WO2023179723A1 - 亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用 - Google Patents

亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用 Download PDF

Info

Publication number
WO2023179723A1
WO2023179723A1 PCT/CN2023/083443 CN2023083443W WO2023179723A1 WO 2023179723 A1 WO2023179723 A1 WO 2023179723A1 CN 2023083443 W CN2023083443 W CN 2023083443W WO 2023179723 A1 WO2023179723 A1 WO 2023179723A1
Authority
WO
WIPO (PCT)
Prior art keywords
adc
group
compound
circle
residue
Prior art date
Application number
PCT/CN2023/083443
Other languages
English (en)
French (fr)
Inventor
黄蔚
唐峰
曾悦
Original Assignee
中国科学院上海药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海药物研究所 filed Critical 中国科学院上海药物研究所
Publication of WO2023179723A1 publication Critical patent/WO2023179723A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a class of affinity fragment-guided cleavable fragments, their design, synthesis and application in the preparation of site-directed drug conjugates.
  • Antibody-drug conjugates are a type of targeted drugs that connect biologically active cytotoxic drug molecules to antibodies through chemical links, and use the targeting effect of antibodies to transport small cytotoxic molecules to target cells to exert their effects. Since random coupling of small molecule drugs is not conducive to the study of pharmacokinetics and pharmacodynamics, and it is difficult to obtain uniform data for clinical evaluation, and the quality of different batches during the production process is also difficult to be consistent, many coupling methods for the quantitative introduction of small molecule drugs at fixed points have been developed. The connection strategy shows good application prospects.
  • the antibodies in antibody-drug conjugates are mainly IgG antibodies with variable Fab regions and constant crystallizable Fc domains. Directly connecting small molecule drugs to the Fc domain does not affect antibody-antigen recognition, and at the same time shows better efficacy and stability. Therefore, site-specific coupling of small molecule drugs in the Fc domain is an important research direction.
  • the purpose of the present invention is to provide an affinity fragment-guided cleavable fragment, its design, synthesis and application in the preparation of site-directed drug conjugates.
  • the present invention provides a type of thioester structure. By utilizing the thioester structure, a thioester-based acyl transfer reagent is prepared. The reagent can achieve fixed-point and quantitative modification of an antibody under the guidance of an affinity fragment.
  • the present invention relates to The design, synthesis and use of the fragments.
  • a conjugate with a ligand affinity directing group is provided, characterized in that the conjugate is as shown in formula I: AT-CL-R(I)
  • AT is the affinity part for the target protein (TP);
  • CL is a cleavable fragment; and there are bivalent fragments shown below in the CL
  • a 1 is each independently an optionally substituted C 1-10 alkylene group (preferably, a C 1-3 alkylene group), an optionally substituted C 6-10 aryl group, an optionally substituted 5 to 10-membered heteroaryl:
  • R is a group that needs to be modified to the target protein (the group can be any desired group or a group containing functional substances.
  • the R group can include a single desired group, or it can include 2 or more identical or different required groups or functional substances);
  • substitution means that one or more H in the group is substituted with a substituent selected from the following group: halogen (such as F, Cl, Br, I), C 1-6 alkyl (preferably , C 1-4 alkyl, such as methyl, ethyl), C 1-6 haloalkyl (preferably, C 1-4 haloalkyl).
  • halogen such as F, Cl, Br, I
  • C 1-6 alkyl preferably , C 1-4 alkyl, such as methyl, ethyl
  • C 1-6 haloalkyl preferably, C 1-4 haloalkyl
  • the cleavable fragment has self-cleavage reactivity (for example, it can be cleaved under specific reaction conditions/when in contact with specific reactants or reactive groups).
  • the affinity moiety is derived from a small molecule compound that can form a reversible covalent bond with the target protein, or is derived from a protein or polypeptide structure that has affinity for the target protein.
  • A1 is the end close to AT, and CO is the end close to R.
  • the conjugate is an acyl transfer reagent for site-directed modification of target proteins (such as antibodies or Fc fusion proteins).
  • the affinity portion for the protein refers to a portion that has affinity for the target protein or is capable of reversible binding (capable of reversible covalent binding).
  • the target protein is an antibody or fusion protein.
  • the antibody is an Fc domain-containing antibody or fusion protein.
  • the target protein is an antibody
  • the antibody is an antibody containing an Fc domain
  • the corresponding AT is a small molecule compound that can reversibly covalently bind to the antibody, or a protein with affinity or Polypeptide structure.
  • the antibody includes: a monoclonal antibody, a bifunctional antibody, a monoclonal antibody, a Nanobody containing an Fc fragment, an Fc fusion protein, or a combination thereof.
  • the selected antibodies include: trastuzumab, pertuzumab, rituximab, cetuximab, morolumab, gemtuzumab, abciximab anti, daclizumab, adalimumab, palivizumab, basiliximab, bevacizumab, panitumumab, nimotuzumab, denitizumab, dixituximab monoclonal antibody, rammonitumab, nexituzumab, ipilimumab, daratumumab, brentuximab, alemtuzumab, elotuzumab, blinatumomab Antibodies, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, toripalimab, catumaxomab, belintuzumab,
  • the antibodies include: Trastuzumab, Rituximab, Pertuzumab, Bevacizumab, Terrepro Toripalimab, Nivolumab (IgG4), Panituzumab, IgG2) or combinations thereof.
  • AT is a part derived from a small molecule compound that can reversibly bind to the target protein (preferably, a small molecule compound that forms a reversible covalent bond), or derived from a protein with affinity for the target protein or part of the polypeptide.
  • AT is a moiety derived from a small molecule compound that can reversibly bind to an antibody (preferably, a small molecule compound that forms a reversible covalent bond), or derived from a protein or polypeptide that has affinity for the antibody (preferably, a small molecule compound that forms a reversible covalent bond).
  • it is derived from an Fc-binding peptide; more preferably, it is derived from a portion of a polypeptide fragment derived from ProteinA or ProteinG and having binding ability to Fc (Fc segment).
  • AT is derived from a sequence in Protein A (ProteinA with affinity to Fc).
  • AT is derived from a binding peptide with affinity to the antibody Fab.
  • polypeptide is a cyclic peptide.
  • AT is derived from a peptide selected from the following group: Fc-III peptide (such as DCAWHLGELVWCT (SEQ ID No. 2)), Fc-binding peptide (such as GPDCAYHRGELVWCTFH (SEQ ID No. 3), RGNCAYHRGQLVWCTYH (SEQ ID No. 3), No.4), CDCAWHLGELVWCTC (SEQ ID No.5), etc.), or combinations thereof.
  • Fc-III peptide such as DCAWHLGELVWCT (SEQ ID No. 2)
  • Fc-binding peptide such as GPDCAYHRGELVWCTFH (SEQ ID No. 3), RGNCAYHRGQLVWCTYH (SEQ ID No. 3), No.4
  • CDCAWHLGELVWCTC SEQ ID No.5
  • AT is a core sequence derived from an Fc-binding polypeptide comprising the sequence shown in Formula II: (X s1 ) t1 -Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Y s2 ) t2 (II)
  • the position of X is the N-terminus of the polypeptide, and the position of Y is the C-terminus of the polypeptide;
  • s1 0, 1, 2 or 3;
  • s2 0, 1, 2 or 3;
  • X and Y are each independently an amino acid residue
  • t1 and t2 are each independently an integer from 0 to 10 (preferably, t1 and t2 are each independently 0, 1, 2 or 3, more preferably, 1 and t2 are each independently 0 or 1);
  • Cys is a cysteine residue
  • Aa1, Aa2, Aa3, Aa5, Aa7, Aa8 and Aa9 are each independently an amino acid residue;
  • Aa4 and Aa6 are each independently an amino acid residue, and at least one of Aa4 and Aa6 is an amino acid residue containing an NH group in the side chain (such as a lysine residue (K), etc.) or a -COOH group in the side chain group of amino acid residues (such as aspartic acid residue (D), glutamic acid residue (E), etc.).
  • an NH group in the side chain such as a lysine residue (K), etc.
  • a -COOH group in the side chain group of amino acid residues such as aspartic acid residue (D), glutamic acid residue (E), etc.
  • Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, Aa8 and Aa9 are not cysteine residues.
  • the derivatization means that the side chain -NH 2 in Aa4 or Aa6 removes an H to form -NH-, or the side chain -COOH and the amino group form -CONH- (that is, AT passes through Aa4 or Aa6 The side chain is connected to CL).
  • Aa6 is an amino acid residue containing an NH 2 group in the side chain or an amino acid residue containing a -COOH group in the side chain.
  • the derivatization means that the side chain -NH2 in Aa6 removes an H to form -NH-, or the side chain -COOH and the amino group form -CONH- (that is, AT passes through the side chain of Aa6 and CL connection).
