Classifications

• • 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
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/14—Scaffolds; Matrices
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture

Definitions

• the present disclosure relates to cyclic peptides, cell scaffolding materials, cell separating materials, and media.
• Integrin is a cell adhesion molecule and is a heterodimer protein consisting of two subunits, ⁇ chain and ⁇ chain. Integrins play an important role not only in cell adhesion but also in cell extension, cell migration, cell proliferation, tissue formation, cancer metastasis, tissue repair, blood coagulation, and the like.
• Japanese Patent Application Laid-Open No. 2005-507376 discloses a cyclic peptide that binds to an integrin and is cyclized by a disulfide bond.
• Other cyclic peptides having an affinity for integrin are known.
• Japanese Patent Application Laid-Open No. 6-509551 describes a cyclic peptide obtained by cyclizing Tyr-Arg-Gly-Asp as a platelet aggregation inhibitor.
• Bioconjugate Chem, 1995, 6, p. 269-277 also describes techniques for cyclizing peptides and / or binding to carrier proteins or glass coverslips using (bromoacetyl) diaminopropionic acid. ..
• the problem to be solved by one embodiment of the present disclosure is a cyclic peptide having excellent integrin binding property and molecular stability, for example, alkali resistance, and a cell scaffolding material, a cell separating material and a cell separating material containing the cyclic peptide.
• a cell scaffolding material for example, alkali resistance
• the means for solving the above problems include the following aspects. ⁇ 1> A cyclic peptide having an amino acid sequence represented by the following formula (1).
• X a and X b and X c and X d each independently represent amino acid residues crosslinked via a thioether bond.
• X 1 to X 5 independently represent amino acid residues.
• R represents an arginine residue
• G represents a glycine residue
• D represents an aspartic acid residue
• m1 to m5 each independently represent an integer of 0 or more.
• the total number of amino acid residues of X a , X b , X c and X d , and the amino acid residues represented by X 1 , X 3 and X 4 and RGD is 7 to 16.
• X 3> The cyclic peptide according to ⁇ 2>, wherein the immobilized functional group is an amino group or a thiol group.
• the amino acid having an immobilized functional group in the side chain is at least one selected from the group consisting of L-lysine, D-lysine, L-cysteine, D-cysteine, L-homocysteine and D-homocysteine.
• cyclic peptide according to ⁇ 2> or ⁇ 3> which is a species amino acid.
• Either one of X c and X d in the above formula (1) is (2S) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2R) -2-amino-3.
• ⁇ 1> to ⁇ 4> which are amino acid residues derived from the amino acids to be produced, and the other is an amino acid residue derived from L-homocysteine, D-homocysteine, L-peniciramine, or D-peniciramine.
• the cyclic peptide according to any one of the above. ⁇ 6> Any of ⁇ 1> to ⁇ 5>, wherein X b in the above formula (1) is an amino acid residue derived from L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine.
• the cyclic peptide according to one. ⁇ 7> The cyclic peptide according to any one of ⁇ 1> to ⁇ 6>, wherein the amino acid sequence represented by the above formula (1) is the amino acid sequence represented by the following formula (2).
• X a and X b and X c and X d each independently represent an amino acid residue crosslinked via a thioether bond
• X 1 and X 2 independently represent an amino acid residue.
• R represents an arginine residue
• G represents a glycine residue
• D represents an aspartic acid residue
• m1 and m2 each independently represent an integer of 1 or more.
• the total number of amino acid residues of X a , X b , X c and X d , and the amino acid residue represented by X 1 and RGD is 7 to 14.
• Either one of X c and X d in the above formula (2) is (2S) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2R) -2-amino-3. -[(2-Acetyl) amino] propanoic acid, (2S) -2-amino-4-[(2-acetyl) amino] butanoic acid, (2R) -2-amino-4-[(2-acetyl) amino ] Selected from the group consisting of butanoic acid, N- ⁇ -acetyl-L-ornithin, N- ⁇ -acetyl-D-ornithine, N- ⁇ -acetyl-L-lysine, and N- ⁇ -acetyl-D-lysine.
• X b in the above formula (2) is an amino acid residue derived from L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine.
• Cyclic peptide. ⁇ 10> X a in the above formula (2) is an amino acid residue represented by the following structural (r), the cyclic peptide of any one of ⁇ 7> to ⁇ 9>.
• R r represents a hydrogen atom or a monovalent organic group
• L1 represents an integer of 0 or more and 10 or less
• nr1 represents an integer of 0 or more
• * represents a binding site with an adjacent amino acid residue. Represented, ** represents the bonding site with the sulfur atom in the thioether bond.
• X b in the above formula (2) is an amino acid residue derived from L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine, and X a is (2S) -2.
• X a is an amino acid residue derived from an arbitrary amino acid in which an acetyl group is bonded to ⁇ carbon
• X b is an amino acid residue derived from homocysteine
• X c is an amino acid residue derived from homocysteine.
• Is a group X d is an amino acid residue derived from N- ⁇ -acetyl-ornithin or an amino acid residue derived from N- ⁇ -acetyl-lysine, and (X 1 ) m1 is F as a whole?
• X a is an amino acid residue derived from an amino acid having an acetyl group bonded to ⁇ carbon
• X b is an amino acid residue derived from homocysteine
• X c is N- ⁇ -acetyl-ornithine.
• X d is an amino acid residue derived from homocysteine
• (X 1 ) m 1 is F as a whole, ⁇
• ⁇ 13> A cell scaffold material containing the cyclic peptide according to any one of ⁇ 1> to ⁇ 12> and a base material.
• ⁇ 14> The cyclic peptide according to any one of ⁇ 1> to ⁇ 12>, a retaining material, and the like. Including cell separator.
• ⁇ 15> A medium containing the cyclic peptide according to any one of ⁇ 1> to ⁇ 12> and a culture component.
• a cyclic peptide having excellent integrin binding property and molecular stability for example, alkali resistance
• a cell scaffolding material, a cell separating material and a medium containing the cyclic peptide are provided.
• the numerical range indicated by using "-" in the present disclosure means a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
• the upper limit value or the lower limit value described in a certain numerical range may be replaced with the upper limit value or the lower limit value of another numerical range described stepwise.
• the upper limit value or the lower limit value described in a certain numerical range may be replaced with the value shown in the examples.
• the amount of each component means the total amount of a plurality of kinds of substances when a plurality of kinds of substances corresponding to each component are present, unless otherwise specified.
• the term "process" includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.
• the cyclic peptide according to the present disclosure has an amino acid sequence represented by the following formula (1) (hereinafter, also referred to as "specific amino acid sequence").
• X a and X b and X c and X d each independently represent amino acid residues linked via a thioether bond, and X 1 to X 5 independently represent amino acid residues.
• Represents a group R represents an arginine residue, G represents a glycine residue, D represents an aspartic acid residue, and m1 to m5 each independently represent an integer of 0 or more.
• the total number of amino acid residues represented by X a , X b , X c and X d , and X 1 , X 3 and X 4 is 7 to 16.
• a cyclic peptide having integrin-binding property (hereinafter, also referred to as an integrin-binding cyclic peptide) has an ability to bind to integrin in the cell plasma membrane on the cell surface.
• Integrin is one of the cell adhesion molecules, and the integrin-binding cyclic peptide binds to the integrin on the cell surface. Therefore, the integrin-binding cyclic peptide is a scaffolding material for cell culture and cell separation for cell separation. It is a useful molecule because it can be used as a material, various cell culture media, and the like.
• cyclic peptides may have higher binding and specificity for cell adhesion molecules such as integrins as compared to linear peptides
• the molecular stability of cyclic peptides is higher than that of linear peptides. Tends to be low.
• cyclic peptides tend to have low alkali resistance; acid resistance; resistance to active rays such as X-rays and ⁇ -rays.
• the integrin-binding cyclic peptide was also degraded during long-term use or repeated use due to its low molecular stability, and the desired effect could not be obtained for a long period of time.
• cyclic peptides do not always have higher binding properties than linear peptides, and the integrin binding property changes depending on the amino acid sequence of the cyclic peptide, so that both molecular stability and integrin binding properties of cyclic peptides It was not easy to obtain an excellent integrin-binding cyclic peptide.
• a cyclic peptide having a specific amino acid sequence and a specific structure is excellent in both molecular stability and integrin binding property.
• the reason is not clear, but it is presumed as follows.
• the cyclic peptide having a specific structure according to the present disclosure contains two cyclic sites (hereinafter, also referred to as “cyclic segments”) in which amino acid residues are crosslinked by a thioether bond at a specific site in the amino acid sequence.
• the cyclic peptide according to the present disclosure is, for example, molecularly stable with respect to alkali resistance. It is presumed that it is excellent in and also excellent in integrin binding. The details of the cyclic peptide according to the present disclosure will be described below.
• amino acids are referred to as, in principle, the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology. It is expressed using the names, abbreviations, etc. adopted in the Communication on Biochemical Nomenclature (JCBN)).
• amino acid residue is represented by using the abbreviation of the amino acid from which the amino acid residue is derived.
• the amino acid sequence of a peptide or protein (also referred to as "primary structure”) represents the amino acid residues arranged in a row from the N-terminal to the C-terminal from the left end to the right end.
• a number indicating the number of the amino acid residue from the N-terminal side is added to the right side of the abbreviation of the amino acid residue. May be expressed as.
