WO2021167107A1 - ヒトトランスフェリンレセプター結合ペプチド - Google Patents

ヒトトランスフェリンレセプター結合ペプチド Download PDF

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Publication number
WO2021167107A1
WO2021167107A1 PCT/JP2021/006709 JP2021006709W WO2021167107A1 WO 2021167107 A1 WO2021167107 A1 WO 2021167107A1 JP 2021006709 W JP2021006709 W JP 2021006709W WO 2021167107 A1 WO2021167107 A1 WO 2021167107A1
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Prior art keywords
amino acid
peptide
seq
residue
minutes
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Ceased
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PCT/JP2021/006709
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English (en)
French (fr)
Japanese (ja)
Inventor
高橋　健一
英士 余田
秀彦 橋本
紗希 藤山
政輝 大内
中村　菜穂子
ナセル 加藤 バシルディン
直己 澤井
武 江原
正訓 多久和
圭一 舛屋
慎之介 稲葉
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JCR Pharmaceuticals Co Ltd
Peptidream Inc
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JCR Pharmaceuticals Co Ltd
Peptidream Inc
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Application filed by JCR Pharmaceuticals Co Ltd, Peptidream Inc filed Critical JCR Pharmaceuticals Co Ltd
Priority to AU2021224412A priority Critical patent/AU2021224412A1/en
Priority to IL295797A priority patent/IL295797A/en
Priority to EP21756644.7A priority patent/EP4108676A4/en
Priority to CN202180015934.0A priority patent/CN115151556A/zh
Priority to US17/801,261 priority patent/US20230203098A1/en
Priority to JP2022501104A priority patent/JP7796636B2/ja
Priority to CA3171988A priority patent/CA3171988A1/en
Publication of WO2021167107A1 publication Critical patent/WO2021167107A1/ja
Anticipated expiration legal-status Critical
Priority to JP2025197068A priority patent/JP2026032056A/ja
Ceased legal-status Critical Current

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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
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/56—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
    • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/62—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a peptide capable of binding to the human transferrin receptor (hTfR).
  • the present invention relates to peptides capable of crossing the blood-brain barrier (BBB), peptides having directional muscle tissue, and peptides having cell permeability.
  • BBB blood-brain barrier
  • the present invention relates to a method of delivering an arbitrary substance into the brain or a method of delivering to muscle tissue by using these peptides.
  • BBB blood-brain barrier
  • the blood-brain barrier also limits the exchange of substances between the blood and the tissue fluid of the central nervous system, including the brain and spinal cord, as well as the brain. Due to the presence of the blood-brain barrier, most cells of the central nervous system maintain their biochemical homeostasis without being affected by fluctuations in the concentrations of substances such as hormones and lymphokines in the blood.
  • Patent Document 1 describes a blood-brain barrier shuttle that has an affinity for the transferrin receptor and is capable of binding to the receptor.
  • One invention described herein is intended to provide a novel peptide that binds to the human transferrin receptor (hTfR). Another invention further provides peptides capable of crossing the blood-brain barrier (BBB), peptides having directional muscle tissue and capable of efficiently translocating to muscle tissue, and peptides having cell permeability. The purpose is to do. Another invention aims to provide various uses of the above novel peptides.
  • BBB blood-brain barrier
  • Another invention aims to provide various uses of the above novel peptides.
  • One invention described herein relates to a peptide that binds to a transferrin receptor.
  • This peptide is the amino acid sequence set forth in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or 1 in the amino acid sequence set forth in SEQ ID NO: 1. It is a peptide having an amino acid sequence having substitutions, deletions, additions and / or insertions of amino acid residues of 10 or more.
  • peptides that bind to the human transferrin receptor (hTfR), peptides that can cross the blood-brain barrier (BBB), and peptides that are directional to muscle tissue. , Peptides with cell permeability, etc. can be provided.
  • FIG. 1 is a photograph instead of a drawing showing the results of fluorescence intensity measurement in each tissue.
  • FIG. 1-2 is a photograph instead of a drawing showing the results of fluorescence intensity measurement in each tissue.
  • FIG. 1-3 is a photograph instead of a drawing showing the results of fluorescence intensity measurement in each tissue.
  • FIG. 2 is a photograph that replaces the drawing showing the result of fluorescence intensity measurement in the enlarged brain.
  • FIG. 3 is a photograph that replaces the drawing showing the results of the mouse brain localization confirmation test (single dose).
  • FIG. 4 is a photograph that replaces the drawing showing the results of the mouse brain localization confirmation test (multidose).
  • FIG. 5 is a fluorescence micrograph instead of a drawing showing the transition to human breast cancer cells.
  • One invention described herein relates to a peptide that binds to a transferrin receptor and is capable of crossing the blood-brain barrier.
  • Transferrin receptor is a receptor that is contained in plasma and has the function of binding to transferrin, which is a protein that binds to iron ions, and taking it into cells.
  • the transferrin receptor is expressed on various cells such as reticulocytes, placental trophoblast cells, and lymphocytes, and its expression in tumor cells has been suggested.
  • transferrin receptor since the transferrin receptor has the property of triggering cell endocytosis by stimulating the binding of iron ions in plasma, an antibody that binds to the transferrin receptor is used as DDS to allow the substance to pass through the BBB. Research is underway.
  • the human transferrin receptor is referred to as human TfR, hTfR or simply TfR.
  • Peptide that binds to the transferrin receptor Bound to the transferrin receptor means that it specifically binds to the transferrin receptor.
  • Affinity expressed by the equilibrium constant (KD) for dissociation of the transferase receptor and the binding peptide, is a measure of the binding strength between the transferase receptor and the antigen-binding site on the binding peptide: small KD values.
  • KD equilibrium constant
  • the affinity can be expressed as the affinity constant (KA), which is 1 / KD).
  • affinity can be determined in a manner known per se, depending on the particular antigen of interest. ..
  • Binding activity is a measure of the strength of binding between the transferrin receptor and the binding peptide. Binding activity is related to both the affinity between the transferrin receptor and its binding site on the binding peptide and the number of associated binding sites present on the binding molecule.
  • transtransferase receptor Specific binding of the transtransferase receptor to the binding peptide is, for example, the surface plasmon resonance (SPR) assay, scatterd analysis and / or radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA) and sandwich competition described herein.
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme-linked immunosorbent assay
  • sandwich competition described herein.
  • Competitive binding assays such as assays can be determined in any suitable manner known in the art, including its different variants known in the art.
  • the affinity of the peptide of the invention with the transtransferase is less than 100 nM, preferably less than 50 nM KD.
  • BBB blood-brain barrier
  • Brain-related diseases are diseases caused by some abnormality in the brain, and include, for example, central nervous system (CNS) diseases.
  • CNS central nervous system
  • brain-related diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Prion's disease, Huntington's disease, Lysozome's disease, central neuropathy, central nervous system tumors including brain tumors, cerebral ischemia, diseases associated with cerebral disorders, etc. Traumatic central nervous system disorders, viral and bacterial central nervous system disorders, and psychiatric disorders such as schizophrenia and depression.
  • the muscular tissue having a directivity to the muscular tissue may be any of myocardium, skeletal muscle and smooth muscle. Particularly preferred muscle tissue is myocardium or skeletal muscle. Having directivity in muscle tissue means having the property of migrating to muscle tissue in a specific and efficient manner.
  • Neuromuscular disease refers to diseases that cause motor disorders such as muscle weakness due to nerve or muscle lesions such as the brain, spinal cord, and peripheral nerves.
  • Examples of neuromuscular disease are, but are not limited to, spinal cerebral degeneration, muscular atrophic lateral sclerosis, severe myasthenia, muscular dystrophy, polymyopathy, hereditary myopathy, neuromuscular disease, muscular atrophy, drug-induced Myopathy, acute heart failure, chronic heart failure, myocardial infarction, chronic fatigue syndrome, mitochondrial disease, mitochondrial respiratory chain complex disorder and Gillan Valley syndrome.
  • Peptides with cell permeability are known, for example, as described in Japanese Patent No. 6478632 and Japanese Patent No. 6708770 (peptide having cell permeability). Then, as shown by Examples, the peptide of the present invention binds to the transferrin receptor and is taken up into cells. Therefore, by using the peptide of the present invention or a complex thereof, the target active ingredient can be delivered into the cell, and for example, a nucleic acid drug can be delivered into the cell.
  • Peptides refers to a structure in which multiple amino acids are continuous, and includes polypeptides and proteins in that sense.
  • the amino acid is not only a naturally occurring amino acid (natural amino acid) in which the mRNA is translated into the peptide chain in the cell and is incorporated into the peptide chain, but also a natural amino acid that can form a part of the peptide chain by peptide bond. It also includes amino acids (unnatural amino acids) that do not exist in. Amino acids may be artificially synthesized or naturally occurring.
  • a peptide having a cyclic portion formed by cyclization after synthesis also referred to as a cyclic peptide
  • a peptide obtained by further chemically modifying the peptide a complex of a peptide and a substance bound to the peptide, and a peptide.
  • a complex in which a substance is bound via a linker is also included in the peptide of the present invention and the complex of a peptide and a substance.
  • a cyclic peptide is a peptide in which two amino acids separated from each other via one or more amino acid residues in the amino acid sequence are bound to each other, so that all or part of the peptide becomes cyclic.
  • the bond type between the two amino acids is not particularly limited, but an amide bond between the carboxyl group of one amino acid and the amino group of the other amino acid, and the carboxyl group of one amino acid and the other amino acid.
  • the amide bond is not limited to the one formed by the bonding of the carboxyl group of one amino acid and the amino group of the other amino acid, and is not limited to that formed by the bond of the amino group of the synthetic reaction. As a result, it may be bound by an amide bond.
  • the cyclic peptide may have a linear portion as long as a part thereof forms a cyclic structure.
  • amino acids may be modified due to peptide cyclization. It also includes amino acids with some such modifications. For example, there is a case where a chloroacetyl group is added to an amino acid located at the N-terminal, and the amino acid is bound to a cysteine residue in a peptide to form a cyclization. Various (natural / unnatural) amino acids to which a chloroacetyl group is added. Is also included in the amino acids of the present application.
  • An unnatural amino acid is a compound other than a natural amino acid that has the characteristics of an amino acid.
  • ⁇ -amino acids ⁇ -amino acids, L-amino acids
  • D-amino acids also called D-type amino acids
  • chemically modified amino acids such as amino acid variants and amino acid derivatives
  • norleucine ⁇ - Examples include amino acids such as alanine and ornithine, which are not constituent materials of proteins in the living body.
  • N-methyl amino acid, N-ethyl amino acid, D-amino acid, histidine-like amino acid, amino acid having a structure such as extra methylene or aromatic ring in the side chain, and carboxylic acid functional group amino acid in the side chain are replaced with sulfonic acid groups. Examples thereof include amino acid derivatives having a similar structure. Examples of unnatural amino acids and abbreviations herein are shown below.
  • the CAS reference number or the company name of the supplier is shown in parentheses, and the synthesis example number is shown for the newly synthesized one.
  • the CAS number is one in which the unnatural amino acid alone or a protective group is bonded.
  • the special amino acid is not limited to these, and for example, one or more hydrogen atoms in these molecules are alkyl groups.
  • Substituted structures are also special amino acids.
  • the alkyl group is preferably a methyl group, an ethyl group, and more preferably a methyl group.
  • amino acids having Me or N-Me- before the amino acid name indicate N-methyl amino acids unless otherwise specified.
  • the N-methylated amino acid of alanine (Ala or A) is designated as MeAla, N-MeAla, MeA or N-MeA.
  • Amino acid notation with a one-letter notation and a description of d in front of it indicates a D-amino acid.
  • the D-amino acid of alanine (Ala or A) is indicated by da.
  • Those without a CAS number or purchase destination can be purchased as general reagents.
  • the following amino acids can be used for peptide synthesis by Fmoc-protecting the alphaamino group by a known method.
  • the peptide of the present invention has the amino acid sequence set forth in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or in the amino acid sequence set forth in SEQ ID NO: 1.
  • the number of amino acids substituted, deleted, added and / or inserted may be 1 or more and 10 or less, and the lower limit is 1.
  • the upper limit is 10, 9, 8, 7, 6, 6, 5, 4, 3, 2, and the minimum is one.
  • Such amino acid substitutions are preferably conservative amino acid substitutions.
  • Conservative amino acid substation means substitution with a functionally equivalent or similar amino acid.
  • Conservative amino acid substitutions in a peptide result in static changes in the amino acid sequence of the peptide.
  • one or more amino acids with similar polarities act functionally equivalent, resulting in static changes in the amino acid sequence of such peptides.
  • permutations within a group can be considered conservative in terms of structure and function.
  • the role played by a particular amino acid residue can be determined in terms of its implications for the three-dimensional structure of the molecule containing that amino acid.
  • cysteine residues can take the oxidized (disulfide) form, which is less polar than the reduced (thiol) form.
  • the long aliphatic portion of the arginine side chain can constitute structurally and functionally important features.
  • side chains containing aromatic rings can contribute to ion-aromatic interactions or cation-pi interactions.
  • substituting amino acids with these side chains with amino acids belonging to the acidic or non-polar group may be structurally and functionally conservative.
  • Residues such as proline, glycine, and cysteine (disulfide form) can have a direct effect on the conformation of the main chain and often cannot be replaced without structural distortion.
  • Conservative amino acid substitutions are specific substitutions based on side chain similarity (Leninja, Biochemistry, 2nd revised edition, 1975, pp. 73-75: L. Leniner, Biochemistry, 2nd edition, as shown below. , Pp73-75, Worth Publicer, New York (1975)) and typical substitutions.