  • the amino acid residue is an amino acid residue derived from natural or unnatural amino acids.
  • AT is derived from cyclic peptide.
  • AT is derived from a cyclic peptide formed by forming an -S-S- bond between two Cys in the sequence shown in formula II.
  • Aa1 is an alanine residue (A), a serine residue (S), or a threonine residue (T); and/or
  • Aa2 is a tyrosine residue (Y), a histidine residue (H), or a tryptophan residue (W); and/or
  • Aa3 is a histidine residue (H), phenylalanine residue (F), tyrosine residue (Y), tryptophan residue (W), arginine residue (R) or glycine residue Base (G); and/or
  • Aa5 is glycine residue (G), serine residue (S), asparagine residue (N), glutamine residue (Q), aspartic acid residue (D), glutamic acid residue ( E), phenylalanine residue (F), tyrosine residue (Y), tryptophan residue (W), histidine residue (H), threonine residue (T), Leu An amino acid residue (L), an alanine residue (A), a valine residue (V), an isoleucine residue (I), or an arginine residue (R); and/or
  • Aa7 is a leucine residue (L), an isoleucine residue (I), a valine residue (V), an alanine residue (A), a glutamine residue (Q), or a glutamine residue (Q). amino acid residue (E); and/or
  • Aa8 is valine (V), isoleucine residue (I), or leucine residue (L); and/or
  • Aa9 is a tryptophan residue (W) or a phenylalanine residue (F).
  • amino acid residue containing -NH 2 group in the side chain is as follows
  • amino acid residue containing a -COOH group in the side chain is as follows:
  • the amino acid residue containing a -NH 2 group in the side chain is a lysine residue (K).
  • the amino acid residue containing a -COOH group in the side chain is an aspartic acid residue (D) or a glutamic acid residue (E).
  • Aa4 is an amino acid residue containing a -NH 2 group in the side chain
  • Aa6 is a glutamine residue (Q), a glutamic acid residue (E), or a histidine residue (H ), asparagine residue (N), proline residue (P), aspartic acid residue (D), lysine residue (K), or glycine residue (G); or,
  • Aa6 is an amino acid residue containing -NH 2 group in the side chain
  • Aa4 is an arginine residue (R), a leucine residue (L), a lysine residue (K), an aspartic acid residue (D), glutamic acid residue (E), 2-aminosuberic acid or diaminopropionic acid.
  • Aa4 is an amino acid residue containing a -COOH group in the side chain, 2-aminosuberic acid or diaminopropionic acid
  • Aa6 is a glutamine residue (Q), a glutamic acid residue (E), histidine residue (H), asparagine residue (N), proline residue (P), aspartic acid residue (D), lysine residue (K), Or a glycine residue (G);
  • Aa6 is an amino acid residue containing a -COOH group in the side chain, 2-aminosuberic acid or diaminopropionic acid
  • Aa4 is an arginine residue (R), leucine Acid residue (L), lysine residue (K), aspartic acid residue (D), glutamic acid residue (E), 2-aminosuberic acid or diaminopropionic acid.
  • sequence shown in Formula II is HYTCWVLKGRHYACNGR (SEQ ID No. 6), HYTCWVLDGRHYACNGR (SEQ ID NO. 7), or HYTCWVLEGRHYACNGR (SEQ ID No. 8).
  • AT is a small molecule fragment that has reversible covalent interaction with the protein.
  • AT is selected from the following group:
  • CL is as follows
  • W 1 is selected from the group consisting of: none (single bond), -NH-, -C(O)-, -C(O)-NH- and -NH-C(O)-;
  • W 2 is none (single bond), -NH-, or -C(O)-;
  • L 1 is no (single bond) or divalent connecting group
  • a 1 is as defined before.
  • L 1 is none (single bond) or consists of one or more selected from the following group (preferably, 1-10; more preferably, 1, 2, 3, 4, or 5 (preferably, 1, 2 or 3) divalent linking groups composed of units: amino acid residues, optionally substituted C 1-4 alkylene, -(CH 2 -NH-CO)-, - (CH 2 -CH 2 -CO)-, -(CH 2 -CH 2 -O)-, -(CH 2 -O-CH 2 )-.
  • W 2 is -NH- or -C(O)-.
  • W 1 and L 1 are none.
  • W 1 is -C(O)- and L 1 is C 1-10 alkylene (preferably, C 1-3 alkylene).
  • CL is selected from Group 1, and Group 1 includes the following groups:
  • the conjugate is represented by formula III,
  • R is a moiety containing one or more reactive groups selected from the group consisting of reactive groups that can be further modified, reactive groups that are biologically active and/or detectable.
  • the reactive group that can be further modified refers to an reactive group that can perform bioorthogonal reactions (also known as a bioorthogonal group); preferably, the reactive group that can perform bioorthogonal reactions
  • Reactive active groups include: azide group (-N 3 ), aldehyde group (-CHO), thiol group (-SH), alkynyl group (such as C2-C6 alkynyl group, ring tension alkynyl group BCN, DBCO, etc.) , Alkenyl (such as C2-C6 alkenyl), halogen (such as F, Cl, Br and I), tetrazinyl, nitronyl, hydroxylamine, nitrile, hydrazine, ketone, boronic acid residue, cyano group Benzothiazolyl, allyl, phosphine, maleimide, disulfide, thioester, ⁇ -halocarbonyl, isonitrile, stedon
  • the biologically active and/or detectable active group is any one or more of small molecule drugs, cytotoxics, or other functional molecules.
  • the small molecule drug is a radioactive therapeutic agent or a molecular imaging agent.
  • the biologically active and/or detectable active group is derived from a compound selected from the following group: maytansine, DM-1, DM-4, MMAE (Monomethyl auristatin E), MMAF, SN-38, Dxd, PBD and its analogs, amanitanic acid, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, Ebo Mycin B, nocodazole, colchicine, estramustine, simadodin, agaricate, fluorescent reagents, monosaccharides, disaccharides, oligosaccharides, polyethylene glycol (PEG), Cytotoxic, immune agonists, or radioactive therapeutics, molecular imaging reagents.
  • R is -L 2 -(L 3 -B) o , where the subscript o is an integer from 1 to 10, and L 2 and L 3 are each are independently none or connected fragments, and B are each independently the same or different reactive groups that can be further modified (such as bioorthogonal groups).
  • R is -L 2 -(L 3 -D) o , where the subscript o is an integer from 1 to 10, L 2 and L 3 are each independently none or a connected fragment, and D is each independently Ground is the same or different biologically active and/or detectable active groups (such as small molecule compounds).
  • o is 1.
  • o is 2, 3, 4, or 5.
  • L2 and L3 are each independently none or one or more selected from the following group (such as 1-10, preferably 1, 2, 3, 4, 5 or 6)
  • the unit structure consists of connecting fragments: CH 2 OCH 2 , C1-C4 alkylene, CO, NH, and amino acid residues.
  • L2 and L3 do not contain a connecting group formed by an orthogonal reaction (for example, a connecting group formed by an orthogonal reaction of B as defined in the formula).
  • the conjugate is represented by formula III-A1, III-A2 or III-A3,
  • the circles marked 1, 2, 3, 4, 5, 7, 8 and 9 are as defined in Formula II respectively for Aa1, Aa2, Aa3, Aa4, Aa5, Aa7, Aa8 and Aa9;
  • the end close to the circle marked 1 is the N-terminus of the polypeptide, and the end close to the circle marked 9 is the C-terminus of the polypeptide;
  • Each blank circle is defined as X and Y in Formula II;
  • Circle C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • Circle K is the main chain part of the lysine residue (the amino acid side chain is shown);
  • circle D is the main chain part of the aspartic acid residue (D) (the amino acid side chain is shown),
  • circle E is the main chain portion of the glutamic acid residue (E) (the amino acid side chain is shown);
  • R is as defined before.
  • Blank circles (such as any one of the circles) is independently none (absent) or amino Acid residue (preferably, the amino acid residue is not a lysine residue and a cysteine residue);
  • the numbered circles are each independently an amino acid residue (preferably, the amino acid residue is not are lysine residues and cysteine residues);
  • the circle marked with C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • the circle marked with K is the main chain part of the lysine residue
  • the circle marked with D is the main chain part of the aspartic acid residue (D)
  • the circle marked with E is the main chain part of glutamic acid residue (E);
  • Ac is an N-terminal protecting group or is absent (preferably, it is an acetyl group), connected to the N-terminus of the polypeptide;
  • -CONH 2 is an amide group, which is the C-terminus of the polypeptide
  • any R group or substance can be modified onto the target protein (preferably, the target protein is an antibody).
  • the conjugate is represented by formula III-A1-1, III-A2-1 or III-A3-1,
  • the circle R is the N-terminus of the polypeptide, and the circle H is the C-terminus of the polypeptide;
  • the circle R is an arginine residue
  • G is a glycine residue
  • N is an asparagine residue
  • A is an alanine residue
  • Y is a tyrosine residue
  • H is a histidine residue
  • L is a leucine residue.