• the second ricin from the N-terminal may be represented as Lys2.
• L-form and D-form amino acids are represented using their names and isomers having an enantiomeric relationship, that is, L-form and D-form are present, the distinction between L-form and D-form is explicitly shown. Except for the case, in principle, it may be L-form or D-form.
• "isoleucine” shall represent “L-isoleucine” or "D-isoleucine”, and the same applies to amino acid residues.
• amino acids are represented using their abbreviations (three-letter abbreviations or one-letter abbreviations) and there are isomers related to enantiomers, that is, L-form and D-form, the L-form is also present.
• L-form In principle, it may be either L-form or D-form, unless the distinction between and D-form is explicitly indicated.
• “Lys” shall represent “L-lysine” or “D-lysine”, and the same applies to amino acid residues.
• L-form and D-form can be independently selected for each amino acid and each amino acid residue.
• all RGD sequences existing in the cyclic segment are L-form amino acid residues.
• the amino acid residues existing in the cyclic peptide are all L-form amino acid residues or all D-form amino acid residues, L-form amino acid residues and D-form. Both of the amino acid residues of may be present.
• amino acids are represented using their names and there are isomers related to diastereomers, they are not included in the amino acids specified by their names.
• Diastereomers are treated as different types of amino acids with the prefix "aro". For example, "threonine” does not include “allotreonine”. The same applies to amino acid residues.
• Table 1 shows the names and abbreviations of amino acids (one-letter abbreviations and three-letter abbreviations) for which one-letter abbreviations and three-letter abbreviations are officially recognized.
• amino acid residue contained in the cyclic peptide according to the present disclosure is not limited to the amino acid residue derived from the amino acids listed in Table 1 above, and may be an amino acid residue derived from an abnormal amino acid. Examples of abnormal amino acids are given in Table 2 below, but the abnormal amino acids are not limited thereto.
• Any amino acid residue contained in the cyclic peptide according to the present disclosure may be chemically modified.
• the chemical modification on the amino acid residue is not particularly limited as long as it is a chemical modification usually performed on the amino acid residue.
• Examples of chemical modifications to amino acid residues include N-acetylation, N-formylation, or N-acyllation, PEG (polyethylene glycol) formation of amino groups present in amino acid residues, and in amino acid residues. Examples thereof include amidation and PEGylation of the carboxy group present in.
• the N-terminal amino group residue is not particularly limited, and N-acetylation, It may have undergone N-terminal modification such as N-formylation, N-acylation, PEGylation, or may remain an amino group.
• the C-terminal carboxy group is not particularly limited, and amidation, PEGylation, etc. It may have undergone the C-terminal modification of, or it may remain as a carboxy group.
• the cyclic peptide according to the present disclosure has a specific amino acid sequence, and X a and X b and X c and X d each independently represent an amino acid residue linked via a thioether bond, and X 1 to X. 5 independently represents an amino acid residue.
• the cyclic peptide according to the present disclosure has a specific amino acid sequence, and X c and X d and X a and X b each independently represent an amino acid residue linked via a thioether bond.
• "-S-" in the formula (1) represents that X c and X d and X a and X b were crosslinked via a thioether bond.
• the amino acid residue X c and the amino acid residue X d , and the amino acid residue X a and the amino acid residue X b described later are each cross-linked by a thioether bond.
• the details are not clear, it is presumed that the molecular stability is superior to that of a cyclic peptide having an amino acid residue cross-linked by a disulfide bond because it is structurally irreversible as compared with a disulfide bond. ..
• the amino acid before the formation of the thioether bond is not particularly limited, but for example, one of X c and X d is an amino acid derived from an amino acid having a thiol group, and the other is an organic group having a halogen atom. Examples include combinations of amino acids derived from protected amino acids.
• the amino acid before the formation of the thioether bond is not particularly limited, but for example, one of X a and X b is an amino acid derived from an amino acid having a thiol group, and the other has a halogen atom. Examples include combinations of amino acids derived from amino acids protected by organic groups. Preferable examples of the organic group having a halogen atom include a haloacetyl group.
• the "main chain” in a cyclic peptide refers to a peptide chain having a relatively long stem among the chain portions in the cyclic peptide.
• side chain is meant a chain that binds to the backbone of a cyclic peptide.
• the main chain of an amino acid or amino acid residue corresponds to the "main chain” of a cyclic peptide
• the side chain of an amino acid or amino acid residue is a side chain that binds to the main chain of a cyclic peptide.
• amino acid residues derived from amino acids having a thiol group that can be taken by X a to X d before the formation of a thioether bond will be described.
• amino acid residues derived from amino acids having a thiol group are preferably an amino acid having a thiol group on the side chain from the viewpoint of reactivity. It is preferable that the main chain of the amino acid does not contain a thiol group.
• the amino acid residue having a thiol group on the side chain is not particularly limited, but an amino acid residue represented by the following structure (t-1) or (t-2) is preferably used.
• nt-1 and nt-2 represent an integer of 0 or more.
• the nt-1 or nt-2 carbon atoms in the side chain and the ⁇ -carbon atom are -NH 2 , -SH, -COOH, an alkyl group having 1 to 10 carbon atoms, and 6 to 14 carbon atoms. It may be substituted with at least one substituent selected from the group consisting of aryl groups of.
• the alkyl group having 1 to 10 carbon atoms may be either linear or branched.
• alkyl group having 1 to 10 carbon atoms examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group and the like.
• the aryl group having 6 to 14 carbon atoms may be a monocyclic ring or a condensed ring.
• Examples of the aryl group having 6 to 14 carbon atoms include a phenyl group, a naphthyl group, an anthranyl group, a phenanthrene group and the like.
• nt-1 and nt-2 are preferably an integer of 0 to 10, more preferably an integer of 0 to 6, and an integer of 1 to 4 because of the ease of forming a thioether bond. Is more preferable.
• amino acids having a thiol group on the side chain are derived from cysteine, peniciramine, homocysteine (amino acid derived from 2-amino-4-mercaptobutanoic acid), and 2-amino-5-mercaptopentanoic acid.
• Amino acids and the like can be mentioned.
• the amino acid having a thiol group on the side chain may be an amino acid derived from L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine from the viewpoint of binding stability. preferable.
• either X c or X d before thioether bond formation is an amino acid residue derived from an amino acid having a thiol group, and is on the side chain. It is more preferably an amino acid residue derived from an amino acid having a thiol group, and more preferably an amino acid residue derived from L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine. L-homocysteine or D-homocysteine is more preferable because it is less likely to cause chemistry.
• X a is an amino acid residue derived from an amino acid protected by an organic group having a halogen atom
• X b is an amino acid derived from an amino acid having a thiol group from the viewpoint of bond stability and molecular stability. It is preferably a residue, more preferably an amino acid residue derived from an amino acid having a thiol group on the side chain, to L-homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine. L-homocysteine or D-homocysteine is more preferable because it is preferably an amino acid residue derived from it and is less likely to cause racemization.
• ⁇ of ⁇ -amino acid before thioether binding It may be a thioether bond in which an amino group bonded to carbon or a modified amino group and a thiol group are bonded, and a side chain of an amino acid residue derived from an ⁇ -amino acid before the thioether bond and a thiol before the thioether bond. It may be a thioether bond to which a group is bonded.
• the bond between the amino group and the thiol group can be carried out via a haloacetyl group or the like arranged on the amino group.
• the ⁇ -amino acid before thioether binding It may be a thioether bond in which a carboxy group or a modified carboxy group moiety bonded to ⁇ carbon and a thiol group before thioether bond are bonded, and a side chain of an amino acid residue derived from ⁇ -amino acid before thioether bond. It may be a thioether bond in which a moiety and a thiol group before the thioether bond are bonded.
• amino acid residues derived from amino acids protected by organic groups having halogen atoms As an amino acid residue derived from an amino acid protected by an organic group having a halogen atom, an amino acid in which an organic group having a halogen atom is bonded to an amino group bonded to the ⁇ carbon of the amino acid, or a halogen atom in a side chain of the amino acid. It is preferably an amino acid residue derived from an amino acid having an organic group.
• the organic group having a halogen atom is not particularly limited as long as it is usually used as a protecting group for amino acids, and for example, a haloacetyl group and the like are preferable.
• the halogen atom is desorbed and the sulfur atom is bonded instead, so that a thioether bond is formed and the above amino acid residue is formed.
• Examples of amino acids in which an organic group having a halogen atom is bonded to an amino group bonded to the ⁇ carbon of an amino acid include any amino acid in which a haloalkanoyl group is bonded to an amino group on the ⁇ carbon.
• the haloalkanoyl group is, for example, a haloacetyl group.
• the halogen atom in the haloalkanoyl group is, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
• the amino acid to be bonded to the organic group having a halogen atom may be an L-amino acid or a D-amino acid, and examples thereof include isoleucine, lysine, aspartic acid, glutamic acid, tyrosine, leucine, threonine, and valine.
• the amino acid in which an organic group having a halogen atom is bonded to an amino group bonded to the ⁇ carbon of the amino acid may be an L-amino acid or a D-amino acid, N- ⁇ -chloroacetyl-isoleucine, N- ⁇ -chloro.