  • Preferred examples of this peptide include the following groups: (I) Substitution of the first alanine residue of SEQ ID NO: 1 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) Substitution of the second valine residue of SEQ ID NO: 1 with a basic amino acid residue or a methylated basic amino acid residue; (III) Substitution of the third phenylalanine residue of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an aromatic ring, or an amino acid residue with a condensed ring; (IV) Substitution of the 4th valine residue of SEQ ID NO: 1 with a methylated valine residue; (V) Aromatic amino acid residue, methyl tryptophan residue, methylated aromatic amino acid residue, amino acid residue with an aromatic ring, or amino acid residue with a fused ring added to the 5th tryptophan residue of SEQ ID NO: 1.
  • Non-polar amino acid group alanine (hereinafter referred to as “Ala” or simply “A”), valine (hereinafter referred to as “Val” or simply referred to as “V”), leucine (hereinafter referred to as “Leu” or simply “Leu”).
  • L isoleucine (hereinafter referred to as “Ile” or simply “I”), proline (hereinafter referred to as “Pro” or simply referred to as “P”), phenylalanine (hereinafter referred to as “Phe” or simply referred to as “F”). ), Tryptophan (hereinafter referred to as “Trp” or simply “W”), methionine (hereinafter referred to as “Met” or simply referred to as “M”)
  • Gly Glycine
  • Ser serine
  • Thr threonine
  • Thr threonine
  • T cysteine
  • Cys cysteine
  • Tyr tyrosine
  • Asn Glutamine
  • Acidic amino acid group aspartic acid (hereinafter referred to as "Asp” or simply “D”), glutamic acid (hereinafter referred to as “Glu” or simply referred to as “E”)
  • Hydrophobic Amino Acid Group Norleucine, Met, Ala, Val, Leu, Ile
  • Neutral hydrophilic amino acid group Cys, Ser, Thr, Asn, Gln
  • Acidic amino acid group Asp
  • Glu (4) Basic amino acid group: His, Lys, Arg
  • Amino acid groups that affect the direction of the main chain Gly, Pro
  • Aromatic amino acid group Trp, Tyr, Phe In each group, unnatural amino acids such as N-methylated amino acids are also included.
  • a preferred example of this peptide is a peptide having the amino acid sequence set forth in SEQ ID NO: 2 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeTyr-Cys). be.
  • a preferred example of this peptide is in the amino acid sequence set forth in SEQ ID NO: 2, the following groups: (I) Substitution of the first alanine residue of SEQ ID NO: 2 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) Substitution of the second valine residue of SEQ ID NO: 2 with a basic amino acid residue or a methylated basic amino acid residue; (III) Substitution of the third phenylalanine residue of SEQ ID NO: 2 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an aromatic ring, or an amino acid residue with a condensed ring; (IV) Substitution of the 4th valine residue of SEQ ID NO: 2 with a methylated valine residue; (V) Substitution of the fifth tryptophan residue of SEQ ID NO: 2 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an aromatic ring, or an amino acid residue with a fused
  • peptide A set forth in SEQ ID NO: 18 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys). Assuming that the peptide contains an amino acid sequence and has a peptide length of 10 or more and 17 or less, Peptide A, or a peptide having an amino acid sequence having substitutions, deletions, and / or insertions of 1 or more and 6 or less amino acid residues in peptide A.
  • a preferred peptide in this specification is a peptide that can bind to the human transferrin receptor (hTfR) in the same manner as the above-mentioned peptide. Also preferred are peptides that are capable of crossing the blood-brain barrier (BBB), peptides that are directional to muscle tissue, or peptides that are cell permeable.
  • BBB blood-brain barrier
  • peptide A it may be a peptide having an amino acid sequence in which any of the amino acid residues 2, 3, 5, 8 and 10 of SEQ ID NO: 18 is substituted. "Amino acid residues have been replaced” means that a particular amino acid residue has been replaced with another potentially modified amino acid residue.
  • the second amino acid residue of SEQ ID NO: 18 is valine (Val), which may be modified, or glutamic acid (Glu), which may be modified.
  • the third amino acid residue of SEQ ID NO: 18 is phenylalanine (Phe), which may be modified.
  • the fifth amino acid residue of SEQ ID NO: 18 is tryptophan (Trp) which may be modified.
  • the eighth amino acid residue of SEQ ID NO: 18 is tyrosine, which may be modified.
  • the 10th amino acid residue of SEQ ID NO: 18 may be a peptide which is isoleucine (Ile) which may be modified or valine (Val) which may be modified.
  • May be modified means that known amino acid modifications and modifications may be made. Examples of modifications are N-methylation, amino acid modification having an abbreviation described later, modification (conversion) to D type, and conversion of a known amino acid to a derivative.
  • This peptide preferably has a peptide length of 11 or more and 13 or less. Twice
  • This peptide is more preferably a peptide having the following amino acid sequence.
  • the second amino acid residue of SEQ ID NO: 18 is Val or Glu.
  • the third amino acid residue of SEQ ID NO: 18 is Ph or MeF3C.
  • the fifth amino acid residue of SEQ ID NO: 18 is Trp or MeTrp.
  • the 8th amino acid residue of SEQ ID NO: 18 is Tyr or F4OMe.
  • the 10th amino acid residue of SEQ ID NO: 18 may be a peptide of Ile or Val.
  • this peptide is a peptide having the following amino acid sequence at the N-terminal of peptide A.
  • the 11th amino acid residue of SEQ ID NO: 18 is Ser or His.
  • the 12th amino acid residue of SEQ ID NO: 18 may be a peptide of Cys or Hgl.
  • peptide B the first to tenth of SEQ ID NO: 15 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Val-Pro-Arg-Asp-Cys). Assuming that the peptide contains the amino acid sequence described in the second order and has a peptide length of 10 or more and 19 or less. Peptide B, or a peptide having an amino acid sequence having substitutions, deletions, and / or insertions of 1 or more and 5 or less amino acid residues in peptide B.
  • Peptide B may be a peptide having an amino acid sequence in which any of the amino acid residues 2, 3, 5, 8 and 10 of SEQ ID NO: 15 is substituted.
  • the second amino acid residue of SEQ ID NO: 15 is valine (Val), which may be modified, or glutamic acid (Glu), which may be modified.
  • the third amino acid residue of SEQ ID NO: 15 is phenylalanine (Phe) or tryptophan (Trp), which may be modified.
  • the fifth amino acid residue of SEQ ID NO: 15 is tryptophan (Trp) which may be modified.
  • the eighth amino acid residue of SEQ ID NO: 15 is tyrosine, which may be modified.
  • the 10th amino acid residue of SEQ ID NO: 15 may be a peptide which is isoleucine (Ile) which may be modified or valine (Val) which may be modified.
  • These peptides may have a peptide length of 13 or more and 15 or less. Twice
  • This peptide is more preferably a peptide having the following amino acid sequence.
  • the second amino acid residue of SEQ ID NO: 15 is Val or Glu.
  • the third amino acid residue of SEQ ID NO: 15 is Ph, Trp or MeF3C.
  • the fifth amino acid residue of SEQ ID NO: 15 is Trp or MeTrp.
  • the eighth amino acid residue of SEQ ID NO: 15 is Tyr, Phe or F4OMe.
  • a peptide in which the 10th amino acid residue of SEQ ID NO: 15 is Ile or Val may be used.
  • this peptide is a peptide having the following amino acid sequence at the N-terminal of peptide B.
  • the 11th amino acid residue of SEQ ID NO: 15 is Ph.
  • the 12th amino acid residue of SEQ ID NO: 15 is Arg.
  • the 13th amino acid residue of SEQ ID NO: 15 is Glu, Asn, Asp, His, Gln or MeTrp.
  • the 14th amino acid residue of SEQ ID NO: 15 may be a peptide of Cys.
  • peptide C is the first of SEQ ID NO: 214 (MeA-Val-MeF3C-Val-MeW-Asn-Tyr-F4OMe-Ile-Ile-Arg-Arg-Phe-MeY-Cys). Assuming that the peptide contains the amino acid sequence described in the 10th position and has a peptide length of 11 or more and 19 or less. Peptide C, or a peptide having an amino acid sequence having substitutions, deletions, and / or insertions of 1 or more and 5 or less amino acid residues in peptide C.
  • Peptide C may be a peptide having an amino acid sequence in which any of the amino acid residues 1, 3, 5, and 8 of SEQ ID NO: 214 is substituted.
  • the first amino acid residue of SEQ ID NO: 214 is alanine (Ala) which may be modified or glutamic acid (Glu) which may be modified.
  • the third amino acid residue of SEQ ID NO: 214 is phenylalanine (Phe), which may be modified.
  • the fifth amino acid residue of SEQ ID NO: 214 is tryptophan (Trp) which may be modified.
  • the eighth amino acid residue of SEQ ID NO: 214 may be a peptide which is phenylalanine (Phe) which may be modified.
  • These peptides preferably have a peptide length of 15 or more and 18 or less. Twice
  • This peptide is more preferably a peptide having the following amino acid sequence.
  • the first amino acid residue of SEQ ID NO: 214 is Ala, Aib, Abu, Glu, Gly, Ser, Phe, Pro or MeA, particularly preferably Ala or MeA.
  • the third amino acid residue of SEQ ID NO: 214 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, Me3CO, MeF3COO MeF3C, particularly preferably Ph, MeF or MeF3C.
  • the fifth amino acid residue of SEQ ID NO: 214 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1P.
  • the 8th amino acid residue of SEQ ID NO: 214 is Phe, Tyr, Type, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOME, Ye It may be a peptide of Orn, or Nal1, and particularly preferably Tyr or F4OMe.
  • this peptide is a peptide having the following amino acid sequence at the N-terminal of peptide C.
  • the 11th amino acid residue of SEQ ID NO: 214 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, donn, aMeK, A1Me4pip, KCOpipezMe, F4G, Nle, Nva or Orn, particularly preferably Lys or Arg.
  • the 12th amino acid residue of SEQ ID NO: 214 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, particularly preferably Lys, Arg or dr.
  • the 13th amino acid residue of SEQ ID NO: 214 is Ala, Ph, Asn, Tyr, or pHPeG, particularly preferably Ph or Tyr.
  • the 14th amino acid residue of SEQ ID NO: 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph ,, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, in particular. Ph or MeW,
  • the 15th amino acid residue of SEQ ID NO: 214 may be a peptide of Cys or Hgl.
  • peptide D is peptide D, the first of SEQ ID NO: 219 (Ala-Glu-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys). Assuming that the peptide contains the amino acid sequence described in the 10th position and has a peptide length of 11 or more and 19 or less. Peptide D, or a peptide having an amino acid sequence having substitutions, deletions, and / or insertions of 1 or more and 5 or less amino acid residues in peptide D.
  • Peptide D may be a peptide having an amino acid sequence in which any of the amino acid residues 2, 3, 5, 8 and 10 of SEQ ID NO: 219 is substituted.
  • the second amino acid residue of SEQ ID NO: 219 may be modified valine (Val), modified glutamic acid (Glu), modified arginine (Arg), or modified.
  • the third amino acid residue of SEQ ID NO: 219 is phenylalanine (Phe), which may be modified.
  • the fifth amino acid residue of SEQ ID NO: 219 is tryptophan (Trp) which may be modified.
  • the eighth amino acid residue of SEQ ID NO: 219 is tyrosine, which may be modified.
  • the 10th amino acid residue of SEQ ID NO: 219 may be a peptide which is isoleucine (Ile) which may be modified, glutamic acid (Glu) which may be modified, or lysine (Lys) which may be modified.
  • Ile isoleucine
  • Glu glutamic acid
  • Lys lysine
  • These peptides preferably have a peptide length of 15 or more and 18 or less.
  • This peptide is more preferably a peptide having the following amino acid sequence.
  • the second amino acid residue of SEQ ID NO: 219 is Val, Glu, Ala, Arg, Lys, Asp, Phe, Dap, Har, Abu, Nva, AcPr, AtbAhp or Hgl, particularly preferably Gln or Val.
  • the third amino acid residue of SEQ ID NO: 219 is Ph, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3COMe, Me.
  • MeF3F Glu, Epyrl2RCOO, Dpyrl2RCOO or MeF3C, particularly preferably Phe, MeF or MeF3C.
  • the fifth amino acid residue of SEQ ID NO: 219 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1 W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON or W6F, and particularly preferably Trp and MeW.
  • the 8th amino acid residue of SEQ ID NO: 219 is Phe, Tyr, Type, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOME, Ya Orn, or Nal1, particularly preferably Tyr or F4OMe.
  • the 10th amino acid residue of SEQ ID NO: 219 is Ala, Abu, Accr, Ahp, Aib, alI, alT, Atb, Dab, Dap, dorn, Gln, Hly, Ile, Lys, KCOpipezMe, Leu, Nle, Nva, It may be a peptide of Pro, Arg, Ser, Thr, Tbg, Val or Tyr, and particularly preferably Ile or alI.
  • this peptide is a peptide having the following amino acid sequence at the N-terminal of peptide C.
  • the 11th amino acid residue of SEQ ID NO: 219 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, donn, aMeK, A1Me4pip, KCOpipezMe, F4G, Nle, Nva or Orn, particularly preferably Lys or Arg.
  • the 12th amino acid residue of SEQ ID NO: 219 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr.
  • the 13th amino acid residue of SEQ ID NO: 219 is Ala, Ph, Asn, Tyr, or pHPeG, particularly preferably Ph or Tyr.
  • the 14th amino acid residue of SEQ ID NO: 219 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph ,, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, in particular. Ph or MeW,
  • the 15th amino acid residue of SEQ ID NO: 219 may be a peptide of Cys or Hgl.