  • Amino acid residue, V is valine residue
  • W is tryptophan residue
  • T is threonine residue;
  • Circle C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • Circle K is the main chain part of lysine residue
  • circle D is the main chain part of aspartic acid residue (D)
  • Circle E is the main chain part of glutamic acid residue (E);
  • R is as defined before.
  • formula III-A1 is
  • the circles marked with letters respectively represent the amino acid residues represented by the letters.
  • formula III-A2 is
  • the circles marked with letters respectively represent the amino acid residues represented by the letters.
  • formula III-A3 is
  • the circles marked with letters respectively represent the amino acid residues represented by the letters.
  • the conjugate is represented by formula III-B1, III-B2 or III-B3,
  • the circles marked 1, 2, 3, 5, 6, 7, 8 and 9 are as defined in Formula II respectively for Aa1, Aa2, Aa3, Aa5, Aa6, Aa7, Aa8 and Aa9;
  • the end close to the circle marked 1 is the N-terminus of the polypeptide, and the end close to the circle marked 9 is the C-terminus of the polypeptide;
  • Each blank circle is defined as X and Y in Formula II;
  • Circle C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • Circle K is the main chain part of the lysine residue (the amino acid side chain is shown);
  • circle D is the main chain part of the aspartic acid residue (D) (the amino acid side chain is shown),
  • circle E is the main chain portion of the glutamic acid residue (E) (the amino acid side chain is shown);
  • R is as defined before.
  • Blank circles each independently represent none (absence) or an amino acid residue (preferably, the amino acid residue is not a lysine residue and a cysteine residue);
  • the numbered circles are each independently an amino acid residue (preferably, the amino acid residue is not are lysine residues and cysteine residues);
  • the circle marked with C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • the circle marked with K is the main chain part of the lysine residue
  • the circle marked with D is the main chain part of the aspartic acid residue (D)
  • the circle marked with E is the main chain part of glutamic acid residue (E);
  • Ac is an N-terminal protecting group or is absent (preferably an acetyl group), attached to the N-terminus of the polypeptide;
  • -CONH 2 is an amide group, which is the C-terminus of the polypeptide.
  • the conjugate is represented by formula III-C1, III-C2 or III-C3,
  • the circles marked 1, 2, 4, 3, 5, 7, 8 and 9 are as defined in Formula II respectively for Aa1, Aa2, Aa3, Aa4, Aa5, Aa7, Aa8 and Aa9;
  • the end close to the circle marked 1 is the N-terminus of the polypeptide, and the end close to the circle marked 9 is the C-terminus of the polypeptide;
  • Circle C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • Circle K is the main chain part of the lysine residue
  • circle D is the main chain part of the aspartic acid residue (D)
  • circle E is the main chain part of the glutamic acid residue (E).
  • Blank circles each independently represent none (absence) or an amino acid residue (preferably, the amino acid residue is not a lysine residue and a cysteine residue);
  • the numbered circles are each independently an amino acid residue (preferably, the amino acid residue is not are lysine residues and cysteine residues);
  • the circle marked with C is a cysteine residue, and -S-S- represents the disulfide bond formed by the side chain -SH of the cysteine residue;
  • the circle marked with K is the main chain part of the lysine residue;
  • the circle marked with D is the main chain part of the aspartic acid residue (D);
  • the circle marked with E is the glutamic acid residue (E) The main chain part;
  • L2, L3, subscript o, and R are as defined before;
  • Ac is an N-terminal protecting group or is absent (preferably an acetyl group), attached to the N-terminus of the polypeptide;
  • -CONH 2 is an amide group, which is the C-terminus of the polypeptide
  • any L 2 -(L 3 -D) o group or substance can be modified onto the target protein (preferably, the target protein is an antibody).
  • formula III-C1 is
  • the circles marked with letters respectively represent the amino acid residues represented by the letters.
  • formula III-C2 is
  • the circles marked with letters respectively represent the amino acid residues represented by the letters.
  • formula III-C3 is
  • the circles marked with C and E respectively represent the amino acid residues represented by the letters.
  • the circles marked with 1, 2, 3, 4, 5, 6, 7, 8 and 9 are respectively as Aa1, Aa2, Aa3, Aa4, Aa5, Aa6,
  • each blank circle is as defined as X and Y in Formula II.
  • the conjugate is selected from Table A1 and Table A2.
  • an intermediate I is provided, and the intermediate is represented by formula IA
  • R a is H or a protecting group (such as trityl (Trt)); L 1 , W 1 and A 1 are as defined before.
  • an intermediate II is provided, and the intermediate is represented by formula IB
  • R a is H or a protecting group (such as trityl (Trt)) (preferably, R a is H); AT, L 1 , W 1 and A 1 are as defined before.
  • AT is a core sequence derived from an Fc-binding polypeptide comprising the sequence shown in II.
  • a fourth aspect of the present invention provides a method for preparing the conjugate as described in the first aspect, including the steps:
  • a method for site-selective modification of a protein comprising the steps:
  • the modified target protein is modified by one or more of the R groups.
  • the modification is site-selective modification.
  • the amino group includes the side chain amino group of lysine (K).
  • the modification can obtain the modified target protein through a one-step reaction.
  • the modified target protein is a protein modified with one or more active groups selected from the group consisting of: biologically active and/or detectable active groups that can be further modified. active group.
  • the modified target protein is represented by Formula IV TP-(NHCO-R) p (IV)
  • R is as defined before
  • TP is the target protein part
  • subscript p is 1-8.
  • TP is an amino acid residue containing an NH 2 group in the side chain of the target protein (preferably, a lysine residue; more preferably, a lysine residue located in the Fc region; optimally Specifically, the remaining part after NH 2 in the Fc region (lysine residues 246-248) is connected to -CO-R in the conjugate to form -NHCO-R.
  • the target protein is an antibody or Fc fusion protein.
  • the target protein is an antibody
  • the modified target protein is represented by Formula V Ab-(NHCO-R) p (V).
  • R and subscript p are as defined before, and Ab is an antibody.
  • Ab is an amino acid residue containing NH 2 groups in p side chains of the antibody (preferably, a lysine residue; more preferably, a lysine residue located in the Fc region; optimally Specifically, the remaining part after NH 2 in the Fc region (lysine residues 246-248) is connected to -CO-R in the conjugate to form -NHCO-R.
  • the target protein has fragments or sequences that are compatible with the complex shown in Formula I.
  • the antibody or Fc structure is derived from human IgG.
  • the modified position of the target protein is the Fc region.
  • the modified position of the target protein is a region containing 80% homologous sequence to the Fc of human IgG.
  • the original Fc sequence of human IgG is shown in SEQ ID No. 1:
  • the modified position of the target protein is position 246 - in the Fc region of human IgG.
  • the covalently modified region that interacts with the conjugate of Formula I is a region consisting of consecutive 1-10 amino acid residues, and this region includes positions 246-248 in the Fc region of human IgG The target region formed by the amino acid residues.
  • the target region is a lysine residue at position 246 or 248 in the Fc region of human IgG.
  • the selectivity of the region of the method is ⁇ 90%.
  • the modification site is the 248th lysine residue in the Fc region of human IgG.
  • a site-selectively modified protein as shown in formula IV is provided, TP-(NHCO-R) p (IV)
  • TP, R and subscript p are as defined before.
  • R is -L 2 -(L 3 -D) o ; wherein, L 2 , L 3 , D, and subscript o are as defined before.
  • R is -L 2 -(L 3 -B) o ; wherein, L 2 , L 3 , B, and subscript o are as defined before.
  • R is -L 2 -(L 3 -B'-L 4 -D) o ; wherein, L 2 , L 3 , B, and subscript o are as defined before; B' is B and L4 '-D is a connecting group formed by orthogonal reaction, L4 is a connecting fragment, L4' is a group with an active group that can perform orthogonal reaction with B and can form L4 after the reaction.
  • the site-selectively modified protein is an antibody drug conjugate, that is, TP is an antibody and R is -L 2 -(L 3 -D) o or R is -L 2 - (L 3 -B'-L 4 -D) o .
  • TP is an antibody
  • site-selectively modified protein is represented by Formula V; Ab-(NHCO-R) p (V)
  • Ab is an antibody
  • R and subscript p are as defined before.
  • the site-selectively modified protein is represented by formula IV-A; TP-(NHCO-L 2 -(L 3 -D) o ) p (IV-A)
  • TP, L 2 , L 3 , D, subscript o and subscript p are as defined before.