• Acetyl-lysine N- ⁇ -chloroacetyl-aspartic acid, N- ⁇ -chloroacetyl-glutamic acid, N- ⁇ -chloroacetyl-tyrosine, N- ⁇ -chloroacetyl-leucine, N- ⁇ -chloroacetyl-threonine, Examples thereof include N- ⁇ -chloroacetyl-valine.
• the amino acid residue protected by an organic group having a halogen atom in the side chain is an amino acid having an organic group having a halogen atom at the end of the side chain of the amino acid residue from the viewpoint of easiness of forming a thioether bond. Is preferable.
• Amino acid residues having such an aspect preferably include amino acid residues represented by the following structures (p-1) or (p-2).
• halogen represents a halogen atom
• L1 represents an integer of 0 or more and 10 or less
• np1 and np2 each independently represent an integer of 0 or more
• * represents an adjacent amino acid residue.
• Each of np1 and np2 preferably represents an integer of 0 to 6, more preferably an integer of 0 to 4, and even more preferably an integer of 1 to 3.
• the halogen is bonded to the left end (the terminal on the alkyl side when L1 is 1 or more).
• the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
• a bromine atom or a chlorine atom is preferable, and a chlorine atom is more preferable, from the viewpoint of reactivity, ease of formation of a thioether bond, and safety.
• the np1 or np2 carbon atom in the side chain and the carbon atom at the ⁇ -position are composed of an alkyl group having 1 to 10 carbon atoms and an aryl group having 6 to 14 carbon atoms. It may be substituted with at least one substituent selected from the group.
• L1 is preferably an integer of 0 to 10, more preferably an integer of 1 to 6, and even more preferably an integer of 1 to 3.
• Specific examples of the alkyl group having 1 to 10 carbon atoms and the aryl group having 6 to 14 carbon atoms include the alkyl group having 1 to 10 carbon atoms and carbon in the above structures (t-1) and (t-2). Similar to the aryl groups of numbers 6-14.
• the amino acid residue before the thioether bond is selected from the viewpoint of easiness of forming a thioether bond. It is preferably an amino acid residue having an organic group having a halogen atom in the ⁇ -amino group, and more preferably an amino acid residue represented by the following structure (r-1).
• R r represents a hydrogen atom or a monovalent organic group
• halogen represents a halogen atom
• L1 represents an integer of 0 or more and 10 or less
• nr1 represents an integer of 0 or more
• * is adjacent. Represents the binding site with the amino acid residue.
• L and L1 are synonymous with L and L1 in the structures (p-1) and (p-2), and so are preferred embodiments.
• nr1 has the same meaning as np1 and np2 in the structures (p-1) and (p-2), and so does the preferred embodiment.
• the monovalent organic group in R r is not particularly limited, and examples thereof include an aliphatic hydrocarbon group that can be taken as a side chain of an amino acid.
• Examples of the monovalent organic group that can be taken as the side chain of the amino acid include the monovalent organic group that is the side chain of the amino acid shown in Table 1 or Table 2.
• the aliphatic hydrocarbon group is composed of at least one substituent selected from the group consisting of -NH 2 , -SH, -COOH, an alkyl group having 1 to 10 carbon atoms, and an aryl group having 6 to 14 carbon atoms. It may be replaced.
• the alkyl group having 1 to 10 carbon atoms may be either linear or branched. Examples of the alkyl group having 1 to 10 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group and the like. Further, the aryl group having 6 to 14 carbon atoms may be a monocyclic ring or a condensed ring. Examples of the aryl group having 6 to 14 carbon atoms include a phenyl group, a naphthyl group, an anthranyl group, a phenanthrene group and the like.
• amino acid residue X a When the amino acid residue X a is located at the N-terminal of the specific amino acid sequence (when m5 of X 5 is 0) and is an amino acid residue derived from ⁇ -amino acid, it is designated as the amino acid residue X a. Is preferably an amino acid residue derived from the amino acid represented by the following structure (r).
• R r represents a hydrogen atom or a monovalent organic group
• L1 represents an integer of 0 or more and 10 or less
• nr1 represents an integer of 0 or more
• * represents a binding site with an adjacent amino acid residue.
• ** represent the bonding site with the sulfur atom in the thioether bond.
• R r, L and nr1 is synonymous with structure (r-1 in) R r, L and nr1, preferable embodiments thereof are also the same.
• an amino acid residue derived from an amino acid protected by an organic group having a halogen atom before the thioether bond is not particularly limited, but is 2-amino-3-[(2-haloacetyl) amino].
• Amino acid residues derived from acids, N- ⁇ -haloacetylhomolicin and the like can be mentioned.
• halogen atom in haloacetyl in these amino acid residues include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like, and among them, a chlorine atom is preferable.
• Amino acid residues derived from amino acids protected by an organic group having a halogen atom before the thioether bond include amino acids derived from amino acids having a chloroacetyl group in the side chain from the viewpoint of bond stability and molecular stability. It is preferably a residue, preferably (2S) -2-amino-3-[(2-chloroacetyl) amino] propanoic acid, (2R) -2-amino-3-[(2-chloroacetyl) amino] propane.
• (2R) -2-amino-4-[(2-chloroacetyl) amino] butanoic acid N- ⁇ - Chloroacetyl-L-ornithin, N- ⁇ -chloroacetyl-D-ornithin, N- ⁇ -chloroacetyl-L-lysine, N- ⁇ -chloroacetyl-D-lysine, N- ⁇ -chloroacetyl-L-homolicin , N- ⁇ -chloroacetyl-D-homolicin
• X c or X d is an amino acid residue derived from an amino acid protected by an organic group having a halogen atom, and a chlorine atom is used. It is more preferably an amino acid residue having an organic group having an organic group on the side chain, further preferably an amino acid residue having a chloroacetyl group on the side chain, and (2S) -2-amino-3-[().
• X b is an amino acid residue derived from an amino acid having a thiol group
• X a is a halogen atom on the amino group bonded to the ⁇ carbon of the amino acid from the viewpoint of bond stability and molecular stability. It is preferable that the amino acid residue is derived from an amino acid to which an organic group having is bonded, or the amino acid residue is derived from an amino acid protected by an organic group having a halogen atom, and chlorine is added to the amino group bonded to the ⁇ carbon of the amino acid.
• it is an amino acid residue derived from an amino acid to which an organic group having an atom is bonded, or an amino acid residue having an organic group having a chlorine atom on a side chain, and an amino group in which a chloroacetyl group is bonded to ⁇ carbon. It is more preferably an amino acid residue on the upper or side chain, N- ⁇ -chloroacetyl-isoleucine, N- ⁇ -chloroacetyl-lysine, N- ⁇ -chloroacetyl-aspartic acid, N- ⁇ -chloro.
• amino acid selected from the group consisting of acetyl-glutamic acid, N- ⁇ -chloroacetyl-tyrosine, N- ⁇ -chloroacetyl-leucine, N- ⁇ -chloroacetyl-threonine, and N- ⁇ -chloroacetyl-valine.
• amino acid residues X a to X d after thioether bond formation At amino acid residue X a ⁇ X d after thioether bond formation, after the thioether bond is formed, the structure of the amino acid residues derived from the protected amino acid with an organic group attached to the sulfur atom, the following structure ( Examples thereof include a structure represented by q-1) or (q-2).
• L and L1 are synonymous with L and L1 in the structures (p-1) and (p-2), and so are preferred embodiments.
• xq1 and xq1 are synonymous with np1 and np2 in the structures (p-1) and (p-2), and so are preferred embodiments.
• amino acid residues derived from amino acids protected by organic groups attached to sulfur atoms after thioether bonds have been formed are 2-amino-3- [acetylamino] propanoic acid, 2-.
• Examples thereof include amino acid residues derived from amino-4- [acetylamino] butanoic acid, N- ⁇ -acetylornithine, N- ⁇ -acetyllysine, N- ⁇ -acetylhomolicin and the like.
• N- ⁇ -acetyl-isoleucine and N- ⁇ -acetyl can be formed.
• N- ⁇ -acetyl-isoleucine and N- ⁇ -acetyl can be formed.
• -Ricin, N- ⁇ -acetyl-aspartic acid, N- ⁇ -acetyl-glutamic acid, N- ⁇ -acetyl-tyrosine, N- ⁇ -acetyl-leucine, N- ⁇ -acetyl-threonine, N- ⁇ -acetyl- Amino acid residues derived from valine and the like can also be mentioned.
• the acetyl group portion of these amino acid residues is attached to the sulfur atom of the thioether bond. If, of the two amino acid residues involved in the thioether bond, the amino acid residue that does not supply the sulfur atom of the thioether bond is replaced by an alkanoyl group such as an acetyl group, the thioether bond is not specified unless otherwise specified.
• the sulfur atom of is bonded to the alkanoyl group, and it can be said that the alkanoyl group (for example, acetyl group) is an S-linked alkanoyl group (for example, S-acetyl group).
• the notation of "S-bond" in such an S-bonded alkanoyl group is omitted, and it is simply described as an alkanoyl group (for example, an acetyl group).
• amino acid residue having a thiol group after the thioether bond is formed on the side chain
• amino acid residue having a thiol group after the thioether bond is formed on the side chain
• cysteine residues are cysteine residues, peniciramine residues, homocysteine residues (residues derived from 2-amino-4-mercaptobutanoic acid), or 2-amino-.
• examples include amino acid residues derived from 5-mercaptopentanoic acid.