  • peptide E Another preferred example of this peptide is peptide E, first of SEQ ID NO: 296 (Ala-Val-MeF-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys). Assuming that the peptide contains the amino acid sequence described in the 15th position and has a peptide length of 15 or more and 18 or less. Peptide E, or a peptide having an amino acid sequence having substitutions, deletions, and / or insertions of 1 or more and 5 or less amino acid residues in peptide E.
  • Peptide E may be a peptide having an amino acid sequence in which any of the amino acid residues 3, 5, 7, 8, 11, 12, and 13 of SEQ ID NO: 296 is substituted.
  • the third amino acid residue of SEQ ID NO: 296 is phenylalanine (Phe), which may be modified.
  • the fifth amino acid residue of SEQ ID NO: 296 is tryptophan (Trp) which may be modified.
  • the seventh amino acid residue of SEQ ID NO: 296 is tyrosine, which may be modified.
  • the eighth amino acid residue of SEQ ID NO: 296 is tyrosine, which may be modified.
  • the 11th amino acid residue of SEQ ID NO: 296 is arginine (Arg) which may be modified or alanine (Ala) which may be modified.
  • the 12th amino acid residue of SEQ ID NO: 296 is arginine (Arg) which may be modified or lysine (Lys) which may be modified.
  • the 13th amino acid residue of SEQ ID NO: 296 may be a peptide in which either tyrosine (Tyr) may be modified or phenylalanine (Phe) may be modified.
  • the third amino acid residue of SEQ ID NO: 296 is Ph, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3COMe, Me. MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO or MeF3C, particularly preferably Phe, MeF or MeF3C.
  • the fifth amino acid residue of SEQ ID NO: 296 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1 W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON or W6F, and particularly preferably Trp and MeW.
  • the 7th amino acid residue of SEQ ID NO: 296 is Tyr, 3Py6OMe, Ala, Ahp, Phe, F3H, F4C, Nal1, Arg or Trp, particularly preferably Tyr.
  • the 8th amino acid residue of SEQ ID NO: 296 is Phe, Tyr, Type, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOME, Ya Orn, or Nal1, particularly preferably Tyr or F4OMe.
  • the 11th amino acid residue of SEQ ID NO: 296 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, donn, aMeK, A1Me4pip, KCOpipezMe, F4G, Nle, Nva or Orn, particularly preferably Lys or Arg.
  • the 12th amino acid residue of SEQ ID NO: 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr.
  • the 13th amino acid residue of SEQ ID NO: 296 may be Ala, Phe, Asn, Tyr, or pHPeG, and particularly preferably a peptide of Phe or Tyr.
  • this peptide is a peptide having the following amino acid sequence at the N-terminal of peptide C.
  • the 11th amino acid residue of SEQ ID NO: 296 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, donn, aMeK, A1Me4pip, KCOpipezMe, F4G, Nle, Nva or Orn, particularly preferably Lys or Arg.
  • the 12th amino acid residue of SEQ ID NO: 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr.
  • the 13th amino acid residue of SEQ ID NO: 296 is Ala, Ph, Asn, Tyr, or pHPeG, particularly preferably Ph or Tyr.
  • the 14th amino acid residue of SEQ ID NO: 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph ,, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, in particular. Ph or MeW,
  • the 15th amino acid residue of SEQ ID NO: 296 may be a peptide of Cys or Hgl.
  • the third amino acid residue of SEQ ID NO: 296 is phenylalanine (Phe), methylated phenylalanine (MeF), or N- ⁇ -methyl-N- ⁇ -chloroacetyl-3-chloro-L-phenylalanine (MeF3C).
  • the fifth amino acid residue of SEQ ID NO: 296 is tryptophan (Trp) or methylated tryptophan (MeW).
  • the 7th amino acid residue of SEQ ID NO: 296 is tyrosine.
  • the eighth amino acid residue of SEQ ID NO: 296 is tyrosine (Tyr) or (S) -2-amino-3- (4-methoxyphenyl) propanoic acid (F4OMe).
  • the 11th amino acid residue of SEQ ID NO: 296 is arginine (Arg) or lysine (Lys).
  • the 12th amino acid residue of SEQ ID NO: 296 is arginine (Arg) or D-arginine (dr).
  • a peptide in which the 13th amino acid residue of SEQ ID NO: 296 is tyrosine (Tyr) or phenylalanine (Phe) may be used.
  • a preferred example of this peptide is a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 3 to 200, or an amino acid sequence having an N-terminal of chloroacetyl-Ala in any of the amino acid sequences of SEQ ID NOs: 3 to 200. ..
  • a preferred example of this peptide is any of the peptides described above, which is a cyclic peptide.
  • Preferred examples of this peptide include the 1st to 10th amino acid sequences of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 552 or a complex of an amino acid sequence and a linker, and the amino acid sequence site has a cyclic structure. It is a peptide.
  • this peptide are the amino acid sequence set forth in any of SEQ ID NOs: 2, 9, 21 to 148, 159 to 200, 213 to 448, 450 to 552, or the first to the complex of the amino acid sequence and the linker. It is a peptide consisting of the 15th amino acid sequence and having a cyclic structure at the amino acid sequence site.
  • Cyclic peptide refers to a peptide in which two amino acids are bound and all or part of them are cyclic.
  • those in which amino acids in the peptide form a crosslinked structure those in which a cyclic structure is formed by lactam ring formation or macrocyclization reaction, those having a lassopeptide-like structure, and the like are also included. That is, in the present application, the cyclic peptide may have a linear portion as long as a part thereof forms a cyclic structure.
  • Peptides generally have poor metabolic stability in vivo and have a problem that they are difficult to permeate through cell membranes due to their large size. To solve such a problem, a method of cyclizing the peptide has been adopted. Cyclization of the peptide suggests that protease resistance is improved, metabolic stability is improved, and conformational change is also restricted, resulting in increased rigidity and improved membrane permeability and affinity with target proteins. It has been.
  • Cyclization method Peptide cyclization can be carried out according to a known method.
  • a cyclic structure can be formed by a disulfide bond after being translated.
  • a peptide having a chloroacetyl group at the N-terminal was synthesized by the reprogramming technique of the genetic code according to the method of Goto et al. , It can also be cyclized by placing a cysteine residue in the peptide.
  • the mercapto group spontaneously nucleophilically attacks the chloroacetyl group after translation, and the peptide is cyclized by a thioether bond.
  • amino acid combinations that bind to form a ring may be placed within the peptide to form a ring by genetic code reprogramming techniques. It can also be cyclized by synthesizing a peptide having a cycloamide at the N-terminal and arranging an Hgl residue in the peptide. As described above, any known cyclization method can be used without particular limitation.
  • a preferred example of this peptide is any of the above peptides, which consists of 15 amino acid residues.
  • Peptide length The number of amide bonds (number and length of amino acids) of the peptide and peptide site is not particularly limited, but when the total amino acid residue (the substance bound to the peptide or the linker that binds the substance to the peptide contains amino acids) , Those amino acids are not included) is preferably within 20 residues. Amino acids are preferably 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 or more, and preferably amino acids are 19 or less, 18 or less, 17 or less, 16 or less, and 15 or less.
  • This complex is a complex containing any of the above-mentioned peptides, a linker bound to this peptide, and a substance bound to this linker. At a minimum, it is preferably a complex in which the substance can cross the blood-brain barrier.
  • the complex as a whole may be able to cross the blood-brain barrier.
  • the complex is preferably directional in muscle tissue.
  • the complex is preferably capable of transporting at least the substance to the muscle tissue.
  • the complex preferably has cell permeability.
  • the complex is preferably capable of transporting at least the substance to the cell.
  • linker is one in which the amino acid length of the linker is 1 or more and 15 or less, and the linker contains one or more of glycine (Gly) or serine (Ser).
  • Gly glycine
  • Ser serine
  • a preferred example of this linker is cysteine (Cys), which may be N-terminally modified, or lysine (Lys), which may be modified.
  • linker is one that has an amino acid length of 1 or more and 5 or less and contains either or both of D-form glutamic acid (de) and methylated glycine (MeG).
  • de D-form glutamic acid
  • MeG methylated glycine
  • a preferred example of this linker is a complex in which the N-terminus is optionally modified cysteine (Cys) or optionally modified lysine (Lys).
  • linker is polyethylene glycol (PEG) or a PEG linker containing a derivative of polyethylene glycol.
  • Derivatives of polyethylene glycol include all known as PEG linkers. It is preferable that the PEG linker further contains glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), or lysine (Lys).
  • Gly glycine
  • Ser serine
  • Glu glutamic acid
  • Arg arginine
  • Lys lysine
  • a preferred example of this linker is a complex in which the N-terminus is optionally modified cysteine (Cys) or optionally modified lysine (Lys).
  • linker is one in which the linker has the sequence indicated by any of SEQ ID NOs: 201, 553 to 644.
  • linker Polyethylene glycol (PEG), G-linker, which is a peptide linker consisting of Gly or MeG, GS linker, which is a peptide linker consisting of Gly or MeG and Ser, Alternatively, it is that of a linker having an amino acid sequence shown in any of SEQ ID NOs: 201, 553 to 644.
  • PEG Polyethylene glycol
  • G-linker which is a peptide linker consisting of Gly or MeG
  • GS linker which is a peptide linker consisting of Gly or MeG and Ser
  • a linker (also referred to as a cross-linker), herein, indicates an intermolecular link between a peptide that binds to a transferrin receptor and a substance that is desired to be delivered to the brain, and may be any known or described herein. ..
  • the linker is, for example, a chemical linker, a fatty acid linker, a peptide linker (polypeptide linker). It may also be a complex of, for example, a chemical linker and a peptide linker. For example, it may be a linker structure having both PEG and an amino acid residue or a peptide moiety shown in SEQ ID NO: 616, SEQ ID NO: 627 and the like.
  • the linker may be, for example, one that deviates or separates depending on the environment and conditions, or one that maintains a stable structure.
  • the linker may be a chemical linker.
  • Chemical linkers include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene and / or. Includes substituted or unsubstituted heteroarylene.
  • Peptides and linkers can also be conjugated via sulfhydryl groups, amino groups (amines), and / or carbohydrates or any suitable reactive group. Homobifunctional and heterobifunctional crosslinkers (conjugation agents) are available from many commercial sources.
  • the crossed linker may contain a flexible arm, eg, 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 carbon atoms.
  • cross-linkers include BSS3 ([bis (sulfosuccinimidel) sverate], NHSS / EDC (N-hydroxysuccinimide and N-ethyl- (dimethylaminopropyl) carbodiimide, sulfo-EMCSS ([N-e-].
  • Maleimide caproic acid Hydrazide, hydrazide, SSATA (N-succinimidel-SS-acetylthioacetic acid) and the like.
  • a preferred example of a chemical linker is a PEG (Polyethyleneglycol) linker.
  • the PEG linker may be a PEG linker consisting of 1 to 24 ethylene glycol units.
  • the linker may be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid.
  • the fatty acid linker may be a linker having 12-aminododecanoic acid.
  • a peptide linker contains at least one amino acid (eg, a peptide of at least 2,3,4,5,6,7,10,15,20,25,40 or 50 amino acids).
  • the linker is a single amino acid (eg, any natural amino acid such as Cys).
  • the sequence [Gly-Gly-Gly-Gly-Ser] n (where n is 1,2,3,4,5) as described in US Pat. No. 7,271,149.
  • a glycine-rich peptide such as a peptide having (or 6) is used.
  • a serine-rich peptide linker as described in US Pat. No. 5,525,491, is used.
  • the linker is a single amino acid (eg, any amino acid such as Gly or Ala).
  • An invention described herein relates to a prophylactic or therapeutic agent for brain-related diseases.
  • the prophylactic or therapeutic agent for this brain-related disease contains the above-mentioned complex, and the above-mentioned substance is the active ingredient.
  • a substance is a substance that is delivered to the brain.
  • the substance may be any substance desired by those skilled in the art as long as it is a substance desired to be delivered to the brain.
  • BBB binds to the transferrin receptor and depends on the mechanism of endocytosis, transcytosis, etc.
  • a substance that is too large to be transported by those mechanisms is not preferable.
  • Examples of such materials include, but are not limited to: Compound: Not only a small molecule compound and a medium molecule compound, but any compound that can be introduced by the cytosis mechanism of cells may be used. For example, known small molecule drugs can be mentioned.
  • Peptide A peptide that binds to a target in the body and exerts some effect, for example, a cyclic peptide.
  • RI Any compound that can be labeled with a radioisotope, such as a small molecule, medium molecule compound or antibody labeled with a radioisotope.
  • Protein Any protein such as an antibody or an enzyme that exhibits a useful function in the body may be used.
  • enzymes used in enzyme replacement therapy can be mentioned.
  • Nucleic acid Anything containing a base sequence such as DNA and RNA may be used.
  • a nucleic acid drug can be mentioned.
  • DDS It may be a DDS molecule such as a liposome or a micelle. The DDS molecule may further contain a compound such as a pharmaceutical product inside. And, it may be a complex thereof listed above.
  • a certain invention described in this specification relates to a method for producing a prophylactic or therapeutic agent for a brain-related disease.
  • This method is a method for producing a prophylactic or therapeutic agent for a brain-related disease, which comprises the step of obtaining the above-mentioned complex.
  • linker Polyethylene glycol (PEG), G linker, GS linker Alternatively, it is a linker having the amino acid sequence shown in any of SEQ ID NOs: 201, 553 to 644.
  • One invention described herein relates to a diagnostic agent for brain-related diseases, including the complexes described above.