  • the site-selectively modified protein is represented by formula VA; Ab-(NHCO-L 2 -(L 3 -D) o ) p (VA)
  • the antibody drug conjugate is selected from: ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC -9, ADC-10, ADC-11, ADC-12, ADC13, ADC-14, ADC-15, ADC-16, ADC-17, ADC-18, ADC-19, ADC-20 and ADC-21.
  • the seventh aspect of the present invention there is provided a use of the conjugate described in the first aspect of the present invention for site-selective modification of proteins.
  • an eighth aspect of the present invention there is provided a use of the conjugate described in the first aspect of the present invention for preparing an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the use does not rely on bioorthogonal reactions.
  • the conjugate can directly modify the drug molecules that need to be modified to the target protein to the antibody in a site-selective manner through a one-step reaction.
  • a site-selectively modified antibody as shown in formula V is provided, Ab-(NHCO-R) p (V)
  • Ab is an antibody
  • R and subscript p are as defined before.
  • an antibody drug conjugate (ADC) is provided, and the antibody drug conjugate is represented by formula VA: Ab-(NHCO-L 2 -(L 3 -D) o ) p (VA)
  • the antibody-drug conjugate is a site-specific antibody-drug conjugate prepared in one step without relying on bioorthogonal reactions, and the antibody is site-specifically modified.
  • Ab is an antibody
  • o is an integer from 1 to 10
  • L 2 and L 3 are connecting fragments
  • D is small Molecular drugs; when o is 2-10, D can be the same small molecule drug, or it can be independently different small molecule drugs.
  • the site-directed antibody-drug conjugate, D (such as a small molecule drug), is site-specifically coupled to the antibody constant region (Fc region).
  • the site-directed antibody drug conjugate, D (such as a small molecule drug), is site-specifically coupled to the region consisting of amino acid residues 246-248 in the Fc region of human IgG.
  • the antibody drug conjugate is selected from: ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC -9, ADC-10, ADC-11, ADC-12, ADC13, ADC-14, ADC-15, ADC-16, ADC-17, ADC-18, ADC-19, ADC-20 and ADC-21.
  • the antibody drug conjugate is selected from: ADC-5, ADC-6, ADC-7, ADC-8, ADC-9, ADC-10, ADC-11, ADC-12, ADC13 , ADC-14, ADC-15, ADC-16, ADC-17, ADC-18, ADC-19, ADC-20 and ADC-21.
  • the method for preparing the above-mentioned site-directed ADC is: combining natural antibodies with affinity fragments shown in formula (III, III-A1-3, III-B1-3, III-C1-3) - cleavable After the linker fragment-functional fragment complex is incubated, the carbonyl group contained on the functional fragment directly covalently forms an amide bond with the lysine residue in the Fc region of the antibody, and the site-specific ADC compound is prepared in a one-step reaction without relying on bioorthogonal reactions.
  • a site-specific, bivalent or multivalent bioorthogonal group-modified antibody as shown in formula VB is provided, which is characterized in that, Ab-(NHCO-L 2 -(L 3 -B) o ) p (VB)
  • Ab is an antibody
  • B is a bioorthogonal group. Multiple Bs can be the same or independently different, preferably from: azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen residues, tetrazine residues residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide Amine residues, disulfide residues, thioester residues, ⁇ -halocarbonyl residues, isonitrile residues, stedone residues, selenium residues, conjugated diene residues, phosphate residues, cycloalkynes residues and cycloalkene residues.
  • bioorthogonal groups are site-specifically coupled to the antibody constant region (Fc region);
  • bioorthogonal groups are site-specifically coupled to the region consisting of amino acid residues 246-248 in the human IgG Fc region;
  • a site-specific, bivalent or multivalent drug-modified antibody represented by formula VC is provided, which is characterized in that, Ab-(NHCO-L 2 -(L 3 -B'-L 4 -D) o ) p (VC)
  • L 2 , L 3 , B, and the subscript o are as defined before; and B' is the connecting group formed by the orthogonal reaction between B and L4'-D, L4 is the connecting fragment, and L4' is the linking group that can be combined with B Reactive groups that undergo orthogonal reactions and can form L4 groups after the reaction.
  • Ab is an antibody
  • o is an integer from 2 to 10
  • L 2 and L 3 is the connecting fragment
  • B' is the connecting fragment generated by the reaction between the drug fragment and the above-mentioned bioorthogonal group B
  • B' is the same or different
  • L 4 is the linker connecting B' and drug D
  • D is as described above
  • the structure of D here is the same or different.
  • bioorthogonal groups are site-specifically coupled to the antibody constant region (Fc region);
  • bioorthogonal groups are site-specifically coupled to the region consisting of amino acid residues 246-248 in the Fc region of human IgG.
  • Figures 1a-e show the characterization results of biotinylated trastuzumab Ab-1 prepared in Example 47. Among them, Figures 1a, 1b and 1c show the ESI-TOFMS measurement results, and Figure 1d shows the trypsin-digested trastuzumab peptide consisting of 33 amino acid residues containing a modification site to lysine. Figure 1e shows the MS spectrum of the peptide fragment (THTCPPCPAPELLGGPSVFLFPPKPK(C 10 H 14 N 2 O 2 S)DTLMISR). After analysis by Mascot 2.3 software, it was determined that the modification occurred at lysine residue 251 with high selectivity.
  • Figures 2a-c show the characterization results of the azide group-coupled trastuzumab Ab-2 prepared in Example 48. Among them, Figures 2a, 2b and 2c show the ESI-TOFMS measurement results.
  • Figure 3 shows the ESI-TOFMS measurement results of trastuzumab Ab-3 before and after conjugation with biofluorescein FITC in Example 49.
  • Figure 4 shows the ESI-TOFMS measurement results of trastuzumab Ab-4 before and after coupling the orthogonal group cyclopropene in Example 50.
  • Figure 5 shows the ESI-TOFMS measurement results of rituximab Ab-5 before and after coupling with the orthogonal group DBCO in Example 51.
  • Figure 6 shows the ESI-TOFMS measurement results of rituximab Ab-6 before and after coupling with the orthogonal group DBCO in Example 52.
  • Figures 7a-e show the characterization results of ADC-1 prepared in Example 53. Among them, Figures 7a, 7b and 7c show the ESI-TOFMS measurement results, Figure 7d shows the hydrophobic chromatography analysis results, and Figure 7e shows the size exclusion chromatography analysis results.
  • Figure 8a-e shows the characterization results of ADC-2 prepared in Example 54.
  • Figures 8a, 8b and 8c show the ESI-TOFMS measurement results
  • Figure 8d shows the hydrophobic chromatography analysis results
  • Figure 8e shows the size exclusion chromatography analysis results.
  • Figures 9a-e show the characterization results of ADC-3 prepared in Example 55. Among them, Figures 9a, 9b and 9c show the ESI-TOFMS measurement results, Figure 9d shows the hydrophobic chromatography analysis results, and Figure 9e shows the size exclusion chromatography analysis results.
  • Figure 10 shows the ESI-TOFMS measurement results of ADC-4 prepared in Example 56.
  • Figures 11a-e show the characterization results of ADC-5 prepared in Example 57. Among them, Figures 11a, 11b and 11c show the ESI-TOFMS measurement results, Figure 11d shows the hydrophobic chromatography analysis results, and Figure 11e shows the size exclusion chromatography analysis results.
  • Figures 12a-e show the characterization results of ADC-12 prepared in Example 58. Among them, Figures 12a, 12b and 12c show the ESI-TOFMS measurement results, Figure 12d shows the hydrophobic chromatography analysis results, and Figure 12e shows the size exclusion chromatography analysis results.
  • Figures 13a-e show the characterization results of ADC-7 prepared in Example 59. Among them, Figures 13a, 13b and 13c show the ESI-TOFMS measurement results, Figure 13d shows the hydrophobic chromatography analysis results, and Figure 13e shows the size exclusion chromatography analysis results.
  • Figures 14a-e show the characterization results of ADC-8 prepared in Example 60. Among them, Figures 14a, 14b and 14c show the ESI-TOFMS measurement results, Figure 14d shows the hydrophobic chromatography analysis results, and Figure 14e shows the size exclusion chromatography analysis results.
  • Figure 15 shows the ESI-TOFMS measurement results of rituximab before and after coupling to drug linker D6 in Example 61.
  • Figure 16 shows the ESI-TOFMS measurement results of Pertuzumab before and after coupling with drug linker D6 in Example 62.
  • Figure 17 shows the ESI-TOFMS measurement results of bevacizumab before and after coupling to drug linker D6 in Example 63.
  • Figure 18 shows the ESI-TOFMS measurement results of ADC-12 of toripalimab before and after coupling to drug linker D6 in Example 64.
  • Figure 19 shows the ESI-TOFMS measurement results of panitumumab before and after coupling to drug linker D6 in Example 65.
  • Figure 20 shows the ESI-TOFMS measurement results of nivolumab before and after coupling to drug linker D6 in Example 66.
