖鎖付加ポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、糖鎖、糖鎖付加アミノ酸、または糖鎖付加ポリペプチドであり、R7は、水素原子(H)、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記生理活性物質は、少なくとも1つのカルボキシ基を有しており、
前記方法は、以下のステップ、
(a)前記糖鎖付加リンカーにおける脱離基を有する酸素原子(O)または硫黄原子(S)と、前記生理活性物質のカルボキシ基との間で、エステル結合またはチオエステル結合が形成されるように縮合反応を行うステップを含む、
製造方法。 - 請求項20に記載の化合物またはその塩の製造方法であって、
前記縮合反応のステップは、前記糖鎖付加リンカーが固相合成用の樹脂に結合した状態で行われる(ただし、前記糖鎖付加リンカーが、糖鎖付加アミノ酸または糖鎖付加ポリペプチドを有するときに限る)、
製造方法。 - 糖鎖付加リンカー部分と生理活性物質部分とを含む化合物またはその塩の製造方法であって、
ここで、前記生理活性物質は、少なくとも1つのカルボキシ基を有しており、
前記方法は、以下のステップ、
(a)樹脂に、下記式(B)で表わされるリンカーを結合するステップであって、
前記リンカーは、下記式(B)で表わされ、
X-R1-Y-R2 (B)
[式(B)中、
Xは、脱離基を有する酸素原子(O)または脱離基を有する硫黄原子(S)を意味し、
R1は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、置換もしくは非置換のC2~C5アルケニル、もしくは、置換もしくは非置換のC2~C5アルキニルであるか、または、R1は、-R3-R4-、-R4-R5-、もしくは-R3-R4-R5-を意味し、ここで、R3およびR5は、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC2~C5アルケニル、または、置換もしくは非置換のC1~C5アルキニルであり、R4は、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、または硫黄原子(S)であり、
Yは、存在していても存在していなくてもよく、Yが存在する場合には、Yは、-CO-、または-CONH-(ただし、CがR1と結合し、NがR2と結合する)を意味し、
R2は、アミノ酸、もしくはポリペプチドであるか、または、R2は、-R6-R7を意味し、ここで、R6は、アミノ酸、またはポリペプチドであり、R7は、水素原子(H)、-NH2、置換もしくは非置換のC1~C5アルキル、置換もしくは非置換のC5~C16アリール、置換もしくは非置換のC5~C16ヘテロアリール、核酸、またはPEGである。]
前記リンカーにおけるR2のアミノ酸またはポリペプチドのカルボキシ基と前記樹脂とを結合するステップと、
(b)前記樹脂に結合した前記リンカーと前記生理活性物質とを結合するステップであって、前記リンカーは、前記酸素原子(O)または硫黄原子(S)における脱離基が脱離することにより、前記生理活性物質のカルボキシ基とエステル結合またはチオエステル結合を形成し、前記生理活性物質と結合するステップと、
(c)前記リンカーにおけるR2のアミノ酸またはポリペプチドの側鎖に、糖鎖を付加するステップと
を含む、製造方法。 - 請求項21または22に記載の製造方法により得られうる、化合物またはその塩。
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Cited By (1)
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CN111527214A (zh) * | 2017-12-20 | 2020-08-11 | 阿雷斯贸易股份有限公司 | 用聚醚离子载体调整蛋白质甘露糖基化状况的方法 |
Also Published As
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TW201438743A (zh) | 2014-10-16 |
US10202469B2 (en) | 2019-02-12 |
JP6219308B2 (ja) | 2017-10-25 |
TWI642444B (zh) | 2018-12-01 |
US20150306235A1 (en) | 2015-10-29 |
CN104936613A (zh) | 2015-09-23 |
KR102227919B1 (ko) | 2021-03-15 |
EP2926829B1 (en) | 2018-07-25 |
EP2926829A1 (en) | 2015-10-07 |
EP2926829A4 (en) | 2016-11-09 |
CN104936613B (zh) | 2018-05-22 |
JPWO2014084110A1 (ja) | 2017-01-05 |
KR20150102009A (ko) | 2015-09-04 |
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