  • An invention described in this specification is a prophylactic or therapeutic agent for neuromuscular diseases containing the above-mentioned complex.
  • the substance is the active ingredient in a prophylactic or therapeutic agent for neuromuscular disease.
  • Certain inventions described herein include the complexes described above.
  • One invention described herein relates to a diagnostic agent for neuromuscular disease, including the complexes described above.
  • the peptide of the present invention can be produced by a known peptide production method such as a liquid phase method, a solid phase method, a chemical synthesis method such as a hybrid method combining a liquid phase method and a solid phase method; and a gene recombination method. Twice
  • the hydroxyl group of a resin having a hydroxyl group is subjected to an esterification reaction with the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) in which the ⁇ -amino group is protected by a protecting group.
  • esterification catalyst known dehydration condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used.
  • the protective group of the ⁇ -amino group of the first amino acid is eliminated, and the second amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group.
  • the first and second amino acids are bound.
  • the ⁇ -amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group to activate the second and second amino acids. Bind the third amino acid. This is repeated to deprotect all functional groups once the peptide of the desired length has been synthesized.
  • Resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Link amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEG, etc. Can be mentioned. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).
  • a solvent dimethylformamide (DMF), 2-propanol, methylene chloride, etc.
  • Protecting groups for the ⁇ -amino group include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Allloc). ) Group etc.
  • the Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc.
  • the Boc group can be deprotected by trifluoroacetic acid (TFA)
  • the Fmoc group can be deprotected by treatment with piperidine.
  • methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like can be used.
  • the hydroxy group of serine or threonine can be protected with a benzyl group or tert-butyl group, and the hydroxy group of tyrosine is protected with a 2-bromobenzyloxycarbonyl dilute or tert-butyl group.
  • the amino group of the lysine side chain and the carboxy group of glutamic acid and aspartic acid can be protected in the same manner as the ⁇ -amino group and the ⁇ -carboxy group.
  • Activation of the carboxy group can be performed using a condensing agent.
  • the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), and (1H-benzotriazole-1-yloxy) tris.
  • DCC dicyclohexylcarbodiimide
  • DIPCDI diisopropylcarbodiimide
  • EDC or WSC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • (1H-benzotriazole-1-yloxy) tris examples thereof include (dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexaflu
  • Cleavage of the peptide chain from the resin can be performed by treating with an acid such as TFA or hydrogen fluoride (HF). Twice
  • the peptide can be produced by the gene recombination method (translational synthesis system) using the nucleic acid encoding the peptide of the present invention.
  • the nucleic acid encoding the peptide of the present invention may be DNA or RNA.
  • the nucleic acid encoding the peptide of the present invention can be prepared by a known method or a method similar thereto. For example, it can be synthesized by an automatic synthesizer. A restriction enzyme recognition site may be added to insert the obtained DNA into the vector, or a base sequence encoding an amino acid sequence for excising the resulting peptide chain with an enzyme or the like may be incorporated.
  • the nucleic acid when the peptide of the present invention is fused with a membrane-permeable peptide or the like, the nucleic acid also includes a nucleic acid encoding a membrane-permeable peptide.
  • a chimeric protein expression method in which the target peptide is expressed as a chimeric peptide with another peptide can also be used.
  • the nucleic acid a nucleic acid encoding a target peptide and a peptide that binds to the target peptide is used as the nucleic acid.
  • an expression vector is prepared using the nucleic acid encoding the peptide of the present invention.
  • the nucleic acid can be inserted as it is, digested with a restriction enzyme, added with a linker, or the like, and inserted downstream of the promoter of the expression vector.
  • Vectors include Escherichia coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), bacteriophage-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pC194, pC194, etc.).
  • Escherichia coli-derived plasmids pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.
  • bacteriophage-derived plasmids pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.
  • yeast-derived plasmids pC194, pC194, etc.
  • bacteriophage e-phage, M13 phage, etc.
  • viruses retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), Califlower mosaic virus, tobacco mosaic virus, etc.) , Bacteriophage, etc.
  • Cosmid etc. Twice
  • the promoter can be appropriately selected according to the type of host.
  • a promoter derived from SV40 simian virus 40
  • CMV cytomegalovirus
  • Escherichia coli a trp promoter, a T7 promoter, a lac promoter, or the like can be used.
  • the expression vector encodes a DNA replication origin (ori), a selectable marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a poly A addition signal, and a tag (FLAG, HA, GST, GFP, etc.). It is also possible to incorporate a nucleic acid or the like.
  • the host can be appropriately selected in relation to the vector, and for example, Escherichia coli, Bacillus subtilis, Bacillus), yeast, insects or insect cells, animal cells and the like are used.
  • animal cells for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be performed according to a known method such as a lipofection method, a calcium phosphate method, an electroporation method, a microinjection method, or a particle gun method, depending on the type of host. By culturing the transformant according to a conventional method, the target peptide is expressed. Twice
  • the obtained peptide may be converted from a free form to a salt or from a salt to a free form by a known method or a method similar thereto.
  • the translation synthesis system may be a cell-free translation system.
  • Cell-free translation systems include, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF) elongation factor (EF), termination factor (RF), and ribosomal proteins. Includes regeneration factor (RRF), as well as other factors required for translation.
  • Escherichia coli extract or wheat germ extract may be added to increase the expression efficiency.
  • rabbit red blood cell extract or insect cell extract may be added.
  • a system containing RNA polymerase may be used to perform transcription from gene DNA at the same time.
  • Escherichia coli-derived systems include RTS-100 (registered trademark) GeneFrontier Corp. PURESYSTEM and NEW ENGLAND Biolabs PUREXpress In Vitro Protein Synthesis Kit, etc.
  • RTS-100 registered trademark
  • PURESYSTEM registered trademark
  • NEW ENGLAND Biolabs PUREXpress In Vitro Protein Synthesis Kit, etc.
  • As a system using the wheat germ extract those of Zoegene, CellFree Science, etc. can be used. According to the cell-free translation system, the expression product can be obtained in a highly pure form without purification.
  • an artificial aminoacyl-tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to the tRNA may be used instead of the aminoacyl-tRNA synthesized by the natural aminoacyl-tRNA synthase.
  • Such aminoacyl-tRNA can be synthesized using an artificial ribozyme.
  • Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A. Ohta, H. Ashigai, H.
  • Flexizymes are also known as prototype flexizymes (Fx) and modified dinitrobenzyl flexizymes (dFx), enhanced flexizymes (eFx), aminoflexizymes (aFx) and the like.
  • a desired codon can be translated in association with the desired amino acid or hydroxy acid.
  • a special amino acid may be used.
  • the unnatural amino acids required for cyclization described above can also be introduced into the binding peptide by this method.
  • the chemical synthesis of the large cyclic peptide and its analogs of the present invention is widely used in various technical fields including stepwise solid phase synthesis, semisynthesis of peptide fragments undergoing coordinated support religation, and chemical ligation. Can be synthesized using the above method.
  • the synthesis of peptides and their analogs described herein can be described, for example, in K. et al. J. Jensen, P.M. T. Shelton, S.A. L. It is a chemical synthesis using various solid phase technologies as described in Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013 and the like.
  • a preferred strategy is based on a combination of an Fmoc group that temporarily protects the ⁇ -amino group and allows selective removal by a base, and a protecting group that temporarily protects the side chain functional group and is stable under de-Fmoc conditions. .. Selection of such a general peptide side chain is described in Peptide Synthesis and Applications, 2nd edition and G.I. B. Fields, R.M. L. Noble, Solid Phase, Peptide Synthesis, Utilizing 9-Fluorolenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res.
  • preferred peptide side chain protecting groups are Boc group and Mtt group for amino groups such as lysine, and tert-butyl group for carboxyl group of glutamate and aspartic acid.
  • Trt and Mmt groups are also Trt and Mmt groups relative to the thiol group of cysteine.
  • the peptides and analogs thereof described in the present invention can be synthesized by a stepwise method on the above-mentioned solid phase resin.
  • the C-terminal amino acids used and all amino acids and peptides used in the synthesis must be selectively stripped of ⁇ -amino protecting groups during the synthetic process.
  • the above-mentioned solid phase resin is used, and the C-terminal carboxyl group of an appropriately protected peptide such as Fmoc at the N-terminal or the C-terminal carboxyl group of an amino acid protected with Fmoc is used as an activated ester with an appropriate reagent and then solidified. It is started by adding to the amino group on the phase resin.
  • Subsequent elongation of the peptide chain can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and then the condensation of the protected amino acid derivative according to the amino acid sequence of the peptide of interest. In addition, these can release the target peptide at the final stage.
  • Fmoc group N-terminal protecting group
  • these can release the target peptide at the final stage.
  • TFA Water / TIS / DODT (volume ratio 92.5: 2.5: 2.5: 2.5).
  • peptide analogs described herein can be synthesized by using a single or multi-channel peptide synthesizer, for example, a Liberty Blue synthesizer from CEM or a Siro I synthesizer from Biotage.
  • a single or multi-channel peptide synthesizer for example, a Liberty Blue synthesizer from CEM or a Siro I synthesizer from Biotage.
  • Activation of the carboxy group can be performed using a condensing agent.
  • the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), and (1H-benzotriazole-1-yloxy) tris.
  • DCC dicyclohexylcarbodiimide
  • DIPCDI diisopropylcarbodiimide
  • EDC or WSC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • (1H-benzotriazole-1-yloxy) tris examples thereof include (dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexaflu
  • the medicament includes the above-mentioned peptides, pharmaceutically acceptable salts or solvates thereof (for simplicity, these are also simply referred to as peptides below).
  • This medicine preferably contains the above-mentioned peptide as an active ingredient in an effective amount.
  • the administration form of the pharmaceutical composition is not particularly limited, and may be oral administration or parenteral administration.
  • Parenteral administration includes, for example, intramuscular injection, intravenous injection, subcutaneous injection, and other injections, transdermal administration, and transmucosal administration (nasal, oral cavity, eye, lung, vagina, and rectum). Etc. can be raised. Twice
  • the above-mentioned pharmaceutical composition can be modified in various ways in view of the property that the polypeptide is easily metabolized and excreted.
  • polyethylene glycol (PEG) or a sugar chain can be added to a polypeptide to prolong the residence time in blood and reduce the antigenicity.
  • sustained-release bases such as biodegradable polymerized compounds such as polylactic acid / glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, and nanospheres are used. It may be used as an inclusion of a polypeptide.
  • a weak electric current can be applied to the surface of the skin to allow it to penetrate the stratum corneum (iontophoresis method). Twice
  • the active ingredient may be used as it is, or the pharmaceutical composition may be formulated by adding a pharmaceutically acceptable carrier, excipient, additive or the like.
  • Dosage forms include, for example, liquids (for example, injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, troches, etc. Examples include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.
  • Formulations include, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjustments. It can be carried out by a conventional method with appropriate use of agents, preservatives, antioxidants, etc.
  • ingredients used in formulation include purified water, saline solution, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose.
  • the above-mentioned pharmaceutical composition may contain an absorption enhancer for improving the absorption of a poorly absorbable drug in view of the fact that the peptide is difficult to be absorbed through the mucosa.
  • absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate and saponin; bile acids such as glycocholic acid, deoxycholic acid and taurocholic acid; chelating agents such as EDTA and salicylic acids; Fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitrogen monoxide donors, etc. Can be used.
  • Pills or tablets can also be coated with sugar-coated, gastric-soluble, enteric-soluble substances.
  • the injection may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols and the like.
  • wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizing agents, preservatives and the like can be added.
  • the dosage is determined by symptoms, patient age, sex, etc. It depends on the body weight, sensitivity difference, administration method, administration interval, type of active ingredient, type of preparation, and is not particularly limited, but for example, 30 ⁇ g to 100 g, 100 ⁇ g to 500 mg, 100 ⁇ g to 100 mg are administered in one or several divided doses. can do.
  • 1 ⁇ g / kg to 3000 ⁇ g / kg and 3 ⁇ g / kg to 1000 ⁇ g / kg may be administered once or in several divided doses depending on the body weight of the patient. Twice
  • a method for preventing or treating a brain-related disease using the peptide of the present invention can be carried out with reference to the above description of the pharmaceutical composition.
  • the above-mentioned pharmaceutical composition may be used as a preventive or therapeutic agent for neuromuscular diseases containing the above-mentioned complex.
  • This brain-related disease detection agent contains the above-mentioned peptides, salts thereof or solvates thereof.
  • Brain-related disease detection drug and detection kit The present invention also includes a brain-related disease detecting agent containing the peptide of the present invention.
  • the peptides of the invention may be detectable detectable.
  • Peptide labels include, for example, enzymes such as peroxidase and alkaline phosphatase, radioactive substances such as 125I, 131I, 35S and 3H, fluorescein isothiocyanate, rhodamine, dansylloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent material and the like.
  • Antibodies labeled with luminescent substances such as fluorescent substances, luciferase, luciferin, and equolin are used.
  • antibodies labeled with nanoparticles such as colloidal gold and quantum dots can be detected.
  • a complex is prepared from an antibody that binds to a specific target related to a brain-related disease and a peptide of the present invention, a complex labeled with the antibody or the peptide of the present invention is prepared, and the complex is administered and detected. This makes it possible to detect brain-related diseases.
  • the peptide of the present invention can be labeled with biotin, and avidin or streptavidin labeled with an enzyme or the like can be bound and detected.
  • the ELISA method using an enzyme label is preferable because the antigen can be measured easily and quickly.
  • the antibody is immobilized on a solid-phase carrier, a sample is added and reacted, and then a labeled peptide of the present invention is added and reacted. After washing, brain-related diseases can be detected by reacting with the enzyme substrate, developing color, and measuring the absorbance.