  • Figure 21a shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-15 in Example 67
  • Figure 21b shows the hydrophobic chromatography analysis results.
  • Figure 22a shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-16 in Example 68
  • Figure 22b shows the hydrophobic chromatography analysis results.
  • Figure 23a shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-17 in Example 69
  • Figure 23b shows the hydrophobic chromatography analysis results.
  • Figure 24a shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-18 in Example 70
  • Figure 24b shows the hydrophobic chromatography analysis results.
  • Figure 25a shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-19 in Example 71
  • Figure 25b shows the hydrophobic chromatography analysis results.
  • Figure 26 shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-20 in Example 72.
  • Figure 27 shows the ESI-TOFMS measurement results of the raw material trastuzumab and the prepared ADC-21 in Example 73.
  • Figure 28 shows the cell activity and cytotoxicity results of ADC prepared in the embodiment of the present invention.
  • the inventor unexpectedly discovered a conjugate with a unique structure, which has With the cleavable fragment and affinity part (AT) of the bivalent fragment shown, the conjugate of the present invention is very suitable for direct modification of proteins, especially the Fc segment of antibodies or Fc fusion proteins).
  • the conjugate of the present invention there is no need to pre-modify the active groups on the side chains of the protein before modification, and the desired product (such as ADC) modified by the R group can be obtained by direct reaction (such as only one-step direct reaction). wait).
  • the conjugate of the present invention can carry out site-selective modification with extremely high efficiency.
  • the inventor completed the present invention.
  • halogen refers to F, Cl, Br, and I. More preferably, the halogen atoms are selected from F, Cl and Br.
  • alkyl refers to a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (ie, C 1-6 means 1 to 6 carbons).
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. .
  • alkylene by itself or as part of another substituent, refers to a divalent group derived from an alkane , such as -CH2CH2CH2CH2- .
  • Alkyl (or alkylene) groups typically have 1-10 carbon atoms (ie, C 1-10 alkylene). Examples of alkylene include, but are not limited to, methylene and ethylene.
  • aryl means a polyunsaturated (usually aromatic) hydrocarbyl group, which may be a single ring or multiple rings (up to three rings) fused together or covalently linked. Generally, aryl groups have 6-10 ring atoms.
  • heteroaryl refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. .
  • the heteroaryl group has 5-10 ring atoms, that is, a 5-10 membered heteroaryl group, preferably, it has 5-6 ring atoms, that is, a 5-6 membered heteroaryl group, and contains 1, 2, 3 or 4 a heteroatom.
  • a heteroaryl group can be attached to the rest of the molecule through a heteroatom.
  • Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl groups, while non-limiting examples of heteroaryl groups include pyridyl and the like.
  • bonds represented by dotted lines Represents the site at which a group (or fragment or part) is attached to the remainder.
  • substitution means that one or more H in a group is substituted with a substituent selected from the group consisting of: halogen, C 1-6 alkyl, C 1-6 haloalkyl.
  • bioorthogonal group refers to a group containing functional groups useful in bioorthogonal reactions, such as azide-containing ( -N3 ), cyclooctyne, Aldehyde group, ketone carbonyl group, tetrazine, trans-cyclooctene, thiol, halogen, hydrazine, hydroxylamine and other groups.
  • amino acid residue refers to a group formed by removing an H from -NH2 at the N-terminus of an amino acid and removing -OH from -COOH at the C-terminus.
  • chain segment including the N-terminus and C segment of an amino acid (residue) is called the main chain, and the part that determines the specific type of amino acid is called the side chain.
  • amino acids as used herein include natural amino acids or unnatural amino acids, including D- and/or L-type amino acids.
  • amino acids include, but are not limited to, Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H) ), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp (W), Tyr(Y), Val(V).
  • the amino acid is an amino acid selected from the group consisting of L-glycine (L-Gly), L-alanine (L-Ala), ⁇ -alanine ( ⁇ -Ala), L-glutamine Acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L- Lys), L-valine (L-Val), L-serine (L-Ser), L-threonine (L-Thr); in addition, when the amino acid has 2 or more amino groups and/or 2 or above carboxyl group, the term also includes groups formed by -NH 2 not on the same carbon atom removing an H and -COOH removing -OH, such as -NH 2 and non- ⁇ position of glutamic acid The bivalent group -C(O)-(CH 2 ) 2 -C(COOH)-NH- formed after -COOH removes one H respectively
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, they can form a partially folded structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody. Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant region. There are 5 main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable regions which are separated into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • the term "Fc-binding polypeptide” refers to a peptide with the ability to reversibly bind to a target protein, especially the Fc region of an antibody; preferably, it has a core sequence as shown in Formula II (i.e. (X s1 ) t1 - Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Y s2 ) t2 ).
  • the term also includes variant forms of the Fc-binding polypeptide.
  • variant forms include (but are not limited to): deletion and insertion of 1-5 (usually 1-4, preferably 1-3, more preferably 1-2, optimally 1) amino acids and/or substitution, and addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the C-terminus and/or N-terminus.
  • substitutions with amino acids with similar or similar properties generally do not alter the function of the protein.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear as well as non-linear polypeptides (such as cyclic peptides).
  • a preferred class of active derivatives refers to those in which at most 5, preferably at most 3, more preferably at most 2 and most preferably 1 amino acid are replaced by amino acids with similar or similar properties compared to the amino acid sequence of formula I. Replacement to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the invention also provides analogs of the polypeptides of the invention.
  • the difference between these analogs and the natural polypeptide of the present invention may be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both.
  • Analogues also include analogs with residues that differ from natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified forms include chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of the polypeptide during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility properties.
  • glycosylation such as those resulting from glycosylation modifications of the polypeptide during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosy
  • an affinity fragment-directed cleavable fragment also known as the conjugate of the invention
  • a derivative thereof such as a salt thereof
  • this complex can directly couple the R group to the protein and form a new covalent bond with the amino acid at a specific site.
  • AT affinity tag
  • CL cleavable linker
  • R reagent
  • AT is an affinity fragment of a protein, which can be either a small molecule compound that forms a reversible covalent bond with the protein, or a protein or polypeptide structure with affinity.
  • AT can be either a small molecule compound that forms a reversible covalent bond with the antibody, or a protein or polypeptide structure that has affinity with the antibody, such as an Fc-binding peptide, which is derived from ProteinA or ProteinG and has Fc affinity. Polypeptide fragment structure with binding ability, etc.
  • AT can be, for example, an Fc-III peptide (DCAWHLGELVWCT (SEQ ID No. 2)), other Fc-binding peptides (GPDCAYHRGELVWCTFH (SEQ ID No. 3), RGNCAYHRGQLVWCTYH (SEQ No. 4), CDCAWHLGELVWCTC (SEQ ID No.5)), etc., its core structure sequence is as follows (II): (X 0-3 ) t1 -Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Y 0-3 ) t2 (II)
  • C is a cysteine residue
  • X 0-3 and Y 0-3 are any amino acids except lysine and cysteine, and can be 1-3 consecutive identical or different amino acid residues.
  • Base, m and n are independently numbers from 0 to 10.
  • Aa1-Aa3 and Aa5 and Aa7-Aa9 are any amino acids except cysteine and lysine
  • Aa4 or Aa6 are are independently lysine residues, that is, when Aa4 is lysine, Aa6 is any other amino acid except cysteine and lysine; when Aa6 is lysine, Aa4 is any amino acid except cysteine and lysine. Any other amino acid from lysine.
  • the affinity fragment (AT) is or is derived from a cyclic polypeptide fragment that can have affinity with antibody Fc.
  • the preferred structure is:
  • unlabeled or numbered circles represent any amino acid except cysteine and lysine.
  • the unlabeled circle may not exist.
  • C represents cysteine and -S-S- represents the two cysteine.
  • the -S-S- bond formed by the -SH group in the amino acid, and K represent the lysine backbone and depict its side chain structure.
  • a part of the affinity fragment (AT) and the cleavable fragment (CL) is derived from an Fc-binding peptide containing a sulfhydryl group , its preferred modified structure is as follows:
  • AT can also be a binding peptide with affinity to antibody Fab; for example, trastuzumab Fab binding peptide, etc.
  • AT can also be a small molecule fragment that has a reversible covalent interaction with the protein, for example, o-hydroxybenzaldehyde, p-acetylphenylboronic acid, etc. as shown below,
  • CL is a cleavable fragment that can form a new chemical bond under the attack of a nucleophile, covalently connecting the R fragment to a specific site on the protein, and at the same time, the fragment is broken, releasing the affinity FragmentAT.
  • CL is as follows, and the CL fragment can be an acyl transfer reagent such as a thioester fragment, a maleimide ester fragment, or an ester fragment:
  • R1 and R2 are independently hydrogen atoms, methyl, ethyl and other aliphatic or aromatic fragments, or R1 and R2 form a cyclic alkane or alkene structure.