• Amino acid residues derived from amino acids protected by organic groups bonded to sulfur atoms are the amino acid residues X on the N-terminal side in the cyclic segment, respectively. It may be a or X c , or it may be an amino acid residue X b or X d on the C-terminal side in the cyclic segment, respectively.
• the amino acid residue having a thiol group such as the amino acid residue of (t-1) or (t-2) is the amino acid residue X a or X c on the N-terminal side of the cyclic segment. It may be the amino acid residue X b or X d on the C-terminal side of the cyclic segment.
• the amino acid residue represented by the structural formula (p-1) or (p-2) is an amino acid residue having a structure selected from the amino acid residues represented by the following structures (a) to (h). There may be.
• * is a bond with an adjacent amino acid residue
• ** is a bond with the sulfur atom of the amino acid residue of the other party of the thioether bond.
• the amino acid residue X c and the amino acid residue can be obtained from the viewpoint of obtaining good molecular stability. It is preferable to design so that the ⁇ carbon of the other amino acid residue of the group Xd and the cysteine residue are separated by five or more atoms.
• the electron configuration of the thioether bond is stabilized by increasing the carbon chain length from the sulfur atom of the amino acid residue of the thioether bond partner to the main chain of the cyclic peptide. It is presumed that higher binding stability will be obtained.
• one of the amino acid residue X a and the amino acid residue X b is a cysteine residue, and the other is an amino acid residue in which the sulfur atom of the thioether bond is bonded to the amino acid side chain via an organic group. The same is true.
• the total number of carbon atoms on the side chain having a thiol group in the amino acid residue derived from the amino acid having a thiol group among the amino acid residue X a and the amino acid residue X b is preferably 1 to 10, more preferably 1 to 6, further preferably 1 to 4, and particularly preferably 2 to 4.
• the total number of carbon atoms on the side chain having a thiol group is the total number of carbon atoms in the side chain having a thiol group.
• the total number of carbon atoms on the side chain having a thiol group does not include the number of carbon atoms contained in the main chain.
• the total number of carbon atoms on the thiol-bearing side chain at the cysteine residue is 1, and the total number of carbon atoms on the thiol-bearing side chain at the homocysteine residue is 2.
• the total number of carbon atoms on the side chain in the indicated peniciramine residue is 3.
• * in the penicillamine residue shown below indicates a binding site with an adjacent amino acid residue.
• the amino acid residue having a thiol group has a chain length between the main chain of the cyclic peptide and the sulfur atom in the side chain having a thiol group having 1 to 10 carbon atoms. It is preferably 1 to 6, more preferably 1 to 4, and particularly preferably 1 to 3.
• the chain length between the main chain of the cyclic peptide and the sulfur atom in the side chain having a thiol group is counted so as to minimize the number of atoms in the side chain connecting the main chain and the thiol group in the cyclic peptide. It is a value, and the carbon atom contained in the main chain is not included in the number of chain lengths.
• the amino acid residue having a thiol group is the penicillamine residue shown below
• the chain length between the main chain of the cyclic peptide and the sulfur atom in the side chain having a thiol group is 1.
• * in the penicillamine residue shown below indicates a binding site with an adjacent amino acid residue.
• the chain length between the main chain of the cyclic peptide and the sulfur atom in the side chain having a thiol group is It is preferably 1 to 10, more preferably 1 to 6, further preferably 1 to 4, and particularly preferably 1 to 3.
• Chain length between the ⁇ carbon of the amino acid residue X a and the amino acid residue X b that do not supply the sulfur atom of the thioether bond and the sulfur atom of the amino acid residue that supplies the sulfur atom of the thioether bond is preferably separated by 4 or more atoms, more preferably separated by 4 to 9 atoms, and separated by 4 to 8 atoms, from the viewpoint of obtaining higher molecular stability. It is even more preferable that they are separated. However, this does not apply when Xa is an amino acid residue in which the sulfur atom of the thioether bond is bonded to the amino group on the ⁇ -carbon via the organic group.
• the chain length between them is preferably separated by 4 or more atoms, more preferably separated by 4 to 9 atoms, and separated by 4 to 8 atoms. Is more preferable.
• * is the binding site with the adjacent amino acid residue.
• the chain length of the ⁇ carbon and the sulfur atom of the cysteine residue is one nitrogen atom and three carbon atoms. Since it is separated by atoms, it has four atoms.
• the number of atoms (chain length) that separates the ⁇ carbon of the amino acid residue of the thioether bond partner and the sulfur atom of the cysteine residue is the number of atoms on the chain connecting the ⁇ atom and the sulfur atom. This means that atoms that do not participate in the above chain, such as hydrogen atoms that are bonded to the atoms on the chain, are not counted.
• the method for forming a thioether bond in the amino acid residue X c, the amino acid residue X d, the amino acid residue X a, and the amino acid residue X b before the formation of the thioether bond is not particularly limited, and the thioether bond is formed in the peptide.
• Known methods to be used can be mentioned.
• As a thioether bond for example, an amino acid residue having a thiol group and an amino acid residue protected by an organic group having a halogen atom are reacted under neutral or alkaline conditions (pH 7 to pH 9).
• a thioether bond can be formed by bonding the sulfur amino acid in the thiol group to the organic group with the formation of hydrogen halide.
• a protecting group such as a haloacetyl group is preferably mentioned.
• an amino acid residue having a thiol group on the side chain and an amino acid residue in which an organic group having a halogen atom is bonded to an amino group bonded to the ⁇ carbon of the amino acid or an organic group having a halogen are placed on the side chain.
• a method for forming a thioether bond by reacting a thiol group with an organic group having a halogen with an amino acid residue having, for example, a linear peptide before cyclization is neutralized or basic A method of forming a thioether bond by reacting in a buffer solution can be mentioned.
• a method in which an aqueous solution containing a linear peptide is slowly added dropwise to a Tris-HCl (pH 8.5) buffer solution and allowed to stand is preferably mentioned.
• a thioether bond an oligomer in which a plurality of acyclic peptides are linked by an intermolecular bond may be formed in addition to the cyclic peptide depending on the reaction conditions.
• the method for forming the thioether bond preferably includes a purification step from the viewpoint of improving the yield of the cyclic peptide. Examples of the method for purifying the cyclic peptide include known purification methods. As a method for purifying the cyclic peptide, a method for purifying the peptide after the cyclization reaction by reverse phase high performance liquid chromatography or the like is preferable.
• X 1 ⁇ X 5 each independently represent an amino acid residue, it is m1 ⁇ m5 each independently represents an integer of 0 or more.
• the number m1 to m5 of the amino acid residues X 1 to X 5 is the total amino acid residue of the amino acid residues represented by X a , X b , X c and X d , and X 1 , X 3 and X 4 and RGD.
• the radix is not particularly limited as long as it is in the range of 7 to 16.
• the amino acid residue is not particularly limited as long as the cyclic peptide can be formed, and is selected from the group consisting of the amino acids shown in Table 1 (excluding B, Z and X) and the amino acids shown in Table 2. It is preferably an amino acid residue derived from an amino acid, and more preferably an amino acid residue derived from an amino acid selected from the group consisting of the amino acids (excluding B, Z and X) shown in Table 1. In addition, if present, it may be an amino acid residue derived from the enantiomer or diastereomer of these amino acids.
• X 1 to X 5 may form a thioether bond between any two amino acid residues selected from the group consisting of X 1 to X 5 .
• the combination of amino acid residues capable of forming a thioether bond is an amino acid residue derived from an amino acid having a thiol group, and the other is an amino acid residue derived from an amino acid protected by an organic group having a halogen atom.
• Amino acid residues derived from amino acids having a thiol group and amino acid residues derived from amino acids protected by an organic group having a halogen atom are amino acid residues derived from amino acids having an all group in amino acid residues Xa to Xd . It is synonymous with an amino acid residue derived from an amino acid protected by an organic group having a group and a halogen atom, and the preferred embodiment is also the same.
• the amino acid residue may be, for example, an amino acid residue having a carboxy group in the side chain, an amino acid residue having a hydroxy group in the side chain, or the like.
• Amino acid residues having a carboxy group in the side chain include, for example, L-aspartic acid residue, D-aspartic acid residue, L-glutamic acid residue, D-glutamic acid residue, L-homoglutamic acid residue, and D. -Homoglutamic acid residues and the like can be mentioned.
• amino acid residue having a hydroxy group in the side chain examples include L-serine residue, D-serine residue, L-homoserine residue, D-homosine residue, L-tyrosine residue, and D-tyrosine residue.
• Examples include groups, L-threonine residues, D-threonine residues, L-threonine residues, D-threonine residues and the like.
• the amino acid residue may be, for example, a hydrophobic amino acid residue.
• hydrophobic amino acid residue include amino acid residues such as valine, methionine, tryptophan, proline, leucine, phenylalanine, isoleucine, alanine, and glycine.
• the amino acid residue represented by X 1 is preferably at least one of an amino acid residue derived from a hydrophobic amino acid and an amino acid residue having a ring structure from the viewpoint of integrin binding, and tyrosine and phenylalanine. , 3-Iodotyrosine, naphthylalanine, homotyrosine, proline, or isoleucine-derived amino acid residue is more preferable, and phenylalanine or tyrosine-derived amino acid residue is particularly preferable.