  • the unlabeled peptide of the present invention may be added, and the antibody against the peptide of the present invention may be further labeled with an enzyme.
  • DAB 3,3'-diaminobenzidine
  • TMB 3,3', 5,5'-tetramethylbenzidine
  • OPD o-phenylenediamine
  • NPP p-nitrophenyl phosphatase
  • the "solid phase carrier” is not particularly limited as long as it is a carrier capable of immobilizing an antibody, and is a microtiter plate made of glass, metal, resin, etc., a substrate, beads, a nitrocellulose membrane, a nylon membrane, PVDF. Examples thereof include membranes, and the target substance can be immobilized on these solid-phase carriers according to a known method. Twice
  • the test kit according to the present invention includes, but is not limited to, the reagents and instruments necessary for the above detection (including, but not limited to, the peptide, antibody, solid phase carrier, buffer solution, enzyme reaction terminator, microplate reader, etc. of the present invention). including. Twice
  • Another embodiment disclosed herein is a brain-related disease detection kit containing the above-mentioned brain-related disease detection agent, a brain-related disease, and a tool for elucidating various cell functions and biological phenomena associated therewith. Can also be considered for use as.
  • This specification also provides the use of peptides for the manufacture of pharmaceuticals for the prevention or treatment of brain-related diseases.
  • the peptide in this case may be any of the above.
  • the specification also provides for the use of peptides to produce pharmaceuticals for the prevention or treatment of neuromuscular diseases.
  • the peptide in this case may be any of the above.
  • This specification comprises the step of administering an effective amount of a peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a complex thereof to a subject who is a human, a non-human mammal or a bird. Also provides preventive or therapeutic methods for.
  • a peptide the above-mentioned peptide can be appropriately used. Examples of non-human mammals are non-human primates, pigs, cows, dogs, cats, horses, sheep, rats and mice.
  • This specification comprises the step of administering to a human, non-human mammal or avian subject an effective amount of a peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a complex thereof. Also provides preventive or therapeutic methods for.
  • the extension of the peptide chain in the solid-phase resin was carried out using the resins described in the respective examples as starting materials, and using the commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions.
  • the reaction was carried out using a CEM Liberty Blue, which is an automatic peptide synthesizer, according to the manufacturer's manual. Common amino acids used are listed below and side chain protecting groups are shown in parentheses.
  • Fmoc-Trp Boc
  • Fmoc-Thr (tBu) -OH
  • Fmoc-Asp OtBu
  • Fmoc-N-Me-Phe H
  • Fmoc-Ala H
  • Fmoc-N-Me-Ala H
  • Fmoc-His Trt
  • Fmoc-HidPro (tBu) -OH
  • Fmoc-Cys Trt) -OH
  • Fmoc-Lys (Mtt) -OH
  • Fmoc-N-Me-Ser tBu
  • the Fmoc group of the ⁇ -amino group is removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then chloroacetic acid (3rd grade, etc.) is introduced. Amount) is added by adding 3 equal amounts of N, N'-diiropropylcarbodiimide DMF solution (0.5 M) and 3 equal amounts of HOAt DMF solution (0.5 M), and shaking at room temperature for 40 minutes. rice field.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF and methylene chloride 5 times each and dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 150 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, and the solution component was recovered from the frit and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the peptide cyclization reaction is carried out by dissolving the peptide in DMSO so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, adding 6 equal amounts of triethylamine, and stirring at room temperature for about 16 hours. rice field.
  • the obtained reaction solution was acidified with acetic acid and concentrated under reduced pressure using Biotage (registered trademark) V-10 (Biotage Japan).
  • the purity of the chemically synthesized peptide in the present invention was determined by any of the following analytical methods.
  • Analysis condition A Column; CORETCS (registered trademark) UPLC (registered trademark) C18 volume (Japan Waters Corp.), 90 ⁇ , 1.6 ⁇ m, 2.1 x 100 mm
  • Mobile phase 0.025% TFA inMeCN / 0.025% TFA inH 2 O
  • Temperature 40 ° C
  • Gradient 5-95% MeCN / 0.025% TFA in H 2 O in 5.56 min; linear gradation Flow rate 0.4 mL / min
  • Detection method UV 220 nm
  • the hTfR-binding peptide was identified by the screening method described in WO2014 / 119600, WO2012 / 033154, or WO2007 / 066627. ..
  • the peptide was chemically synthesized for the purpose of confirming whether or not the peptide actually has a binding activity to hTfR.
  • the sequence of the synthesized peptide is shown in Table 1.
  • the peptide of interest was synthesized, starting with the removal of Fmoc according to the general method described above.
  • Liberty Blue HT manufactured by CEM was used as a solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual.
  • the basic condition of the condensation reaction was to react at 75 ° C. for 10 minutes.
  • the basic condition for de-Fmocization was to react in 20% piperidine in DMF at 75 ° C. for 3 minutes.
  • the Fmoc group of the ⁇ -amino group is removed from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 5 equal amounts of chloro are introduced.
  • a DMF solution of acetic acid (0.2 M), a DMF solution of 5 equal volumes of HATU (0.5 M), and a DMF solution of 10 equal volumes of DIPEA (1 M) were added and shaken at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure.
  • a reactant cocktail (a mixture of TFA / H 2 O / TIS / DODT in a volume ratio of 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin, and the temperature was changed to room temperature. Was shaken for 90 minutes. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. Addition of this filtrate to excess diethyl ether / hexane (1/1) cooled to 0 ° C. resulted in a cloudy precipitate.
  • the mixture was centrifuged (10000 rpm, 1 min) and the solution was decanted.
  • the obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and the obtained solid was dried and used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 6 equal amounts of triethylamine was added, and the mixture was stirred at room temperature for about 16 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVac.
  • the obtained target product was analyzed under the above-mentioned analysis condition A or analysis condition B, and the structure was confirmed by ESI-MS (+) in the mass spectrum analysis method.
  • Table 1 shows the obtained ESI-MS (+) observed value and the value of X when shown as the number of proton additions (M + XH) X + in that case.
  • a 350 mM EDTA solution was reacted at a flow rate of 10 ⁇ L / min for 60 seconds, a 0.5 mM NiCl 2 solution (Kishida Chemistry) was reacted at a flow rate of 10 ⁇ L / min for 60 seconds, and then a 3 mM EDTA solution (Nacalai Tesque, Inc.) was reacted at a flow rate of 10 ⁇ L / min.
  • the NTA sensor chip was washed with min for 60 seconds.
  • a 1.0 M aqueous ethanolamine solution (Global Life Science Technologies Japan Co., Ltd.) was reacted at a flow rate of 10 ⁇ L / min for 420 seconds for capping.
  • the peptide solution prepared to 10 mM in the DMSO solution was diluted with a running buffer solution so as to have a final concentration of 10 ⁇ M, and then 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM peptide solutions were prepared.
  • the kinetics of the peptide for hTfR was obtained by SPR measurement.
  • the kinetic evaluation model was Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evolution Software Version 3.0 (Global Life Science Technologies Japan Co., Ltd.). Curve fitting by the least squares method was performed on the obtained sensorgram, and the binding of the peptide to hTfR was evaluated by determining the KD value.
  • the KD value when the KD value is less than 1 nM, it is expressed as A, when it is 1 nM or more and less than 100 nM, it is expressed as B, when it is 100 nM or more and less than 1 ⁇ M, it is expressed as C, and when it is 1 ⁇ M or more, it is expressed as D. Shown in 1.
  • hTfRNo Cyclic peptides with 894 and similar amino acid sequences have been shown to have significant hTfR binding potential.
  • hTfRNo The chemical structure of 894 is as follows.
  • hTfR-Binding Peptide-PEG11-Vivotag 750 Conjugate hTfRNo. 894-Vivotag 750
  • hTfRNo a compound in which Vivotag 750 (VivoTag-S TM 750, PerkinElmer), which is a near-infrared fluorescent labeling substance, was bound via a PEG11 linker was synthesized as a payload (hTfRNo.894-vivotag750, or hTfR_000894_PEG11_). VivoTag)) (SEQ ID NO: 146).
  • the chemical structure of the hTfR-binding peptide-PEG11 is as follows, and the compound described was synthesized by binding Vivo-tag750 to the compound. Details are described below.
  • the 11th and 12th residues were repeatedly reacted twice at 25 ° C. for 20 minutes.
  • the 13th and 14th residues were repeatedly reacted twice at 75 ° C. for 10 minutes.
  • the 15th residue was reacted once at 25 ° C. for 20 minutes.
  • the 16th residue was reacted once at 25 ° C. for 60 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • Fmoc removal was carried out by reacting at 25 ° C. for 5 minutes and then reacting for 10 minutes.
  • the introduction of the chloroacetyl group is carried out with respect to the resin obtained in the previous step, 5 equal amounts of chloroacetic acid in DMF solution (0.2 M), 5 equal amounts of HATU DMF solution (0.5 M), and 10 equal amounts of DIPEA.
  • DMF solution (1M) was added to the solid phase resin and shaken at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether and hexane cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (10000 rpm, 1 min) and the supernatant was decanted.
  • the obtained solid was washed with ice-cooled diethyl ether and then dried.
  • the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction is carried out by dissolving in DMSO so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, adding 6 equal amounts of triethylamine, shaking at room temperature overnight, and then using a Savant Explorer SpeedVac. After concentration under reduced pressure, it was purified by reverse phase HPLC.
  • the resulting peptide (26.1mg, 9.30umol) was dissolved in DMSO / H 2 O (9/1) , 0.91 eq of VivoTag-NHS, 4.5 eq of DIEA was added stirred for 45 minutes bottom. AcOH was added to the reaction solution and quenched.
  • the purity of the target product was 97.1% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • ESI-MS (+) Observed value m / z 1226.4 (M + 3H) 3+
  • Control peptide conjugate hTfRNo. Synthesis of 894FLIP-vivotag750 hTfRNo. As a control of 894-vivotag750, No. A cyclic peptide having the following amino acid sequence in which the sequence of 894 was inverted and a conjugate in which vivotag 750 was bound via a PEG11 linker were synthesized (denoted as hTfRNo.894 FLIP-vivotag 750 or FLIP_000894_PEG11_K_FITC).
  • hTfRNo Brain transfer evaluation test using 894-vivotag750 hTfR-KI mice [preparation of test solution] Hydroxypropyl- ⁇ -cyclodextrin (Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 20 w / v%, and this was prepared as a 20% hydroxypropyl- ⁇ -cyclodextrin solution.
  • hTfRNo 894-vivotag 750 conjugate administration solution (No. 894 administration solution): hTfRNo. Synthesized in Example 2. To 6 ⁇ L of a 5 mM solution of 894-vivotag750 conjugate, 4.8 ⁇ L of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added, and 66 ⁇ L of a 20% hydroxypropyl- ⁇ -cyclodextrin solution was added and mixed well. To this, 48 ⁇ L of polyethylene glycol 400 was further added and mixed, and the mixture was No. The 894-administration solution was used. No. The hTfRNo. In the 894 administration solution.
  • DMSO dimethyl sulfoxide
  • the 894-FITC conjugate concentration is 250 ⁇ M. hTfRNo.
  • 894FLIP-vivotag750 conjugate administration solution (No. 894NC administration solution): The above-mentioned hTfRNo. To 6 ⁇ L of a 5 mM solution of 894FLIP-vivotag750 conjugate, 66 ⁇ L of a 20% hydroxypropyl- ⁇ -cyclodextrin solution was added and mixed well. To this, 48 ⁇ L of polyethylene glycol 400 was further added and mixed, and the mixture was No. The 894NC administration solution was used. No. 894FLIP administration solution hTfRNo. The 894FLIP-vivotag 750 conjugate concentration is 250 ⁇ M.
  • a DNA fragment having a base sequence in which a resistance gene was arranged and a targeting vector having a 5'arm sequence and a 3'arm sequence were prepared.
  • the prepared targeting vector was introduced into mouse ES cells by an electroporation method.
  • Mouse ES cells after gene transfer were selectively cultured in the presence of neomycin, and mouse ES cells in which the targeting vector was integrated into the chromosome by homologous recombination were selected.
  • the obtained transgenic mouse ES cells were injected into an 8-cell stage embryo (host embryo) of an ICR mouse and transplanted into a pseudo-pregnant mouse (recipient mouse) obtained by mating with a mouse subjected to vas deferens ligation. bottom. Hair color was determined for the obtained offspring (chimeric mice), and individuals in which ES cells contributed to the formation of a living body with high efficiency, that is, individuals in which white hair occupies a large proportion of total hair were selected.
  • mice in which the mouse transferrin receptor gene was heterozygously replaced with chimeric hTfR on the chromosome were designated as TfR-KI mice.
  • mice Brain migration evaluation test using TfR-KI mice
  • No. 2 for TfR-KI mice female, 12 weeks old
  • hTfR. 894 administration solution or No. 894NC administration solution was rapidly administered 100 ⁇ L each into the tail vein.
  • mice were anesthetized with isoflurane and perfused with saline from the left ventricle for 4 to 5 minutes for blood removal treatment.
  • tissues for measuring fluorescence intensity were collected and stored in physiological saline so as not to dry.
  • tissues for measuring fluorescence intensity were collected and stored in physiological saline so as not to dry.
  • the fluorescence intensity was measured according to the instruction manual.
  • Brain shows a picture of the brain
  • Live shows a picture of the lateral left lobe area of the liver
  • Kidney shows a picture of the kidney
  • (4) Lung shows a picture of the lungs
  • Thermal vertebrae shows a picture of the thoracic spine.