  • CL is as shown in any of the following
  • the carbonyl group of the cleavable fragment is connected to the R fragment, and the S or O part of the cleavable fragment is coupled to the affinity fragment.
  • the CL fragment is an acyl transfer reagent with a thioester as the core, and its structure is shown in the following formula (III):
  • the structure of the linker is a methylene structure of different lengths, amino acid, polyglycine, polyethanol (PEG) structure, etc.; more preferably, the structure of the linker is as follows:
  • n is an integer from 1 to 10.
  • R is derived from a compound to be modified onto the protein, which includes reactive groups that can be further modified such as bioorthogonal groups, etc. or contains or is derived from biotin, fluorescent reagents, small molecule drugs, mRNA , peptide compounds and proteins, etc.
  • the source compound of R itself contains a carboxylic acid fragment or can be derivatized to obtain a carboxylic acid structure.
  • the -CO- structure in the cleavable fragment of CL is formed by carboxylic acid and sulfhydryl group, maleimide, phenol, etc., that is, an acyl transfer reagent.
  • -CO-R is selected from Group 2 (where -CO- is -CO- in CL), and Group 2 includes groups as shown below:
  • n is 0 or any positive integer; preferably, it is a positive integer from 1 to 20.
  • L 3 -D is selected from the following group:
  • p, q and r are 0 or any positive integer; preferably 0 or a positive integer from 1 to 20; more preferably, p and r are each independently 0, 1, 2 or 3, and q is 1, 2, 3, 4, 5 or 6;
  • MMAE/MMAF refers to MMAE or MMAF, and the structures of MMAE and MMAF are as follows:
  • the affinity fragment-directed cleavable fragment represented by Formula III is represented by any one of the following formulas:
  • R and linker are defined as previously defined, and circles 1, 2, 3, 4, 5, 6, 7, 8 and 9 respectively refer to Aa1, Aa2, Aa3, Aa4, Aa5, Aa6, Aa7, and Aa8 as previously defined.
  • the circle C refers to Cys
  • the circle K refers to the main chain part of the lysine residue (i.e., the -NH-C-CO- part of the lysine residue).
  • AT, CL and R are as defined in the first aspect.
  • AT, CL and R are each independently the corresponding group in the conjugates shown in Table A1 and Table A2 or in each specific compound in the examples.
  • the conjugate (or affinity fragment-directed cleavable fragment) is selected from the compounds shown in Table A1 and Table A2 (the side chain structure of K in the cyclic peptide (i.e. -(CH 2 ) 4 -NH-) is shown):
  • Ac is an N-terminal protecting group or does not exist (preferably it is an acetyl group).
  • Ac is an N-terminal protecting group or does not exist (preferably it is an acetyl group).
  • the affinity fragment-guided cleavable fragment can directly transfer the R group to the natural antibody (Ab) to achieve selective modification of the amino acid of the antibody and form a functional modification of the antibody.
  • Fluorescence modification, drug conjugation, peptide coupling, mRNA coupling, etc. form an antibody modified structure shown in the following formula (V): Ab-NHCO-R(V)
  • Ab is an antibody
  • -NHCO- is an amide bond structure
  • R is a modified fragment modified to the antibody.
  • linker 1 is -L 1 -W 1 -A 1 -;
  • PG is any thiol protecting group other than thiol, such as Trt-, Acm-, Mob, etc.;
  • AE is any active ester form, such as NHS ester, acid chloride, Acyl azide, thioester, p-nitrophenol ester, etc.;
  • L 1 , W 1 , A 1 , circles 1-9, circle K, circle C, and R are as defined before.
  • the method includes the steps of:
  • Step 1 Synthesis of affinity fragments: After obtaining the peptide through solid-phase synthesis, dissolve it in DMSO to a final concentration of 5mM, add hydrogen peroxide (10mM) and ammonia (100mM), stir at room temperature overnight, and monitor with LC-MS After the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain the affinity fragment.
  • Step 3 Synthesis of affinity fragment-thiol derivative: The crude affinity fragment-thiol derivative is obtained through the corresponding deprotection strategy.
  • Step 4 Preparation of affinity fragment-guided cleavable fragments: Mix the crude affinity fragment-thiol derivative and the active ester form of the carboxylic acid compound. After monitoring the reaction with LC-MS, use semi-preparative isolation and purification, and lyophilize. Obtain affinity fragment-guided cleavable fragments.
  • the method includes the steps of:
  • Step 1 Synthesis of affinity fragments: After obtaining the peptide through solid-phase synthesis, dissolve it in DMSO to a final concentration of 5mM, add hydrogen peroxide (10mM) and ammonia (100mM), stir at room temperature overnight, and monitor with LC-MS After the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain the affinity fragment.
  • Step 2 Synthesis of cleavable fragments: The active ester form of a carboxylic acid compound is reacted with a sulfhydryl derivative to obtain a cleavable fragment - a thioester compound.
  • Step 3 Preparation of affinity fragment-guided cleavable fragments: Affinity fragment-guided cleavable fragments are obtained through reactions such as condensation and reductive amination between the affinity fragment and the cleavable fragment.
  • a method for preparing a site-directed antibody drug conjugate (or antibody or Fc fusion) based on the above-mentioned affinity fragment-guided cleavable fragment (i.e., directing group-thioester-carrier molecule complex).
  • Synthetic protein site-directed modification method as shown in the following reaction equation:
  • R is the modified structure coupled to the antibody
  • R1 and R2 are bioorthogonal groups
  • R3 is the linker structure formed by the reaction of R1 and R2.
  • the preparation method or site-directed modification method includes incubating the complex represented by formula (I) (as described above) with an antibody or Fc fusion protein to achieve lysine at a specific site in the Fc region of the antibody or Fc fusion protein.
  • Fixed point modification is preferred.
  • the antibody or Fc fusion protein has an affinity fragment or sequence that forms a complex with formula (I).
  • the antibody or Fc structure is of human IgG origin
  • the covalently modified region formed by interacting with the complex shown in (I) is a region composed of continuous 1-10 amino acid residues, wherein the target region consists of positions 246-248 in the Fc region of human IgG A region formed by amino acid residues.
  • the target region is a lysine residue at position 246 or 248 in the Fc region of human IgG.
  • the selectivity of the region of the antibody or Fc fusion protein is above 90%.
  • the antibody drug conjugate or the site-directed modified antibody or Fc fusion protein is selected from the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the group consisting of the site-directed modified antibody or Fc fusion protein
  • the conjugate provided by the invention has high modification efficiency (>95%) when used to modify proteins such as antibodies.
  • the conjugate provided by the present invention can be directly modified on the side chain amino group of the amino acid residue at the site to be modified in the protein without the need to modify the side chain amino group in advance.
  • the conjugate provided by the invention is easy to operate on antibody modification, and has no strict requirements on pH and concentration (pH 5.5-8.0, antibody concentration 0.5mg/mL-10mg/mL), which facilitates technology promotion.
  • the method constructed in the present invention has a wide range of uses and can be used to construct a rapid antibody modification platform, that is, diversified modification of antibodies and preparation of antibody drug conjugates (ADC).
  • ADC antibody drug conjugates
  • the method constructed in the present invention can be used to prepare site-specific modified ADC in one step, and is expected to construct a large-capacity ADC library for systematic screening and optimization of ADC drug linkers.
  • the thioester fragment provided by the present invention is used as an acyl transfer reagent and is connected to a specific site of the Fc-binding peptide, which can realize "one-step" efficient site-specific coupling of small molecule drugs and is compatible with a variety of drug linker structures.
  • the material has wide applicability.
  • Mobile phase A 0.1% FA in water
  • mobile phase B 0.1% FA in acetonitrile
  • Method A for mass spectrometry analysis of small molecule compounds.
  • the liquid phase program is 0-0.2-1.5-1.8-2.0-2.5 minutes and the corresponding mobile phase B is 10-10-70-70-10- 10%, detection wavelength is 214nm, column temperature: 35°C.
  • Method B for mass spectrometry analysis of antibodies and ADC molecules.
  • C4 column ACQUITY UPLC Protein BEH C4, 1.7 ⁇ m, 2.1mm x 50mm
  • the liquid phase program is 0-2.0-6.0-7.2-7.3-7.6-7.7-8.0-8.1-10.0 minutes
  • the corresponding mobile phase B is 5-5-90-90-5-90-5-5%
  • detection wavelength is 280nm
  • column temperature 80°C.
  • the detection wavelength is 280nm.
  • Step 1 Weigh 2-(tritylthio)acetic acid (15.7mg, 0.047mmol) and dissolve it in DMF. HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-1 (20mg, 0.0094mmol) were added one at a time and stirred at room temperature for 1 hour. After the reaction was monitored by LC-MS, semi-preparative separation and purification was performed and frozen. After drying, compound 3 was obtained as a white powder (yield 92%).