• the number m1 of amino acid residues represented by X 1 is an integer of 0 or more, preferably 1 or more and 6 or less, more preferably 1 or more and 3 or less, and 1 or 2 from the viewpoint of cost. Is preferable.
• the amino acid residues represented by X 3 and X 4 are not particularly limited, but from the viewpoint of integrin binding, amino acid residues of proline, threonine, or asparagine, serine, homoserine, valine, or alanine can be used. It is preferable to have.
• the number m3 and m4 of amino acid residues represented by X 3 and X 4 are each independently an integer of 0 or more. From the viewpoint of cost, m3 and m4 are each independently preferably 0 or more and 2 or less, more preferably 0 or more and 1 or less, and further preferably both m3 and m4 are 0. If the number m3 and m4 of amino acid residues represented by X 3 and X 4 is 2 or more, X 3 and X 4 may be the same amino acid residues with each other, a different amino acid residues May be good.
• Amino acid residues represented by X 2 and X 5 are each independently an integer of 0 or more. From the viewpoint of cost, m5 is preferably 0. From the viewpoint of molecular stability and the ease of forming a covalent bond by reacting with a functional group on a base material or a retaining material described later, m2 is preferably 1 or more, preferably 1 or more and 6 or less. More preferably, it is 1 or more and 4 or less. If m5 is 0, the amino acid residue represented by X a may be an amino group or modified amino group. When the number m2 of amino acid residues represented by X 2 is 2 or more, X 2 may be the same amino acid residues or different amino acid residues, but X 2 may be mutual. It is preferably the same amino acid residue.
• Amino acids with immobilized functional groups in the side chain are immobilized functional groups on the side chains because of their molecular stability and the ease with which they can react with functional groups on the substrate or retaining material described below to form covalent bonds. It is preferable to include an amino acid residue having (hereinafter, also simply referred to as “amino acid residue having an immobilized functional group”).
• immobilized functional group refers to a functional group capable of forming a covalent bond by reacting with a functional group on a base material or a retaining material described later.
• immobilized functional group examples include an amino group, a carboxy group, a hydroxy group, a thiol group, an aldehyde group (formyl group), a carbamoyl group, an azide group, an alkynyl group and the like.
• the immobilized functional group in the amino acid having the immobilized functional group is preferably selected from the group consisting of an amino group, a thiol group, and an aldehyde group from the viewpoint of reactivity with the functional group on the base material or the retaining material. Is at least one group, more preferably at least one group selected from the group consisting of an amino group and a thiol group.
• the amino group When the amino group is used as the immobilized functional group, the amino group can be bonded to the carboxy group on the base material or the retaining material via an amide bond, and the cyclic peptide according to the present disclosure can be bonded to the base material or the holding material. Can be easily fixed on top.
• the thiol group When the thiol group is used as the immobilized functional group, the thiol group can be bonded to the epoxy group on the base material or the retaining material via a covalent bond, and the cyclic peptide according to the present disclosure can be bonded to the base material or the holding material. It can be easily fixed on the material.
• amino acid residues having an amino group in the side chain examples include L-lysine residues and D-lysine residues.
• amino acid residue having a thiol group in the side chain examples include an L-cysteine residue and a D-cysteine residue. Since the amino acid residue having an amino group in the side chain and the amino acid residue having a thiol group in the side chain can be introduced at a relatively low cost, the production cost of the cyclic peptide according to the present disclosure can be suppressed. Therefore, the use of the above amino acid residues is preferable from an economical point of view.
• the amino acid having an immobilized functional group in the side chain includes a group consisting of L-lysine, D-lysine, L-cysteine, D-cysteine, L-homocysteine and D-homocysteine from the viewpoint of molecular stability. It is preferably at least one amino acid selected from, and more preferably at least one amino acid selected from the group consisting of L-lysine and D-lysine.
• At least one of X 5 (N-terminal of cyclic peptide) and X 2 (C-terminal of cyclic peptide) has a lysine residue as an amino acid residue having an immobilized functional group from the viewpoint of immobilization with a substrate or the like. It is preferable to include a lysine residue as an amino acid residue in which X 2 has an immobilized functional group. From the viewpoint of immobilization with a substrate or the like, at least one of X 5 (N-terminal of cyclic peptide) and X 2 (C-terminal of cyclic peptide) has a lysine residue as an amino acid residue having an immobilized functional group. It is preferable to include one or more consecutively, more preferably 2 to 10 consecutively contained, and further preferably 2 to 5 consecutively contained.
• X 5 N-terminal of cyclic peptide
• X 2 C-terminal of cyclic peptide
• X 5 and X 2 may be composed only of amino acid residues having continuous immobilized functional groups (preferably lysine residues), but are fixed. It may contain an amino acid residue other than the amino acid residue having a chemical functional group. Amino acid residues other than amino acid residues having an immobilized functional group include 1 to 20, 1 to 10, 1 to 5, or 1 to 3, alanine residue, ⁇ -alanine residue, and glutamic acid residue. It may be an amino acid residue selected from a group, an aspartic acid residue, and a glycine residue. Further, a lysine residue may be further sandwiched between X a or X b and an amino acid residue other than the amino acid residue having the immobilized functional group.
• the combination of the immobilized functional group of the cyclic peptide according to the present disclosure and the functional group on the base material or the retaining material is not particularly limited, but is limited to a combination of an amino group and a carboxy group, and an amino group and an aldehyde group.
• Combination with, combination of amino group and epoxy group, combination of hydroxy group and epoxy group, combination of carboxy group and hydroxy group, combination of thiol group and epoxy group, combination of azide group and alkynyl group, etc. Can be mentioned.
• the cyclic peptide according to the present disclosure is immobilized on the base material or the holding material by reacting the immobilized functional group of the cyclic peptide according to the present disclosure with the functional group on the base material or the holding material to form a covalent bond. Will be done. It is sufficient that at least a part of the immobilized functional groups of the cyclic peptide according to the present disclosure react with the functional groups on the base material or the retaining material to form a covalent bond, and all the immobilized functional groups are the base material or It does not have to react with the functional groups on the retainer.
• RGD sequence The specific amino acid sequence contained in the cyclic peptide according to the present disclosure has an RGD sequence, and the RGD sequence is arranged between X c and X d , which are amino acid residues cross-linked by a thioether bond. , Excellent integrin binding and molecular stability.
• the "RGD sequence” includes three amino acid residues of "R (arginine)", “G (glycine)", and “D (aspartic acid)" from the N-terminal side to the C-terminal side. Shows the sequence. Since the "RGD sequence" is involved in cell adhesion, the cyclic peptide according to the present disclosure can enhance the binding property to a cell adhesion molecule such as integrin by having the "RGD sequence".
• the number of "RGD sequences" may be 2 or more, but is preferably 1.
• the RGD sequence is located between the amino acid residue X c and the amino acid residue X d from the viewpoint of integrin binding.
• the position of the RGD sequence may be a position adjacent to the amino acid residue X c and the amino acid residue X d , or may be a position not adjacent to the amino acid residue X d with the amino acid residue X c. Good. From the viewpoint of integrin binding, when counting the amino acid residue to the C-terminal side X 5 at the N-terminal as the first amino acid residue at a particular amino acid sequence, corresponding to 3-5 amino acid residues Is preferable.
• the total number of amino acid residues of X a , X b , X c and X d , and the amino acid residues represented by X 1 , X 3 and X 4 and RGD is 7 to 16.
• the total number of amino acid residues X a , X b , X c and X d , and the total number of amino acid residues represented by X 1 , X 3 and X 4 and RGD that is, the most cross-linked with a thioether bond in the cross-linked peptide.
• amino acid residues located between amino acid residues (X a) in the N-terminal to amino acid residue (X b) in the most C-terminal (hereinafter, both "amino acid residues of the outermost annular segment"
• the intramolecular strain of the cyclic peptide does not become too large, the higher-order structure such as ⁇ -helix is stabilized, and the integrin binding property is excellent.
• the number of amino acid residues in the outermost cyclic segment is preferably 7 to 14, and more preferably 7 to 13.
• the number of amino acid residues between amino acid residues (X d ) is preferably 5 to 14, and is preferably 5 to 10. More preferably, it is more preferably 5 to 8.
• the total number of amino acid residues contained in the amino acid sequence of the cyclic peptide according to the present disclosure is not particularly limited and may be 7 to 50 amino acid residues or 7 to 30 amino acid residues. It may be an 8 to 20 amino acid residue or a 9 to 15 amino acid residue. The shorter the total length of the cyclic peptide, the lower the risk of antigenicity and the easier the peptide synthesis.
• amino acid sequence represented by formula (2) The amino acid sequence (specific amino acid sequence) represented by the above formula (1) contained in the cyclic peptide according to the present disclosure is an amino acid sequence represented by the following formula (2) from the viewpoint of integrin binding property and molecular stability. Is preferable.
• X a and X b and X c and X d each independently represent an amino acid residue crosslinked via a thioether bond
• X 1 and X 2 independently represent an amino acid residue.
• the group represents a group
• R represents arginine
• G represents glycine
• D represents aspartic acid
• m1 and m2 each independently represent an integer of 1 or more.
• the total number of amino acid residues of RGD and the amino acid residues represented by X a , X b , X c and X d and X 1 is 7 to 14.
• X a , X b , X c , X d , X 1 , X 2 , m 1 and m 2 are X a , X b , X c , X d , X 1 in the above formula (1).