  • Color scale the lowest value for (1) is 5.00 ⁇ 10 7, the maximum value of 1.3 ⁇ 10 8, (2) and (3) a minimum value for the 6.00 ⁇ 10 8, the maximum The value is 2.4 ⁇ 10 10 , the minimum value is 4.5 ⁇ 10 8 and the maximum value is 5.4 ⁇ 10 9 for (3) and (4).
  • FIG. 1-1 (1) Heart shows a photograph of the heart, (2) Spleen shows a photograph of the spleen, and (3) Symus shows a photograph of the thymus.
  • the color scale is the lowest value of 4.83 ⁇ 10 7, the maximum value indicates a 4.8 ⁇ 10 9.
  • FIG. 1-3 (1) Quadriceps shows a photograph of the quadriceps femoris, and (2) Femur shows a photograph of the femur.
  • the color scale is the lowest value of 1.5 ⁇ 10 8, the maximum value indicates a 1.5 ⁇ 10 9.
  • FIG. 2 is an enlarged photograph of FIG. 1-1 (1) brain. From FIGS. 1-1, 1-2, 1-3 and FIG. 2, hTfRNo. The hTfRNo.
  • Tissues in which stronger fluorescence was detected than in the 894FLIP-vivotag750 conjugate group were kidney, thoracic spine, heart, femur, quadriceps and brain.
  • hTfRNo The hTfRNo.
  • the tissues in which stronger fluorescence was detected than in the 894-vivotag750 conjugate group were the liver and spleen.
  • mice were anesthetized with isoflurane and perfused with saline from the left ventricle for 4 to 5 minutes for blood removal treatment. Then, the brain whose fluorescence intensity was measured was collected and stored in physiological saline so as not to be dried. Using this brain tissue, immunohistochemical staining was performed using an anti-FITC antibody (MLB Bioscience).
  • Immunohistochemical staining of the anti-FITC antibody in the brain tissue was performed by a known method and observed with a fluorescence microscope. The results are shown in FIG. In the figure, the red arrow points to the cerebral capillaries, and the arrowhead points to Purkinje cells. (1) shows a photograph of the # 894-FITC-administered group, and (2) shows a photograph of the group not administered # 894-FITC as a control.
  • hTfR-binding special cyclic peptide No. was administered at a dose of 3.7 mg / kg every 10 minutes, for a total of 6 doses. It was administered to hTfR-KI mice conjugated with 894 and the fluorescent substance FITC, and the localization of the conjugate in the brain tissue was confirmed by immunohistochemical staining. The results are shown in FIG. In the figure, the red arrow points to the cerebral capillaries, the arrowhead points to Purkinje cells, and the yellow arrow points to dendrites. (1) shows a photograph of the # 894-FITC-administered group, and (2) shows a photograph of the group not administered # 894-FITC as a control.
  • Transferrin receptor (hTfR) by SPR and hTfRNo Intermolecular interaction evaluation test with variant peptide, linker-added peptide and payload conjugate of 894 [hTfRNo. 894 variants, synthesis of linker-added peptides] hTfRNo.
  • a peptide having a sequence in which several amino acids were inserted, deleted, or substituted in the amino acid sequence of 894 also referred to as a variant
  • a peptide to which various linkers were bound were synthesized, and the binding ability to hTfR by SPR was similarly confirmed.
  • Variant peptides were synthesized according to Example 1 unless otherwise stated in Example 9 or 10.
  • the linker-added peptide was synthesized according to Example 1 except that Fmoc-NH-SAL-PEG resin (Watanabe Chemistry) was used as a resin for peptide synthesis when the linker was PEG.
  • Peptides, fatty acids, and peptides to which a linker of PEG and a complex thereof was added were also synthesized according to Example 1 unless otherwise described in Example 9 or 10.
  • hTfRNo It has been shown that 894 and peptides in which several amino acids are inserted, deleted or substituted in the amino acid sequence and various linkers are bound have the ability to bind to hTfR.
  • Collagen Type I (Corning) was diluted with 20 mmol / L acetic acid to 50 ⁇ g / mL. One sterilized cover glass was placed in each well of the 24-well plate, diluted Collagen Type I solution was added, and the mixture was kept warm at 37 ° C. for 1 hour. Collagen Type I solution was removed and washed 3 times with PBS. 1 ⁇ 10 5 human breast cancer cells BT-549 per well were seeded and cultured overnight at 37 ° C. under 5% CO 2 conditions.
  • the 11th and 12th residues were reacted twice at 25 ° C. for 20 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th residue was reacted at 25 ° C. for 20 minutes.
  • the basic condition for de-Fmocification was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the Fmoc groups at the 2nd and 13th residues were removed by reacting at 25 ° C. for 5 minutes and then reacting for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 10 eq of chloroacetic acid and 10 eq.
  • DIPCI and 10 eq of HOSu were stirred in DCM and the same amount of DMF as that of DCM was added to prepare a DCM / DMF solution of ClAcOSu, which was added to the solid phase resin and shaken at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • F-FAM-NHS prepared from 1.1 eq 5-FAM (Funakoshi), 1.2 eq EDC, and 1.2 eq HOSU was added to the reaction solution, and the mixture was stirred at room temperature for 3 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 95.3% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • HTfR_894_variant03_PEG4dk5FAM similarly synthesized the synthesis hTfR_894_3m_PEG4dk5FAM of Flip894_variant61_PEG4dk5FAM as hTfR_894_variant61_PEG4dk5FAM and negative controls, HTfR_894_3m_PEG4 cyclized, HTfR_894_variant03_PEG4, to hTfR_894_variant61_PEG4 and Flip894_variant61_PEG4, likewise the F-FAM-NHS was adjusted from 5-FAM was added, the conjugate Obtained. Regarding the various synthesized conjugates, binding to hTfR was confirmed by SPR in the same manner as in Example 2. Only Flip894_variant91_PEG4 was not bound to hTfR.
  • a dilution medium (RPMI 1640 medium containing 0.5% bovine serum albumin and 20 ⁇ g / mL human transferrin holo form) was used to dilute the sample.
  • the sample was diluted with a dilution medium to 100 nmol / L.
  • the cells were washed twice with RPMI 1640 medium.
  • RPMI 1640 medium containing 0.5% bovine serum albumin was added at 500 ⁇ L / well and allowed to stand on ice for 15 minutes, and then a diluted sample solution was added at 500 ⁇ L / well under 37 ° C. and 5% CO 2 conditions. It was allowed to stand for 3 hours.
  • the cover glass was taken out from the 24-well plate, sealed on a slide glass using a Fluorescent Monitoring Medium (Agilent), and allowed to stand overnight at room temperature in the dark.
  • An inverted fluorescence microscope DMI6000B (Leica Microsystems, Inc.) was used for observation, and observation was performed at wavelengths for FITC and DAPI detection. The results are shown in FIG.
  • the scale bar in the figure indicates 50 ⁇ m. As can be confirmed in FIG.
  • hTfR_894_3m_PEG4dk5FAM, hTfR_894_variant03_PEG4dk5FAM, and hTfR_894_variant61_PEG4dk5FAM were confirmed to be intracellularly transferred, but hTfR did not have intracellular transfer to hTfR. From this result, it was shown that the variant having the ability to bind to hTfR_894 and hTfR translocates into the cell via the binding to hTfR.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Siever amide resin (Watanabe Kagaku, 0.52 mmol / g, 0.19 g). At that time, Styrene manufactured by Biotage was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The basic condition of the condensation reaction was to repeat the reaction at 75 ° C. for 20 minutes twice. However, when the 15th and 16th residues were introduced, the reaction was carried out at room temperature for 60 minutes and 15 minutes, respectively. Further, when PEG was introduced, the reaction was carried out once, and when the 11th amino acid was introduced, the condensation reaction was carried out three times.
  • the basic condition for de-Fmocification was to react in a DMF solution of piperazine (5%) and Oxima pure (0.2M) at 50 ° C. for 5 minutes, and then react again for 15 minutes.
  • the Fmoc group at the 15th and 16th residues was removed by reacting at 25 ° C. for 5 minutes and then reacting again for 15 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then adding 5 equal amounts of ClAcOSu. This was done by adding a DMF solution and shaking at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC. Subsequently, using the obtained solid-phase resin, side chain deprotection, excision from the solid-phase resin, and cyclization reaction were carried out according to the above-mentioned general method.
  • the purity of the target product was 91.5% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • Analytical condition B: Retention time 12.8 minutes: Gradient (% B conc); 20-60% over 20 minutes, then 60-95% over 1 minute, then 95% over 5 minutes.
  • ESI-MS (+) Observed value m / z 899.6 (M + 3H) 3+
  • the resin was mixed 3 times with DMF (10 mL, Kishida Chemistry), 3 times with DCM (10 mL, Kishida Chemistry), and 3 times with diethyl ether (10 mL, Kishida Chemistry). After washing, it was dried under reduced pressure to obtain Fmoc-CyS [CH 2 COO-Trt (2-Cl) -resin] -PEG8Me (1.686 g, 94%).
  • the peptide of interest was synthesized by starting with the removal of the Fmoc group by the above-mentioned general method.
  • Fmoc was removed by reacting with a 20% piperidine DMF solution at room temperature for 5 minutes and then reacting again for 15 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then adding a triequal amount of chloroacetic acid.
  • 3 equal volumes of HCTU DMF solution (0.43 M) and 3 equal volumes of DIPEA DMF solution (1.57 M) to the solid phase resin and shaking at room temperature for 30 minutes. went.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-HFIP / DCM (1/4) was added to the reaction vessel containing the solid phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken with a cocktail for cutting, the solution component was recovered from the frit, and the operation of mixing with the above-mentioned filtrate was repeated three times.
  • the condensation reaction was carried out twice at 25 ° C. for 30 minutes.
  • the 13th and 14th residues were performed twice at 75 ° C. for 10 minutes.
  • the 15th residue was performed once at 25 ° C. for 30 minutes.
  • the 16th residue was subjected to once for 120 minutes at 25 ° C. using Fmoc-AA-OH / HATU / DIEA (3eq / 3eq / 6eq).
  • the basic condition for de-Fmocization was to react in 20% piperidine in DMF at 75 ° C. for 3 minutes. However, the Fmoc group at the 13th and 15th residues was removed by reacting at 25 ° C. for 5 minutes and then for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then using 10 eq of chloroacetic acid. 10 eq of DIPCI and 10 eq of HOSu are stirred in DCM and the same amount of NMP as DCM is added to prepare a DCM / NMP solution (0.2 M) of ClAcOSu, which is added to the solid phase resin and shaken at room temperature for 60 minutes. rice field.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure. Subsequently, a reactant cocktail (a mixture of TFA / H 2 O / TIS / DODT in a volume ratio of 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin, and the temperature was changed to room temperature. Was shaken for 60 minutes. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • a reactant cocktail a mixture of TFA / H 2 O / TIS / DODT in a volume ratio of 92.5: 2.5: 2.5: 2.5
  • the purity of the target product was 77.0% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the reaction was carried out twice at 75 ° C. for 30 minutes.
  • the 9th residue was reacted twice at 90 ° C. for 10 minutes.
  • the 11th and 12th residues were reacted twice at 50 ° C. for 15 minutes.
  • the 13th residue was reacted twice at 90 ° C. for 3 minutes.
  • the 15th residue was reacted once at 50 ° C. for 15 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the removal of Fmoc at the 2nd residue, the 4th residue and the 13th residue was repeated twice at 25 ° C. for 5 minutes.
  • the Fmoc group of the ⁇ -amino group was removed from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step was retained by the method described above, and then 5 eq of chloroacetate in DMF, This was performed by adding 5 eq HATU in DMF and 10 eq DIEA in DMF to the solid phase resin and shaking at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 60 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether / hexane (1/1) cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (9000 rpm, 1.5 min) and the solution was decanted.
  • the obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and then dried under reduced pressure.
  • the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 10 equal amounts of triethylamine was added, and the mixture was shaken at room temperature for about 24 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 93.1% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 1st and 3rd residues were reacted once at 75 ° C. for 20 minutes.
  • the second and fourth residues were reacted twice at 75 ° C. for 30 minutes.
  • the 9th residue, the 10th residue, and the 11th residue were reacted twice at 75 ° C. for 20 minutes.
  • the 12th residue was reacted twice at 25 ° C. for 30 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th residue was reacted once at 25 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • the removal of Fmoc at the 2nd residue, the 4th residue and the 13th residue was repeated twice for 5 minutes at 25 ° C.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 5 eq of ClAcOH in DMF, 5 eq. HATU in DMF and 10 eq of DIEA in DMF were added to the solid phase resin and shaken at 25 ° C. for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 60 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 10 equal amounts of triethylamine was added, and the mixture was shaken at room temperature for about 16 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the second and fourth residues were reacted twice at 75 ° C. for 30 minutes.
  • the 10th and 11th residues were reacted twice at 90 ° C. for 10 minutes.
  • the 12th residue was reacted twice at 50 ° C. for 15 minutes.
  • the 13th residue was reacted twice at 90 ° C. for 3 minutes.
  • the 15th residue was reacted once at 50 ° C. for 15 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the removal of Fmoc at the 2nd residue, the 4th residue and the 13th residue was repeated twice at 25 ° C. for 5 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then using 10 eq of chloroacetic acid and 5 eq. DIPCI, 5 eq of HOSu was stirred in DCM and the same amount of DMF as DCM was added to prepare a DCM / DMF solution (0.2 M) of ClAcOSu, which was added to a solid phase resin and shaken at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 5 eq of triethylamine was added, and the mixture was shaken at room temperature for about 14 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 87.5% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • Fmoc-D-Glu (OtBu) -wang resin (Watanabe Kagaku, 0.68 mmol / g, 184 mg)
  • the peptide of interest was synthesized by starting with the removal of Fmoc according to a general method.