  • Step 2 Weigh compound 3 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 1.
  • HRMS calculated for C 95 H 137 N 31 O 23 S 3 :[M+H] + 2176.9744,[M+3H] 3+ 726.3300,[M+4H] 4+ 544.9995, measured 726.3278,545.0035.
  • Step 1 Weigh 3-(tritylthio)propionic acid (16.36mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT in sequence. -1 (20 mg, 0.0094 mmol), stirred at room temperature for 1 h, after LC-MS monitoring of complete reaction, semi-preparative isolation and purification was used, and lyophilized to obtain compound 4 as a white powder (yield 89%).
  • Step 2 Weigh compound 4 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 2.
  • HRMS calculated for C 96 H 139 N 31 O 23 S 3 :[M+H] + 2190.9900,[M+3H] 3+ 731.0019,[M+4H] 4+ 548.5034, measured 731.0054,548.5102.
  • Step 1 Weigh NaH (34.5 mg, 1.44 mmol) and dissolve it in DMF (5 mL). Under N2 protection, 0°C, and ice bath conditions, add triphenylmethylmercaptan (200 mg, 0.72 mmol), and ice bath. After stirring for 30 minutes, 4-bromobutyric acid (60 mg, 0.36 mmol) was added. After the temperature slowly rose to room temperature, the reaction was stirred overnight and separated and purified on a silica gel column to obtain compound 5 (yield 76%).
  • Step 2 Weigh compound 5 (17mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-1 (20mg, 0.0094mmol) in order, at room temperature. After stirring for 1 hour, LC-MS monitored that the reaction was complete, the mixture was separated and purified using semi-preparative method, and then lyophilized to obtain compound 6 as a white powder (yield 91%).
  • Step 3 Weigh compound 6 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 3.
  • HRMS calculated for C 97 H 141 N 31 O 23 S 3 :[M+H] + 2205.0057,[M+3H] 3+ 735.6738,[M+4H] 4+ 552.0073, measured 735.6742,552.0153.
  • Step 1 Weigh 2-(tritylthio)acetic acid (334 mg, 1 mmol) and dissolve it in 5 mL DMF. Add N-hydroxysuccinimide (138 mg, 1.2 mmol) and 1-(3-dimethylamino) Propyl)-3-ethylcarbodiimide Hydrochloride (230 mg, 1.2 mmol), stir at room temperature overnight. Then glycine (75 mg, 1 mmol) was added and the reaction was carried out at room temperature for 2 h.
  • Step 2 Weigh compound 7 (18.4mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-1 (20mg, 0.0094mmol) in sequence. Stir at room temperature for 1 hour. After LC-MS monitors that the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain compound 8 as a white powder (yield 87%).
  • Step 3 Weigh compound 8 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 4.
  • HRMS calculated for C 97 H 140 N 32 O 24 S 3 :[M+H] + 2233.9955,[M+3H] 3+ 745.3372,[M+4H] 4+ 559.2548, measured 745.3356,559.2573.
  • Step 1 Weigh 2-(tritylthio)acetic acid (15.7mg, 0.047mmol) and dissolve it in DMF, then add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT- 2 (19.5 mg, 0.0094 mmol), stirred at room temperature for 1 h, after LC-MS monitoring of complete reaction, semi-preparative isolation and purification was used, and lyophilized to obtain compound 9 as a white powder (yield 87%).
  • Step 2 Weigh compound 9 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 5.
  • HRMS calculated for C 94 H 133 N 29 O 24 S 3 :[M+H] + 2148.9319,[M+3H] 3+ 716.9825,[M+4H] 4+ 537.9888, measured 716.9867,537.9872.
  • Step 1 Weigh 3-(tritylthio)propionic acid (16.36mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT in sequence. -2 (19.5 mg, 0.0094 mmol), stirred at room temperature for 1 h, after LC-MS monitoring of complete reaction, semi-preparative isolation and purification was used, and lyophilized to obtain compound 10 as a white powder (yield 89%).
  • Step 2 Weigh compound 10 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 6.
  • HRMS calculated for C 95 H 134 N 29 O 24 S 3 :[M+H] + 2162.9475,[M+3H] 3+ 721.6544,[M+4H] 4+ 541.4928, measured 721.6523,514.4953.
  • Step 1 Weigh compound 5 (17mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-2 (19.5mg, 0.0094mmol) in sequence. Stir at room temperature for 1 hour. After LC-MS monitors that the reaction is complete, use semi-preparative isolation and purification, and freeze-dry to obtain compound 11 as a white powder (yield 92%).
  • step Step 2 Weigh compound 11 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 7.
  • HRMS calculated for C 96 H 137 N 29 O 24 S 3 :[M+H] + 2176.9632,[M+3H] 3+ 726.3263,[M+4H] 4+ 544.9967, measured 726.3248,544.9913.
  • Step 1 Weigh compound 7 (18.4mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-2 (19.5mg, 0.0094mmol) in sequence. , stirred at room temperature for 1 h, after LC-MS monitoring of complete reaction, semi-preparative separation and purification was used, and then lyophilized to obtain compound 12 as a white powder (yield 87%).
  • Step 2 Weigh compound 12 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 8.
  • HRMS calculated for C 96 H 136 N 30 O 25 S 3 :[M+H] + 2205.9533,[M+3H] 3+ 735.9897,[M+4H] 4+ 552.2442, measured 735.9910,552.2418.
  • Step 1 Weigh NaH (34.5 mg, 1.44 mmol) and dissolve it in DMF (5 mL). Under N2 protection, 0°C, and ice bath conditions, add triphenylmethylmercaptan (200 mg, 0.72 mmol), and ice bath. After stirring for 30 minutes, 6-bromocaproic acid (70 mg, 0.36 mmol) was added. After the temperature slowly rose to room temperature, the reaction was stirred overnight and separated and purified on a silica gel column to obtain compound 13 (yield 82%).
  • Step 2 Weigh compound 13 (18.3 mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-2 (19.5mg, 0.0094mmol) in sequence. , stirred at room temperature for 1 h, after LC-MS monitoring the reaction was complete, the reaction was separated and purified using semi-preparation, and then lyophilized to obtain compound 14 as a white powder (yield 87%).
  • Step 3 Weigh compound 14 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 9.
  • HRMS calculated for C 98 H 141 N 29 O 24 S 3 :[M+H] + 2204.9945,[M+3H] 3+ 735.6700,[M+4H] 4+ 552.0044, measured 735.6712,552.0019.
  • Step 1 Weigh 2-(tritylthio)acetic acid (334 mg, 1 mmol) and dissolve it in 5 mL DMF. Add N-hydroxysuccinimide (138 mg, 1.2 mmol) and 1-(3-dimethylamino) Propyl)-3-ethylcarbodiimide hydrochloride (230 mg, 1.2 mmol), stirred at room temperature overnight. Then 4-aminobutyric acid (103 mg, 1 mmol) was added, and the reaction was carried out at room temperature for 2 h to obtain compound 15 (yield 78%). HRMS, calculated for C 25 H 25 NO 3 S:[M+H] + 420.1633, found 420.1649.
  • Step 2 Weigh compound 15 (19.7mg, 0.047mmol) and dissolve it in DMF. Add HATU (7.14mg, 0.0188mmol), DIPEA (4.84 ⁇ L, 0.028mmol) and compound AT-2 (19.5mg, 0.0094mmol) in sequence. , stirred at room temperature for 1 h, after LC-MS monitored that the reaction was complete, it was separated and purified using semi-preparation, and then lyophilized to obtain compound 16 as a white powder (yield 82%).
  • Step 3 Weigh compound 16 (15 mg) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 to obtain compound ATS- 10.
  • HRMS calculated for C 98 H 140 N 30 O 25 S 3 :[M+H] + 2233.9846,[M+3H] 3+ 745.3334,[M+4H] 4+ 559.2520, measured 745.3316,559.2561.
  • Step 1 Weigh CH 3 O-PEG 24 -COOH (58 mg, 0.05 mmol) and dissolve it in acetonitrile. Add NHS (6.8 mg, 0.06 mmol) and EDC (7.9 ⁇ L, 0.06 mmol). After reacting at room temperature overnight, add Fmoc -Lys(NH 2 )-OH (18.4 mg, 0.05 mmol) in acetonitrile solution, stirred at room temperature for 2 h, LC-MS monitored the reaction to be complete, used semi-preparative separation and purification, and lyophilized to obtain a white powder to obtain compound 18 (yield 78% ). HRMS, calculated for C 73 H 126 N 2 O 30 : [M+H] + 756.4275, measured 756.4218.