• m1 and m2 dove synonymous, preferable embodiments thereof are also the same.
• either X c or X d in the formula (2) is (2S) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2R).
• amino acid residues derived from homocysteine, D-homocysteine, L-penicillamine, or D-penicillamine more preferably amino acid residues derived from L-homocysteine or D-homocysteine. ..
• X b in the formula (2) is an amino acid residue derived from L-homocysteine, D-homocysteine, L-peniciramine, or D-peniciramine
• X a is (2S) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2R) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2S) -2.
• R r represents a hydrogen atom or a monovalent organic group
• L1 represents an integer of 0 or more and 10 or less
• nr1 represents an integer of 0 or more
• * represents a binding site with an adjacent amino acid residue.
• ** represent the bonding site with the sulfur atom in the thioether bond.
• R r, L and nr1 is synonymous with structure (r-1 in) R r, L and nr1, preferable embodiments thereof are also the same.
• either X c or X d in the formula (2) is (2S) -2-amino-3-[(2-acetyl) amino] propanoic acid, (2R). ) -2-Amino-3-[(2-acetyl) amino] propanoic acid, (2S) -2-amino-4-[(2-acetyl) amino] butanoic acid, (2R) -2-amino-4- [(2-Amino) amino] butanoic acid, N- ⁇ -acetyl-L-ornithin, N- ⁇ -acetyl-D-ornithine, N- ⁇ -acetyl-L-lysine, N- ⁇ -acetyl-D-lysine , N- ⁇ -acetyl-L-homolicin, and N- ⁇ -acetyl-D-homolicin, which is an amino acid residue derived from an amino acid having an acetyl group in the
• Xb in formula (2) is L-homocysteine, D-homocysteine, L-peniciramine.
• X a is an amino acid residue represented by the above structure (r) (more preferably, (2S) -2-amino-3-[(2-2-).
• the total number of amino acid residues of the amino acid residues represented by X a , X b , X c and X d and X 1 and RGD is 7-14. From the viewpoint that the intramolecular strain of the cyclic peptide does not become too large, the higher-order structure such as ⁇ -helix is stabilized, and the integrin binding property is excellent, it is represented by X a , X b , X c and X d , and X 1 .
• the total number of amino acid residues of the amino acid residues to be added and RGD is more preferably 7 to 13, and more preferably 8 to 10. From the above viewpoint, the total number of amino acid residues of the amino acid residues represented by X c and X d and RGD is preferably 5.
• each amino acid residue may be an L-amino acid residue or a D-amino acid residue.
• X a is preferably the N-terminal amino acid residue of the cyclic peptide, in which case X a is isoleucine, lysine, aspartic acid, glutamic acid, tyrosine, leucine, threonine, or an acetyl group bonded to the ⁇ carbon. It may be an amino acid residue derived from valine.
• amino acid residues derived from aspartic acid in which an acetyl group is bonded to ⁇ carbon and amino acid residues derived from glutamic acid in which an acetyl group is bonded to ⁇ carbon are preferable, and glutamic acid residues in which an acetyl group is bonded to ⁇ carbon are more preferable.
• X b may be an amino acid residue derived from cysteine, an amino acid residue derived from penicillamine, or an amino acid residue derived from homocysteine. Of these, amino acid residues derived from homocysteine are preferable.
• the amino acid residues that do not supply the sulfur atom of the thioether bond are 2-amino-3-[(2-acetyl) amino] propanoic acid and 2-amino-4-[(2-amino-4-[(2-)). It may be an amino acid residue derived from acetyl) amino] butanoic acid, N- ⁇ -acetyl-ornithine, N- ⁇ -acetyl-lysine, or N- ⁇ -acetyl-homolicin.
• amino acid residues derived from N- ⁇ -acetyl-ornithine and amino acid residues derived from N- ⁇ -acetyl-lysine are preferable, and amino acid residues derived from N- ⁇ -acetyl-lysine are more preferable.
• amino acid residue that supplies the sulfur atom of the thioether bond is an amino acid residue derived from cysteine, an amino acid residue derived from peniciramine, or an amino acid residue derived from homocysteine. It may be. Of these, amino acid residues derived from homocysteine are preferable.
• (X 1 ) m1 preferably represents F as a whole, or FX g .
• X g represents an arbitrary amino acid residue, and examples thereof include alanine and arginine.
• (X 2 ) m2 preferably contains K having 3 or more consecutive residues. The upper limit of the number of repetitions of K is not particularly limited, but may be 10, 6, or 4, for example.
• (X 2 ) m2 may represent, for example, A, K, KKK, AKKK, etc. as a whole.
• X a is an amino acid residue derived from any amino acid with an acetyl group attached to the ⁇ carbon
• X b is an amino acid residue derived from homocysteine
• X c is a homocysteine.
• X d is an amino acid residue derived from N- ⁇ -acetyl-ornithine or an amino acid residue derived from N- ⁇ -acetyl-lysine
• (X 1 ) m 1 is the whole. Is F.
• X a is isoleucine bound acetyl group in ⁇ carbon, lysine, aspartic acid, glutamic acid, tyrosine, leucine, threonine, or an amino acid residue derived from a valine are preferred.
• (X 2 ) m2 may be A, K, KKK, or AKKK as a whole.
• X a is an amino acid residue derived from any amino acid with an acetyl group attached to ⁇ carbon
• X b is an amino acid residue derived from homocysteine
• X c is N-.
• X d is an amino acid residue derived from homocysteine
• (X 1 ) m 1 is the whole. Is F.
• X a is isoleucine bound acetyl group in ⁇ carbon, lysine, aspartic acid, glutamic acid, tyrosine, leucine, threonine, or an amino acid residue derived from a valine are preferred.
• (X 2 ) m2 may be A, K, KKK, or AKKK as a whole.
• cyclic peptide according to the present disclosure may be a cyclic peptide having a structure in which an additional amino acid is added to the N-terminal of the cyclic peptide of the above formula (2).
• the structure of such a cyclic peptide can be represented by the following formula (3).
• X 5 and m5 have the same meanings as X 5 and m5 in the formula (1), preferred embodiments are also the same.
• Xa is not an amino acid residue in which the sulfur atom of the thioether bond is bonded to the amino group on the ⁇ -carbon via the organic group.
• examples of the combination of X a and X b and preferable examples include examples of the combination of X c and X d in the description of the formula (2) and amino acid residues mentioned as preferable examples.
• Which of X a and X b is the amino acid residue supplying the sulfur atom of the thioether bond is not particularly limited, but may be, for example, X a .
• (X 5 ) m5 may be A, K, KKK, or AKKK as a whole.
• the cyclic peptide according to the present disclosure has a dissociation constant of 200 nM (M; mol / L) or less measured by the methods described in "(2) Fixation of cyclic peptide" and "(3) Evaluation of integrin binding property" in Examples. It is more preferably 100 nM or less, and even more preferably 50 nM or less. The closer the dissociation constant is to 0 nM, the more preferable, but from a practical point of view, for example, 0.1 nM or 0.5 nM may be used as the lower limit value that can be combined with the above upper limit value.
• the cyclic peptide according to the present disclosure preferably has a residual rate of 30% or more, more preferably 50% or more, as measured by the method described in "(4) Evaluation of molecular stability" in Examples. It is preferably 70% or more, and more preferably 70% or more. The closer the residual rate is to 100%, the more preferable it is. Therefore, 100% may be used as the upper limit value that can be combined with the above lower limit value.
• the integrin in the present disclosure is not particularly limited as long as it is an integrin that recognizes the RGD sequence.
• the binding property is evaluated using integrin ⁇ v ⁇ 5, but the integrin is not limited to this, and the cyclic peptide according to the present disclosure may bind to the integrin that recognizes the RGD sequence of ⁇ V ⁇ 3. it can.
• the molecular stability of the cyclic peptide according to the present disclosure is measured using the alkali resistance as an index, but the molecular stability of the cyclic peptide according to the present disclosure is resistance to stimuli other than alkali, for example, X-ray resistance, ⁇ .
• a carrier for affinity chromatography using the cyclic peptide according to the present disclosure as an affinity ligand is used for cell purification, integrin binding property is maintained even after repeated washing with alkali, and cells are maintained. The separation cost can be reduced.
• Cyclic peptides 1 to 47 shown in Tables 3-1 and 3-2 are all amino acid residues in which all amino acid residues do not have L-amino acid residues or optical isomers such as glycine.
• Hcy represents a homocysteine residue
• Dab acetyl
• Dap acetyl
• Orn represents an N- ⁇ -acetyl-ornithine residue
• K represents an N- ⁇ -acetyl-lysine residue.
• One of the hydrogen atoms on the methyl group in the acetyl group in the amino acid residue containing these acetyl groups is replaced with the bond to the sulfur atom in the amino acid residue to which the thioether bond is bonded, and the thioether bond is formed.
• a bridge is formed. Further, in the column of amino acid residues in the crosslinked portion, the acetyl group is omitted.
• amino acid residues shown in parentheses and the amino acid residues shown in bold typeface indicate crosslinked amino acid residues due to thioether bonds.
• amino acid residues described in the oblique body represent one of the combinations of amino acid residues cross-linked by the thioether bond, and the amino acid residues described in the non-oblique body in bold are due to the thioether bond. It represents one of the combinations of crosslinked amino acid residues.