  • CEM's Liberty Blue HT was used as a solid-phase synthesizer, and the procedure was performed according to the manufacturer's manual.
  • the basic condition of the condensation reaction was that HATU was used as a condensing agent and the reaction was carried out twice at 75 ° C. for 10 minutes. However, the second residue was reacted twice at 75 ° C. for 30 minutes. The fourth residue was reacted twice at 75 ° C. for 30 minutes.
  • the 6th residue was reacted once at 75 ° C. for 10 minutes.
  • the 7th residue was reacted once at 75 ° C. for 10 minutes.
  • the 8th residue was reacted once at 75 ° C. for 10 minutes.
  • the 10th residue was reacted once at 75 ° C. for 10 minutes.
  • the 11th residue was reacted twice at 30 ° C. for 30 minutes.
  • the 12th residue was reacted once at 30 ° C. for 30 minutes.
  • the 15th residue was reacted once at 30 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • Fmoc removal was performed by reacting twice at room temperature for 5 minutes.
  • chloroacetic acid 5 equal amount
  • DIPCI 5 equal amount
  • HOSu 5 equal amount
  • the mixture was shaken at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. Addition of this filtrate to excess diethyl ether / hexane (3/1) cooled to 0 ° C. resulted in a cloudy precipitate.
  • the mixture was centrifuged (9500 rpm, 1 min), the supernatant was decanted, washed with diethyl ether cooled to 0 ° C., and the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 4 mM based on the number of moles of the solid-phase resin, 10 equal amounts of triethylamine was added, and the mixture was shaken at room temperature for about 16 hours.
  • the obtained reaction solution was concentrated under reduced pressure using GenevaC EZ-2 Elite.
  • the purity of the target product was 88.60% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • ESI-MS (+) Observed value m / z 1137.23 (M + 2H) 2+
  • the second and fourth residues were reacted twice at 75 ° C. for 45 minutes.
  • the 11th and 12th residues were repeatedly reacted twice at 50 ° C. for 15 minutes.
  • the reaction was carried out twice at 90 ° C. for 3 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. once for 3 minutes.
  • the removal of Fmoc at the 2nd residue, the 4th residue and the 13th residue was repeated twice at 25 ° C. for 5 minutes.
  • the resin was suspended in DCM, Pd (PPh 3 ) 4 / PhSiH 3 (0.2 eq / 10 eq) was added, and the mixture was shaken at room temperature for 1 hour.
  • Pd (PPh 3 ) 4 / PhSiH 3 0.2 eq / 10 eq
  • the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step was washed 3 times with DMF and 3 times with DCM, and then the Fmoc group of the ⁇ -amino group was removed by the method described above. Then, 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF were added, and the mixture was shaken at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the obtained mixture was dissolved in DMSO (4 mL), HOSU (10 eq) and EDC HCl (10 eq) were added, and the mixture was stirred at room temperature for 2 hours.
  • the purity of the target product was 97.9% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the reaction was carried out twice at 75 ° C. for 45 minutes.
  • the reaction was repeated twice at 50 ° C. for 15 minutes.
  • the reaction was carried out twice at 90 ° C. for 3 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. once for 3 minutes.
  • the removal of Fmoc at the 2nd residue, the 4th residue and the 13th residue was repeated twice at 25 ° C. for 5 minutes.
  • the resin was suspended in DCM, Pd (PPh 3 ) 4 / PhSiH 3 (0.2 eq / 10 eq) was added, and the mixture was shaken at room temperature for 1 hour.
  • the solid phase resin was washed 3 times with DMF and 3 times with DCM, and then dried under reduced pressure.
  • the Fmoc group of the ⁇ -amino group is removed from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 5 eq of chloroacetate in DMF, 5 eq. HATU in DMF of 10 eq of DIEA in DMF was added, and the mixture was shaken at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether / hexane (1/1) cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (10000 rpm, 1 min) and the solution was decanted.
  • the obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and then dried under reduced pressure.
  • the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 10 eq of triethylamine was added, and the mixture was shaken overnight at room temperature.
  • the reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the second and fourth residues were reacted twice at 75 ° C. for 30 minutes.
  • the 11th and 12th residues were reacted twice at 90 ° C. for 10 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th residue was reacted once at 25 ° C. for 20 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • the reaction was carried out at 25 ° C. for 5 minutes and then the reaction was carried out at 25 ° C. for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then chloroacetic acid (10 equal amounts).
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the peptide cyclization reaction was carried out by dissolving the peptide in a 50% MeCN aqueous solution so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, adding 5 equal amounts of triethylamine, and shaking at room temperature for about 14 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 98.2% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then chloroacetic acid (5 equal amounts).
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the purity of the target product was 95.4% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the second and fourth residues were reacted twice at 75 ° C. for 30 minutes.
  • the 11th and 12th residues were repeatedly reacted twice at 25 ° C. for 20 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th and 22nd residues were reacted once at 25 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the Fmoc removal at the 2nd residue, the 4th residue and the 13th residue was carried out at 25 ° C. for 5 minutes and then at room temperature for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then using 10 eq of chloroacetic acid and 10 eq of the solid phase resin.
  • DIPCI and 10 eq of HOSu were stirred in DCM and the same amount of DMF as that of DCM was added to prepare a DCM / DMF solution (0.2 M) of ClAcOSu, which was added to a solid phase resin and shaken at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 90: 2.5: 2.5: 5) was added to the reaction vessel containing the solid phase resin, and 90 at room temperature. Shake for a minute. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 90: 2.5: 2.5: 5
  • the reaction solution was collected by filtration from the frit.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-ment
  • the obtained mixture was dissolved in DMSO, 3 eq of silver acetate was added, and the mixture was shaken for 3 hours. After adding a 2M DTT aqueous solution (11eq), the mixture was centrifuged to collect the supernatant, and then concentrated under reduced pressure to obtain a crude product.
  • the peptide of interest was synthesized, starting with the removal of Fmoc according to a general method.
  • CEM's Liberty Blue HT was used as a solid-phase synthesizer, and the procedure was performed according to the manufacturer's manual.
  • the basic condition of the condensation reaction was to use DIPCI and Oxyma pure as a condensing agent and react once at 90 ° C. for 3 minutes. However, the 11th and 12th residues were repeatedly reacted twice at 50 ° C. for 15 minutes. The 13th residue was repeatedly reacted twice at 90 ° C. for 3 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, for the 13th residue, Fmoc was removed by reacting twice at 25 ° C. for 5 minutes. The obtained resin was washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • this filtrate was added to a mixed solvent of excess diisopropyl ether and hexane cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (9000 rpm, 2 min) and the supernatant was decanted.
  • the obtained solid was washed with ice-cooled diethyl ether and then dried. The obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction is carried out by dissolving the peptide in DMF so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, and then adding 1.5 equal amounts of HATU and 3 equal amounts of triethylamine to about room temperature. It was shaken for 1 hour. The obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 96.8% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 5 equal amounts of chloroacetic acid were added.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the obtained reaction solution was concentrated under reduced pressure using GenevaC EZ-2 Elite.
  • the final concentration of the peptide in the obtained mixture was dissolved in DMF so as to be 25 mM based on the number of moles of the solid phase resin, 1 equivalent HATU and 3 equivalent DIEA were added, and the mixture was shaken at room temperature for about 30 minutes. ..
  • the purity of the target product was 94.8% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the reaction was carried out twice at 90 ° C. for 3 minutes.
  • the reaction was carried out twice at 50 ° C. for 15 minutes.
  • the reaction was carried out once at 50 ° C. for 15 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. once for 3 minutes.
  • the removal of Fmoc at the 2nd residue, the 4th residue, and the 13th residue was repeated twice at 25 ° C. for 5 minutes.
  • the solid phase resin was washed successively with DMF and DCM, dried under reduced pressure, swollen with DCM, 10 equal amounts of PhSiH 3 and 0.2 equal amounts of Pd (PPh 3 ) 4 were added, and the mixture was shaken at room temperature for 1 hour.
  • the solid phase resin was washed 5 times with DCM, 5 times with DMF and 3 times with diethyl ether and dried.
  • the solid phase resin was swollen with NMP, 5 equal volumes of HATU and 5 equal volumes of DIEA were added, and the mixture was shaken at room temperature for 30 minutes. After washing the resin, 5 equal amounts of HATU and 5 equal amounts of DIEA were added again, and the mixture was shaken at room temperature for 30 minutes.
  • the solid phase resin was washed 5 times with DCM, 5 times with DMF and 3 times with diethyl ether.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 5 equal amounts of chloroacetic acid were added.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • Fmoc-Gly-wang resin (Watanabe Kagaku, 0.78 mmol / g, 1.31 g)
  • the peptide of interest was synthesized by starting with the removal of the Fmoc group by the above-mentioned general method.
  • Liberty Blue manufactured by CEM was used as a solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual.
  • Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) with respect to 1 equivalent of the resin, and repeat twice in DMF at 60 ° C. for 15 minutes. The reaction was carried out.
  • the reaction was carried out once at 25 ° C. for 30 minutes.
  • the reaction was repeated twice at 25 ° C. for 20 minutes.
  • the reaction was carried out once at 60 ° C. for 15 minutes.
  • the reaction was carried out once at 60 ° C. for 15 minutes.
  • the reaction was carried out once at 60 ° C. for 15 minutes.
  • the reaction was carried out twice at 60 ° C. for 45 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the purity of the target product was 92.2% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the purity of the target product was 97.3% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 5 equal amounts of chloroacetate in DMF. This was carried out by adding 5 equal amounts of HATU in DMF and 10 equal amounts of DIEA in DMF and shaking at 25 ° C. for 30 minutes. To deprotect the side chains and cut out from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 60 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diisopropyl ether / hexane (1/1) cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (9000 rpm, 2 min) and the solution was decanted.
  • the obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and then dried under reduced pressure.
  • the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 10 equal amounts of triethylamine was added, and the mixture was shaken overnight at room temperature.
  • the reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVac.
  • the obtained solid is dissolved in DMSO so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, and then MePEG4c (1.2 equal amount), HATU (1.1 equal amount), DIEA (3). Equal amount) was added and the mixture was shaken at room temperature for 1 hour.
  • the purity of the target product was 92.3% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, for the 13th and 15th residues, Fmoc removal was carried out by reacting at 25 ° C. for 5 minutes and then reacting for 10 minutes.
  • the introduction of the chloroacetyl group is carried out with respect to the resin obtained in the previous step, 5 equal amounts of chloroacetic acid in DMF solution (0.2 M), 5 equal amounts of HATU DMF solution (0.5 M), and 10 equal amounts of DIPEA.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • this filtrate was added to a mixed solvent of excess diethyl ether and hexane cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (10000 rpm, 1 min) and the supernatant was decanted.
  • the obtained solid was washed with ice-cooled diethyl ether and then dried. The obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 6 equal amounts of triethylamine was added, and the mixture was shaken overnight at room temperature.
  • 1.1 equivalent of FAM-OSu DMSO solution (0.71M) was added to the obtained reaction solution, and the mixture was stirred for 30 minutes.
  • AcOH was added to the reaction solution and quenched, and the solvent was concentrated under reduced pressure.
  • the purity of the target product was 97.1% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 2nd and 13th residues were reacted twice for 10 minutes at 75 ° C.
  • the 11th and 12th residues were repeatedly reacted twice at 25 ° C. for 20 minutes.
  • the 15th and 17th residues were reacted once at 25 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • the Fmoc removal at the 2nd residue, the 13th residue, the 15th residue, and the 17th residue was carried out at 25 ° C. for 5 minutes and then at 25 ° C. for 10 minutes.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 5 equal amounts of chloroacetic acid in. This was performed by adding 5 equal amounts of HATU in DMF and 10 equal amounts of DIEA in DMF to the solid phase resin and shaking at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure.
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether / hexane (1/1) cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (10000 rpm, 1 min), the solution was decanted, the solid was washed with diethyl ether cooled to 0 ° C., and then dried under reduced pressure. The obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 6 equal amounts of triethylamine was added, and the mixture was shaken overnight at room temperature.
  • the obtained reaction solution was subjected to Savant Explorer SpeedVaC. Was concentrated under reduced pressure.
  • the obtained mixture was dissolved in DMSO, 3 equal amounts of silver acetate were added, and the mixture was shaken for 3 hours. After adding 10 equal volumes of DTT, the supernatant was collected by centrifugation.
  • the purified intermediate peptide (29.8 mg, 10.6 ⁇ mol) is dissolved in DMSO (424 ⁇ L), 1.1 equal amounts of fluorescein-5-maleimide and 5 equal amounts of DIEA are added, and the mixture is stirred for 1 hour and then quenched with acetic acid. bottom.
  • the purity of the target product was 82.8% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 12th residue was repeatedly reacted twice at 25 ° C. for 20 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th and 22nd residues were reacted once at 25 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, for the removal of Fmoc at the 4th and 13th residues, the reaction was carried out at 25 ° C. for 5 minutes and then at room temperature for 10 minutes.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 10 equal amounts of chloroacetic acid were added.
  • Stir 10 equal amounts of DIPCI and 10 equal amounts of HOSu in DCM add the same amount of DMF as DCM to prepare a DCM / DMF solution (0.2M) of ClAcOSu, add to solid phase resin, and shake at room temperature for 60 minutes. I went by doing.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 90: 2.5: 2.5: 5) was added to the reaction vessel containing the solid phase resin, and 90 at room temperature. Shake for a minute. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 90: 2.5: 2.5: 5
  • the reaction solution was collected by filtration from the frit.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-ment
  • the obtained mixture was dissolved in DMSO (20 mL), 3 equal volumes of silver acetate were added, and the mixture was shaken for 3 hours. A 2M DTT aqueous solution (11 equal volumes) was added, and the mixture was centrifuged to collect the supernatant and then concentrated under reduced pressure to obtain a crude product.