• alpha-NH 2 amino acid residues, via a chloroacetyl group means that a thioether bond is formed with the amino acid residue to be bound.
• the amino acid residue Xa is "K”: lysine, and lysine is not NH 2 on the side chain, but " ⁇ -NH 2 " is an amino acid to which it binds via a chloroacetyl group. It is shown that a thioether bond is formed with the residue.
• cyclic peptides 3, 11, 14-18, 20, 22-30, 35, 38-40, and 42-47 are preferable, and cyclic peptides 11, 22, 25-30, 35, 38-40, and 42. ⁇ 47 is preferred, and cyclic peptides 29 and 30 are even more preferred.
• the sequence variation may be applied by considering the entire cyclic peptide as a reference sequence. Therefore, an amino acid group in which an amino acid residue is added, deleted or substituted to any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 47 can be used as long as the requirements of the cyclic peptide according to the present disclosure are satisfied. .. However, the RGD region in the cyclic segment must not be modified.
• the total number of amino acid residues added, deleted or substituted shall be 1 to 10. Is preferable, 1 to 5 is more preferable, 1 to 3 is more preferable, and 1 or 2 is particularly preferable.
• the cyclic peptide according to the present disclosure preferably has 70% or more sequence identity with any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 47, and more preferably 80% or more. , 90% or more sequence identity is more preferred.
• the range of the amino acid sequence having 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 includes the amino acid sequence of SEQ ID NO: 1 itself.
• sequence identity of two amino acid sequences is determined as follows.
• (I) Aligning two amino acid sequences For example, alignment algorithms and / or programs such as FASTA and BLAST that can be used by default can be used to align between two sequences.
• (Ii) Calculation of sequence identity Based on the obtained alignment, the sequence identity is calculated by the following equation. Sequence identity [%] (number of matching positions / number of all positions) x 100 [%] The total number of positions is the length of the alignment, and the number of matching positions is the number of positions where the amino acid types match.
• the cell scaffold material according to the present disclosure includes a cyclic peptide according to the present disclosure and a base material. Since cells are supported by an extracellular matrix in vivo, cells can be cultured better by using the cyclic peptide according to the present disclosure as a cell scaffold material that reproduces the same state. ..
• the base material for cell culture include biodegradable polyesters such as polylactic acid, polyglycolic acid and polycaprolactone, glycoproteins such as collagen or its heat-denatured gelatin and fibronectin, or hyaluronic acid, chitin and alginic acid. Examples thereof include a matrix composed of polysaccharides such as.
• the cyclic peptide according to the present disclosure can be bound to the base material.
• the cyclic peptide according to the present disclosure has an amino group of a lysine residue as an immobilized functional group
• the cyclic peptide is used as a substrate by reacting the amino group with a carboxy group on the substrate to form an amide bond. Can be fixed to.
• a cell scaffold material in which the cyclic peptide according to the present disclosure is bound to the surface of a substrate can be obtained.
• the amount of the cyclic peptide according to the present disclosure is not particularly limited, but may be 0.01% by mass to 100% by mass, or 0.1% by mass to 50% by mass, based on the total mass of the base material. May be good.
• the cell scaffold material according to the present disclosure can be applied on any culture tool such as a petri dish, a flask, a plate (for example, a polystyrene well plate), a culture bag, a hollow fiber membrane, and beads. Since the cell scaffold material according to the present disclosure contains the cyclic peptide according to the present disclosure, it has good binding property to integrin, and cells can adhere well to the cell scaffold material.
• the cell to be cultured is not particularly limited as long as it is a cell of an organism expressing integrin, but it may be any animal cell, any vertebrate animal cell, or any mammal cell. It may be a human cell, or it may be a non-human mammal cell.
• Examples of cells include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, perinatal stem cells, sheep water-derived stem cells (AFSC), mesenchymal stem cells of any origin (MSC), any Examples thereof include histologically differentiated progenitor cells or adult cells, mature cells, normal cells, affected cells, tumor cells and the like. More specific examples include liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, bile duct cells, bile tree cells, pancreatic cells and the like. Examples of these cells are the same for the cell separating material and the medium described later.
• the cell separating material according to the present disclosure includes a cyclic peptide according to the present disclosure and a retaining material. Since the cell separating material according to the present disclosure contains the cyclic peptide according to the present disclosure, it can bind to the integrin on the cell surface and capture cells. Therefore, by using the cell separating material according to the present disclosure, for example, as affinity chromatography, cells can be efficiently separated from the cell suspension.
• the cyclic peptide according to the present disclosure can be bound to the holding material by reacting the immobilized functional group in the cyclic peptide according to the present disclosure with the functional group in the holding material.
• the cyclic peptide according to the present disclosure has an amino group of a lysine residue as an immobilized functional group
• the cyclic peptide is held by reacting the amino group with a carboxy group on the retaining material to form an amide bond. Can be fixed to.
• the retaining material is not particularly limited, and is, for example, polysaccharides such as agarose, dextran, starch, cellulose, purulan, chitin, chitosan, cellulose triacetate, cellulose diacetate and derivatives thereof, and polyacrylamide and polymethacrylicamide. , Polyacrylate, polymethacrylate, polyalkylvinyl ether, and a material selected from vinyl-based polymers such as polyvinyl alcohol. These materials may form a crosslinked structure. Crosslinked structures tend to improve mechanical strength.
• the holding material is preferably made of one or more of the above materials. Further, the holding material is preferably porous, more preferably a porous membrane or porous particles, and further preferably porous particles.
• a cell separating material in which the cyclic peptide according to the present disclosure is immobilized on a water-insoluble retaining material can also be used for affinity chromatography.
• the water-insoluble retaining material include polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, crosslinked dextran, and crosslinked purulane, acrylate-based polymers, organic retainers such as styrene-based polymers, and glass.
• examples thereof include beads, an inorganic holding material such as silica gel, and a composite holding material such as organic-organic and organic-inorganic obtained by a combination thereof.
• a polysaccharide or an acrylate-based polymer is more preferable from the viewpoint of alkali resistance, and a polysaccharide such as agarose or cellulose is further preferable.
• Commercially available products that can be used as water-insoluble preservatives include, for example, Cellufine GCL2000 (manufactured by JNC) (CELLUFINE is a registered trademark) and Cellfine MAX (JNC), which are porous cellulose gels.
• TOYOPEARL are registered trademarks
• TOYOPEARL AF-Carboxy-650 Toyopearl Co., Ltd.
• TOYOPEARL GigaCap CM-650 Toyopearl Co., Ltd.
• agarose type Sepharose CL4B manufactured by GE Healthcare
• SEPHAROSE is a registered trademark
• Eupergit C250L manufactured by Sigma Aldrich
• EUPERGIT is a registered trademark
• the water-insoluble retaining material used in the present disclosure preferably has a large surface area and is preferably porous having a large number of pores of an appropriate size in view of the purpose and method of use of the adsorbent material.
• the form of the holding material is not particularly limited, but any form such as a bead shape, a fibrous shape, a film shape, a hollow thread shape, or the like is possible, and any shape can be selected.
• Examples of the method for immobilizing the cyclic peptide according to the present disclosure on a water-insoluble retaining material include, but are not limited to, an immobilization method using an amino group of a lysine residue as described above.
• the method generally adopted when immobilizing a protein or polypeptide on a holding material can be adopted.
• the retaining material is reacted with cyanide bromide, epichlorohydrin, diglycidyl ether, tosilyl lolide, tresil chloride, hydrazine, etc. to activate the retaining material or introduce a reactive functional group on the surface of the retaining material.
• aqueous solvent aqueous dispersion medium
• organic solvent organic dispersion medium
• HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
• acetate buffer solution acetate buffer solution
• phosphate buffer solution citric acid
• examples thereof include a buffer solution and a Tris-hydrochloric acid buffer solution.
• the organic solvent is not particularly limited, but a polar organic solvent is preferable, and DMSO (dimethyl sulfoxide; dimethyl sulfoxide) and DMF (N, N-dimethylformamide; N, N-dimethyl) are particularly preferable.
• Formamide) or alcohol is preferable, for example, methanol, ethanol, IPA (isopropyl alcohol; isopropyl alcohol), TFE (2,2,2-trifluoroethanol; 2,2,2-trifluoroethanol), and HFIP (1). , 1,1,3,3,3-hexafluoro-2-propanol; 1,1,1,3,3,3-hexafluoro-2-propanol) and the like.
• the pH condition for immobilizing the cyclic peptide according to the present disclosure is not particularly limited, and may be any of acidic, neutral, and alkaline, and may be appropriately set according to the solvent (dispersion medium) used, for example. it can.
• a base such as DBU (diazabicycloundecene) or TEA (triethylamine) may be added to DMSO (dimethyl sulfoxide) or alcohol.
• the density of the cyclic peptide according to the present disclosure is not particularly limited, but 0.1 mmol / filler 1 L to 1,000 mmol / filler 1 L is preferable, and 0.1 mmol is preferable. / Filler 1L to 100 mmol / Filler 1L is more preferable, and 0.5 mmol / Filler 1L to 20 mmol / Filler 1L is further preferable.
• the density of the cyclic peptide is within the above range, the amount of the cyclic peptide used according to the present disclosure and the cell separation performance are well-balanced, and cells can be separated efficiently at a lower cost.
• the cell separated by the cell separating material according to the present disclosure is not particularly limited as long as it is a cell of an organism expressing integrin, but may be any animal cell or any vertebrate animal cell. It may be any mammalian cell, a human cell, or a non-human mammalian cell.
• the medium according to the present disclosure contains the cyclic peptide according to the present disclosure and the culture component.
• the cyclic peptide according to the present disclosure is contained in the medium according to the present disclosure, binding of the integrin of the cells cultured in the medium to the cyclic peptide occurs, and cell survival through suppression of apoptosis by signal transduction from the integrin. Effects such as a rate increase can be obtained.
• the culture component refers to a medium component for culturing cells.
• the cell to be cultured is not particularly limited as long as it is a cell of an organism expressing integrin, but may be any animal cell, any vertebrate cell, or any cell.
• the medium containing the culture components may be appropriately selected according to the type of cells to be cultured.
• DMEM Dulbecco modified Eagle's medium
• MEM Eagle's minimum essential medium
• F12 Ham
• RPMI1640 MCDB
• the culture component a medium such as the above-mentioned medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed according to the cell type and cell conditions. Good. Therefore, the culture component is not limited to the one having a known composition, and one or two or more components may be added, removed, increased or decreased.
• the amino acid contained in the culture component is not particularly limited, and examples thereof include known amino acids contained in the culture component.
• the vitamins contained in the culture component are not particularly limited, and for example, calcium D-pantothenate, choline chloride, folic acid, isoinositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B12, adenine, etc. Examples include thimidine.
• the electrolyte contained in the culture component is not particularly limited, and examples thereof include known electrolytes contained in the culture component.
• the electrolytes include CaCl 2 , KCl, sulfonyl 4 , NaCl, NaH 2 PO 4 , NaHCO 3 , Fe (NO 3 ) 3 , FeSO 4 , CuSO 4 , MnSO 4 , Na 2 SiO 3 , (NH 4 ). 6 Mo 7 O 24 , NaVO 3 , NiCl 2 , ZnSO 4 and the like can be mentioned.
• the culture component may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, putrescine, and antibiotics.
• the culture component may or may not contain serum.
• the content of serum in the medium according to the present disclosure is preferably 0% by volume or more and 30% by volume or less, more preferably 0% by volume or more and 10% by volume or less, and 0% by volume or more and 5% by volume or less. It is more preferably 0% by volume or more and 2% by volume or less.
• the content of the cyclic peptide according to the present disclosure in the medium according to the present disclosure is not particularly limited, but is, for example, 0.01 ng / mL to 10 mg / mL, and 0.1 ng / mL to 1 mg / mL. There may be.
• the medium according to the present disclosure unlike the case of the cell scaffolding material and the cell separating material, it is not particularly necessary to immobilize the cyclic peptide.
• a cyclic peptide having excellent integrin binding property and molecular stability for example, alkali resistance
• a cell scaffolding material, a cell separating material and a medium containing the cyclic peptide are provided. be able to.
• M means "mol / L”.
• 0.2 M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and 0.04 M NHS (N-Hydroxysuccinimide; N-hydroxy) 70 ⁇ L of a mixed aqueous solution of (imide succinate) was added. Then, 300 ⁇ L of the sample solution of each of the above cyclic peptides diluted to 0.2 g / L with HEPES buffer was supplied to the sensor chip, then blocked with an ethanolamine solution and washed with an aqueous sodium hydroxide solution. And fixed.
• immobilized sensor chip A the sample solution to be supplied to the sensor chip was 5000 ⁇ L instead of 300 ⁇ L.
• 70 ⁇ L of 0.2 M EDC and 0.04 M NHS mixed aqueous solution was added, and then blocking treatment and washing treatment were performed.
• the obtained immobilized sensor chip is hereinafter referred to as "immobilized sensor chip A”.
• the above-mentioned measurement process consisting of adding integrin for 10 minutes, running a running buffer for 30 minutes, performing measurement with Biacore 3000, and performing regeneration treatment with 0.5 M EDTA aqueous solution is further carried out for 100 nM humans.
• Dissociation constant between cyclic peptide and human integrin ⁇ v ⁇ 5 from the difference between the measured value by Biacore 3000 in the channel where the cyclic peptide was fixed and the value measured by Biacore 3000 in the flow path where the cyclic peptide was not fixed when human integrin ⁇ v ⁇ 5 at each concentration was flowed. was calculated, and the integrin binding property was evaluated according to the following evaluation criteria.
• Evaluation criteria A, B or C are preferable. The evaluation results are shown in Table 5-1 and Table 5-2.
• A The dissociation constant is 50 nM or less.
• B The dissociation constant exceeds 50 nM and is 100 nM or less.
• C The dissociation constant exceeds 100 nM and is 200 nM or less.
• D The dissociation constant exceeds 200 nM.
• the molecular stability of the cyclic peptide was evaluated by analyzing an alkali-treated cyclic peptide aqueous solution by LC / MS (Liquid Chromatography Mass Spectroscopy). Alkaline treatment was performed by the following method. A 500 ⁇ M cyclic peptide aqueous solution was prepared, an equivalent amount of 1 M sodium hydroxide aqueous solution was added to the aqueous solution, and the mixture was incubated at 15 ° C. for 20 minutes to obtain an alkali-treated cyclic peptide aqueous solution.
• the cyclic peptide residual ratio was calculated by setting the total area of all peaks of the cyclic peptide before alkali treatment in LC / MS to 100% and determining the ratio of the total area of all peaks in LC / MS of the aqueous alkali-treated cyclic peptide solution.
• the molecular stability was evaluated according to the following evaluation criteria. Evaluation criteria A, B or C are preferable. The evaluation results are shown in Table 5-1 and Table 5-2.
• A The residual rate of the cyclic peptide is 70% or more.
• B The residual rate of the cyclic peptide is 50% or more and less than 70%.
• C The residual rate of the cyclic peptide is 30% or more and less than 50%.
• D The residual rate of the cyclic peptide is less than 30%.
• LC / MS used for the evaluation of molecular stability was set to the following conditions.
• ⁇ LC equipment Prominence series (pump, column oven, autosampler, detector) (manufactured by Shimadzu Corporation)
• MS detector LC / MS2010EV (manufactured by Shimadzu Corporation)
• Cold Cadenza CD-C18, inner diameter 2.0 mm x length 250 mm, particle diameter 3 ⁇ m (manufactured by Intact Co., Ltd.)
• Eluent A Contains 10 mM ammonium formate as a solute, and the solvent is a solution of 100% water (pH 3).
• the cyclic peptide according to the present disclosure is superior in molecular stability to the cyclic peptide of the comparative example.
• the value of the residual rate of the cyclic peptide was 50% or more (evaluation B or more), whereas in all the comparative examples, the value of the residual rate of the cyclic peptide was 25% or less as an actual value. Met. From the above, it can be seen that the cyclic peptide according to the present disclosure is excellent in both integrin binding property and molecular stability.
• the cell scaffold material according to the present disclosure was prepared using the cyclic peptide obtained above, and an iPS cell culture experiment was conducted using this cell scaffold material.
• CMD coating well Add 9.5 g of distilled water to 0.5 g of sodium carboxymethyl dextran (manufactured by Meito Sangyo Co., Ltd., trade name: "CMD", molecular weight: 1 million, hereinafter also referred to as "CMD"), and stir well. It was dissolved to prepare a 5% by weight CMD solution. Next, 1 mL of distilled water was added to 383.4 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter, also referred to as "EDC”) (manufactured by Nacalai Tesque, Inc.) to prepare an EDC solution. did.
• EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
• N-hydroxysuccinic acid imide hereinafter, also referred to as "NHS" (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare an NHS solution.
• NHS N-hydroxysuccinic acid imide
• 0.05 mL of the EDC solution and 0.05 mL of the NHS solution were added to 10 g of the CMD solution prepared above and stirred, and the obtained CMD-containing coating solution was immediately surface-treated in one well of the polystyrene plate described above. 1 mL each was added dropwise. After allowing the polystyrene plate to stand at room temperature for 1 hour, the wells were thoroughly washed with distilled water to remove the CMD-containing coating liquid to prepare CMD-coated wells.
• a cyclic peptide solution was prepared by adding 1 mL of HBS-N buffer (manufactured by GE Healthcare Japan Co., Ltd.) to 11: 0.2 mg of the cyclic peptide. Then, 1 mL of distilled water was added to 76.7 mg of EDC to prepare an EDC solution. Then, 1 mL of distilled water was added to 11.5 mg of NHS to prepare an NHS solution. Next, 1 mL of distilled water was added to 1 mL of ethanolamine (manufactured by BIO RAD, trade name: "ProteOn ethanolamine HCL”) to prepare an ethanolamine solution.
• HBS-N buffer manufactured by GE Healthcare Japan Co., Ltd.
• a well having a cell scaffold material (hereinafter, also referred to as “ligand coating well”) was obtained.
• mTeSR1 cell dissociation reagent
• the number of cells was measured using a viable cell autoanalyzer (manufactured by Beckman Coulter, product name: Vi-Cell XR), and it was determined whether or not the cells had proliferated. From the above results, it was confirmed that iPS cells proliferated in the cell scaffold material using the peptide of Example 11. From this, it is shown that the cyclic peptide according to the present disclosure has an excellent integrin binding property and therefore has a function as a cell scaffold material.

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