  • the purity of the target product was 94.8% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • Peptides or linker-added peptides were synthesized as follows. The synthesized peptides and linker-added peptides are shown in Table 5, and the linkers are shown in Table 6.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, Liberty Blue manufactured by CEM was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 94.20% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, CEM's Liberty Blue HT was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 97.61% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, Liberty Blue manufactured by CEM was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 97.52% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, Liberty Blue manufactured by CEM was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 98.02% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, Liberty Blue manufactured by CEM was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 96.34% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Sieber amide resin (Watanabe Kagaku, 0.48 mmol / g, 0.52 g). At that time, Liberty Blue manufactured by CEM was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual. The synthesis was carried out in the same manner as in Example 1.
  • the purity of the target product was 96.03% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.
  • the 10th, 11th, and 13th residues were repeatedly reacted twice for 10 minutes at 75 ° C.
  • the 12th residue was reacted twice at 25 ° C. for 20 minutes.
  • the 15th residue was reacted once at 25 ° C. for 20 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the Fmoc removal at the 2nd residue, the 4th residue and the 13th residue was carried out at 25 ° C. for 5 minutes and then at 25 ° C. for 10 minutes.
  • the Fmoc group of the ⁇ -amino group was removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step by the method described above, and then 10 equal amounts of chloroacetic acid were added.
  • Stir 10 equal amounts of DIPCI and 10 equal amounts of HOSu in DCM add the same amount of DMF as DCM to prepare a DCM / DMF solution (0.2M) of ClAcOSu, add to solid phase resin, and shake at room temperature for 60 minutes. I went by doing.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the purity of the target product was 92.8% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • Fmoc-PEG4c-Abu-Alko resin prepared according to more general solid phase synthesis than Fmoc-Abu (4) -Alko resin (Watanabe Kagaku, 1.00 mmol / g, 0.1 g) was used in the above-mentioned general method. Therefore, starting with the removal of Fmoc, a C-terminal carboxylic acid peptide was synthesized. At that time, Liberty Blue manufactured by CEM was used as a solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual.
  • the introduction of the chloroacetyl group is carried out after removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 0.2M chloroacetate.
  • DMF 5 equal amounts
  • 0.5M HATU in DMF 5 equal amounts
  • 1M DIEA in DMF 10 equal amounts
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure.
  • a reactant cocktail (a mixture of TFA / H 2 O / TIS / DODT in a volume ratio of 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin, and the temperature was changed to room temperature. Was shaken for 90 minutes. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. Addition of this filtrate to excess diethyl ether / hexane (1/1) cooled to 0 ° C. resulted in a cloudy precipitate.
  • the mixture was centrifuged (10000 rpm, 1 min) and the solution was decanted.
  • the obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and the obtained solid was dried and used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid-phase resin, 10 equal amounts of triethylamine was added, and the mixture was stirred at room temperature for about 17 hours.
  • the reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVac.
  • the purity of the target product was 87.4% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 1st, 9th, 10th, 13th, and 14th residues were reacted twice at 75 ° C. for 10 minutes.
  • the 11th and 12th residues were reacted twice at 30 ° C. for 20 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes.
  • the reaction was carried out at 25 ° C. for 5 minutes and then at 25 ° C. for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then chloroacetic acid (5 equal amounts).
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 60 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the peptide of interest was synthesized by starting with the removal of the Fmoc group by the above-mentioned general method.
  • Liberty Blue manufactured by CEM was used as a solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual.
  • the basic condition of the condensation reaction was that HATU was used as a condensing agent and the reaction was carried out once at 75 ° C. for 10 minutes. However, when introducing the 15th residue, the reaction was carried out once at 25 ° C. for 20 minutes. When introducing the 13th residue, the reaction was repeated twice at 75 ° C.
  • the DMF solution (0.2 M) of the above was added with 4 equal volumes of the DMF solution of DIPCI (0.5 M) and 4 equal volumes of the DMF solution of HOSu (0.5 M), and the mixture was shaken at room temperature for 60 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the reaction solution was collected by filtration from the frit.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. Addition of this filtrate to excess diisopropyl ether cooled to 0 ° C. resulted in a cloudy precipitate.
  • the mixture was collected by filtration, washed with diethyl ether, and dried under reduced pressure. The obtained solid was used for the next cyclization reaction.
  • peptide cyclization reaction For the peptide cyclization reaction, dissolve in DMSO / IPA / H 2 O (90/5/5) so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, and then add 5 equal amounts of triethylamine. The mixture was stirred at room temperature for about 16 hours. The obtained reaction solution was concentrated under reduced pressure using GenevaC HT-12.
  • the purity of the target product was 90.6% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 3rd, 4th, 8th, 10th, 13th, and 14th residues were reacted twice at 75 ° C. for 10 minutes.
  • the 11th and 12th residues were reacted twice at 50 ° C. for 20 minutes.
  • the second residue was reacted at 75 ° C. for 60 minutes three times.
  • the first residue was reacted twice at 75 ° C. for 20 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, for the 15th residue, the 16th residue, and the 17th residue, Fmoc removal was carried out by reacting at room temperature for 5 minutes and then at 75 ° C. for 3 minutes.
  • the Fmoc group of the ⁇ -amino group was removed from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step was retained by the method described above. Then, chloroacetic acid (5 equal amounts), DIPCI (5 equal amounts), and HOSu (5 equal amounts) were stirred in DCM and the same amount of DMF as DCM was added to prepare a DCM / DMF solution (0.15 M) of ClAcOSu. , And shaken at room temperature for 180 minutes in addition to the solid phase resin.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times and dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 90 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the purity of the target product was 94.1% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the mixture was centrifuged (9000 rpm, 2 min) and the solution was decanted.
  • the obtained solid was washed with diethyl ether cooled to 0 ° C., centrifuged (9000 rpm, 2 min), and the solution was decanted.
  • the purity of the target product was 96.1% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 5th, 6th, 7th, 16th, 17th, 19th, 21st, and 22nd residues are reacted once at 75 ° C for 10 minutes. went.
  • the 11th residue was reacted twice at 50 ° C. for 15 minutes.
  • the 12th, 18th, and 20th residues were reacted once at 50 ° C. for 15 minutes.
  • the 15th residue was reacted once at 50 ° C. for 15 minutes.
  • the basic condition for removing Fmoc was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, for the 2nd and 13th residues, Fmoc removal was performed by reacting twice at room temperature for 5 minutes.
  • chloroacetic acid 5 equal amount
  • DIPCI 5 equal amount
  • HOSu 5 equal amount
  • ClAcOSu's DCM / DMF solution 0. 15M
  • the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 150 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether and hexane cooled to 0 ° C., a cloudy precipitate was formed.
  • the mixture was centrifuged (9500 rpm, 1 min), the supernatant was decanted, washed with diethyl ether cooled to 0 ° C., and the obtained solid was used for the next cyclization reaction.
  • peptide cyclization reaction dissolve in DMSO (5% water content) so that the final concentration of the peptide is 5 mM based on the number of moles of the solid phase resin, add 7 equal amounts of triethylamine, and shake at room temperature for about 3 hours. bottom.
  • DMSO 5% water content
  • the purity of the target product was 95.4% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the peptide of interest was synthesized by starting with the removal of Fmoc using Siever amide resin (Watanabe Kagaku, 0.65 mmol / g, 0.54 g). At that time, CEM's Liberty Blue HT was used as an automatic synthesizer, and synthesis was performed according to the manufacturer's manual.
  • the basic condition for de-Fmocization was to react with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the Fmoc groups at the 2nd and 13th residues were removed by reacting twice at 25 ° C. for 5 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 5 equal amounts of ClAcOH in.
  • the solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate. Addition of this filtrate to excess diisopropyl ether / hexane (1/1) cooled to 0 ° C. resulted in a cloudy precipitate. The mixture was centrifuged (10000 rpm, 1 min) and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0 ° C., and the obtained solid was used for the next cyclization reaction.
  • the peptide cyclization reaction was dissolved in 5% hydrous DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin, 10 equal amounts of triethylamine was added, and the mixture was shaken at room temperature for about 16 hours.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • a DMSO solution 22 mM, 160 ⁇ L
  • DIEA DIEA
  • the purity of the target product was 98.7% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the 15th residue was reacted twice at 25 ° C. for 15 minutes.
  • the 16th residue was reacted at 25 ° C. for 60 minutes.
  • De-Fmocification was carried out by reacting with a 20% piperidine DMF solution at 25 ° C. for 5 minutes and then reacting for 15 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then 5 equal amounts of chloroacetate.
  • the peptide cyclization reaction is carried out by dissolving the peptide in 9% hydrous DMSO so that the final concentration of the peptide is 5 mM based on the number of moles of the solid-phase resin, adding 10 equal amounts of triethylamine, and shaking at room temperature for about 1.5 hours. bottom.
  • the obtained reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.
  • the purity of the target product was 82.7% calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis condition B.
  • the second and fourth residues were reacted twice at 75 ° C. for 30 minutes.
  • the 11th and 12th residues were repeatedly reacted twice at 25 degrees for 20 minutes.
  • the 13th residue was reacted twice at 75 ° C. for 10 minutes.
  • the 15th and 22nd residues were reacted once at 25 ° C. for 30 minutes.
  • the basic condition for removing Fmoc was to react it with a 20% piperidine DMF solution at 75 ° C. for 3 minutes. However, the Fmoc removal at the 2nd residue, the 4th residue and the 13th residue was carried out at 25 ° C. for 5 minutes and then at room temperature for 10 minutes.
  • the introduction of the chloroacetyl group is carried out by removing the Fmoc group of the ⁇ -amino group from the solid phase resin in which the Fmoc-protected peptide obtained in the previous step is retained by the method described above, and then using 5 eq of chloroacetic acid and 5 eq of the solid phase resin.
  • DIPCI and 10 eq of HOSu were stirred in DCM and the same amount of DMF as that of DCM was added to prepare a DCM / DMF solution (0.2 M) of ClAcOSu, which was added to a solid phase resin and shaken at room temperature for 30 minutes.
  • the resin obtained after the chloroacetyl group introduction step was first washed with DMF 5 times and with methylene chloride 3 times, and then dried under reduced pressure. Subsequently, the reactant cocktail-A (a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5) was added to the reaction vessel containing the solid phase resin. , Shake for 20 minutes at room temperature. The reaction solution was collected by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cocktail for cutting, the solution component was recovered from the frit, and mixed with the above-mentioned filtrate.
  • the reactant cocktail-A a mixture of TFA / H 2 O / TIS / DODT by volume 92.5: 2.5: 2.5: 2.5
  • the notation material is 3 instead of 4-((((9H-fluorene-9-yl) methoxy) carbonyl) amino) -5- (terrary butoxy) -5-oxopentanoic acid according to Synthesis Example [1-x]. -((((9H-Fluorene-9-yl) methoxy) carbonyl) amino) -4- (territorial butoxy) -4-oxobutanoic acid was used in the same manner.
  • ESI-MS (+) Observed value m / z 493.30 (M + H) +
  • This invention can be used in the pharmaceutical industry.

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WO2023027125A1 (ja) 2021-08-24 2023-03-02 ペプチドリーム株式会社 ヒトトランスフェリンレセプター結合抗体－ペプチドコンジュゲート
WO2023026994A1 (ja) * 2021-08-21 2023-03-02 武田薬品工業株式会社 ヒトトランスフェリンレセプター結合ペプチド－薬物コンジュゲート
WO2024043341A1 (ja) 2022-08-26 2024-02-29 ペプチドリーム株式会社 シクロアルキン誘導体
WO2024172166A1 (ja) * 2023-02-17 2024-08-22 Ｊｃｒファーマ株式会社 ヒトトランスフェリンレセプター結合ペプチド
WO2024195710A1 (ja) * 2023-03-17 2024-09-26 富士フイルム株式会社 環状ペプチド又はその塩及びそれらの利用
US20240415756A1 (en) * 2022-02-16 2024-12-19 Lipotrue, S.L. Peptides and compositions for use in cosmetics

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EP4626906A1 (en) * 2022-11-29 2025-10-08 Vacino Biotech Co., Ltd. Transporter peptides and application thereof

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WO2023022234A1 (ja) * 2021-08-19 2023-02-23 ペプチドリーム株式会社 ヒトトランスフェリンレセプター結合ペプチド
WO2023026994A1 (ja) * 2021-08-21 2023-03-02 武田薬品工業株式会社 ヒトトランスフェリンレセプター結合ペプチド－薬物コンジュゲート
WO2023027125A1 (ja) 2021-08-24 2023-03-02 ペプチドリーム株式会社 ヒトトランスフェリンレセプター結合抗体－ペプチドコンジュゲート
US20240415756A1 (en) * 2022-02-16 2024-12-19 Lipotrue, S.L. Peptides and compositions for use in cosmetics
WO2024043341A1 (ja) 2022-08-26 2024-02-29 ペプチドリーム株式会社 シクロアルキン誘導体
WO2024172166A1 (ja) * 2023-02-17 2024-08-22 Ｊｃｒファーマ株式会社 ヒトトランスフェリンレセプター結合ペプチド
WO2024195710A1 (ja) * 2023-03-17 2024-09-26 富士フイルム株式会社 環状ペプチド又はその塩及びそれらの利用

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