  • Step 2 Weigh compound 18 (13.5 mg, 8.9 ⁇ mol) and dissolve it in DMF, add HATU (6.8 mg, 17.8 ⁇ mol), DIPEA (4.6 ⁇ L, 27.6 ⁇ mol), compound 17 (10 mg, 8.9 ⁇ mol), and react at room temperature for 2 hours. , LC-MS monitored that the reaction was complete, and the reaction was separated and purified using semi-preparative method, and then freeze-dried to obtain compound 19 as a white powder (yield 86%).
  • HRMS calculated: C 116 H 208 N 12 O 39 : [M+H] + 2394.4741, [M+3H] 3+ 798.8299, measured 798.8312.
  • Step 3 Weigh compound 19 (10 mg, 4.2 ⁇ mol) and dissolve it in DMF, add BCN-O-PNP (1.63 mg, 5 ⁇ mol), triethylamine (1.7 ⁇ L, 12.6 ⁇ mol), stir at room temperature for 2 hours, and monitor with LC-MS The reaction was complete, semi-preparative isolation and purification was used, and compound D3 was obtained by freeze-drying (yield 76%).
  • Step 1 Dissolve H 2 N-PEG 4 -COOH (26.5 mg, 0.1 mmol) in DMF, add 9-fluorenylmethyl-N-succinimidyl carbonate (67.4 mg, 0.2 mmol), and triethylamine (41.6 ⁇ L, 0.3 mmol). After reacting at room temperature for 3 hours, LC-MS monitored the reaction system, which showed that the reaction was complete. Compound 20 was obtained by lyophilization after semi-preparative isolation and purification. HRMS, calculated for C 26 H 33 NO 8 :[M+H] + 488.2284, measured 488.2243.
  • Step 2 Weigh compound 20 (17 mg, 0.035 mmol) and dissolve it in DMF. Add p-nitrophenol (9.7 mg, 0.07 mmol) and EDC (12.7 ⁇ L, 0.07 mmol). After reacting at room temperature for 1 hour, monitor the reaction with LC-MS. The system showed that the reaction was complete. After semi-preparative isolation and purification, compound 21 was obtained by freeze-drying. HRMS, calculated for C 32 H 36 N 2 O 10 :[M+H] + 609.2448, measured 609.2252.
  • Step 3 Weigh compound 21 (10 mg, 0.016 mmol) and dissolve it in DMF, add MMAF (12 mg, 0.02 mmol), HOBt (0.54 mg, 3.2 ⁇ mol), pyridine (36 ⁇ L, 0.45 mmol), react at room temperature for 1 h, LC- After MS monitored that the basic reaction was complete, 20% piperidine was added and reacted for 15 minutes. After semi-preparative separation and purification, compound 22 was obtained by freeze-drying. HRMS, calculated for C 50 H 86 N 6 O 13 :[M+H] + 979.6331, measured 979.6328.
  • Step 4 Weigh compound 22 (10 mg, 0.01 mmol) and dissolve it in DMF, add BCN-O-PNB (9.45 mg, 0.03 mmol), triethylamine (8.3 ⁇ L, 0.06 mmol), react at room temperature for 4 hours, and perform LC-MS The basic reaction was monitored to be complete, and compound D4 was obtained by lyophilization after semi-preparative isolation and purification.
  • HRMS calculated for C 61 H 98 N 6 O 15 : [M+H] + 1155.7168, [M+2H] 2+ 578.3623, measured 578.3619.
  • Step 1 Weigh HOOC-PEG 4 -NH 2 (26.5 mg, 0.1 mmol) and dissolve it in DMF (400 ⁇ L). Add 6-(maleimido)hexanoic acid succinimide ester (30.8 mg, 0.1 mmol), triethylamine (41.6 ⁇ L, 0.3 mmol), stirred at room temperature for 1 h, LC-MS monitored the reaction to be complete, used semi-preparative isolation and purification, and lyophilized to obtain a white powder, which was compound 24 (yield 86%).
  • HRMS calculated for C 20 H 32 N 2 O 10 :[M+H] + 461.2135, measured 461.2169.
  • Step 2 Weigh compound 24 (10 mg, 0.022 mmol) and dissolve it in PB 7.4 (500 ⁇ L), add cytotoxin DM1 (16 mg, 0.022 mmol), stir at room temperature for 1 h, monitor the reaction is complete by LC-MS, use semi-preparative isolation and purification, and freeze Compound D8 was obtained dryly (yield 87%).
  • HRMS calculated for C 55 H 80 ClN 5 O 20 S: [M+H] + 1198.4884, measured 1198.4798.
  • Step 1 Weigh azide acetic acid (250 mg, 2.5 mmol) and dissolve it in THF (5 mL). Add N-hydroxysuccinimide (288 mg, 2.5 mmol). After stirring at 0°C for 10 min, add dicyclohexylcarbodioxide. amine (516 mg, 2.5 mmol), and the reaction solution was stirred at 0°C for 4 h. After filtering to remove DCU, the filtrate was collected, added with diethyl ether (20 mL) to recrystallize, and left to stand at 4°C overnight. After filtration, the filter cake was washed with THF (40 mL) and dried under vacuum to obtain compound 25 as a white powder (yield 82%).
  • HRMS calculated for C 97 H 138 N 34 O 24 S 3 :[M+H] + 2259.9861,[M+3H] 3+ 754.0006,[M+4H] 4+ 565.7524, measured 753.9815,565.7080.
  • Step 1 Weigh 2-methyl-2-cyclopropenecarboxylic acid (0.4 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) was reacted at room temperature overnight to obtain crude compound 26.
  • Step 2 Weigh compound ATS-1 (7 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L). Add it to the reaction solution obtained in step 1, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 30 min, and monitor with LC-MS. After the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-3 (yield 67%).
  • HRMS calculated: C 100 H 141 N 31 O 24 S 3 : [M+H] + 2257.0006, [M+3H] 3+ 753.0054, [M+4H] 4+ 565.0060, measured 753.0013, 565.0059.
  • Step 1 Weigh pentynoic acid (0.4 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-dimethylaminopropyl)-3 -Ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) was reacted at room temperature overnight to obtain crude compound 27.
  • Step 2 Weigh compound ATS-1 (7 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L). Add it to the reaction solution obtained in step 1, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 30 min, and monitor with LC-MS. After the reaction is complete, use semi-preparative isolation and purification, and freeze-dry to obtain a white powder, which is compound ATC-4 (yield 59%).
  • HRMS calculated: C 100 H 141 N 31 O 24 S 3 : [M+H] + 2257.0006, [M+3H] 3+ 753.0054, [M+4H] 4+ 565.0060, measured 753.0051, 565.0086.
  • Step 1 Weigh 2-methyl-2-cyclopropenecarboxylic acid (0.4 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) was reacted at room temperature overnight to obtain crude compound 26.
  • Step 2 Weigh compound ATS-4 (7.05 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add it to the reaction solution obtained in step 1, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 30 min, and perform LC-MS After monitoring the completion of the reaction, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-14 (yield 59%).
  • HRMS calculated: C 102 H 144 N 32 O 25 S 3 : [M+H] + 2314.0221, [M+3H] 3+ 772.0126, [M+4H] 4+ 579.2614, measured 772.0148, 579.2658.
  • Step 1 Weigh pentynoic acid (0.4 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-dimethylaminopropyl)-3 -Ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol), and after reacting at room temperature overnight, a crude compound 27 was obtained.
  • Step 2 Weigh compound ATS-4 (7.05 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add it to the reaction solution obtained in step 1, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 30 min, and perform LC-MS After monitoring the completion of the reaction, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-4 (yield 66%).
  • HRMS calculated: C 102 H 144 N 32 O 25 S 3 : [M+H] + 2314.0221, [M+3H] 3+ 772.0126, [M+4H] 4+ 579.2614, measured 772.0148, 579.2594.
  • Step 2 Weigh compound 28 (1.33 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-dimethylaminopropyl)-3- After reacting with ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) at room temperature overnight, the crude compound 29 was obtained.
  • Step 3 Weigh compound ATS-6 (6.9 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add it to the reaction solution obtained in step 2, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 1 h, and perform LC-MS After monitoring the completion of the reaction, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-20 (yield 75%).
  • HRMS calculated: C 108 H 153 N 31 O 27 S 5 : [M+H] + 2477.0234, [M+3H] 3+ 826.3463, [M+4H] 4+ 620.0117, measured 826.3457, 619.9915.
  • Step 1 Weigh 2-methyl-2-cyclopropenecarboxylic acid (0.4 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) was reacted at room temperature overnight to obtain crude compound 26.
  • Step 2 Weigh compound ATS-8 (7 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add it to the reaction solution obtained in step 1, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 30 min, and monitor with LC-MS After the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-24 (yield 64%).
  • Step 1 Weigh 4-(aminomethyl)benzoic acid (9mg, 0.06mmol) and dissolve it in DMF, add biotin-N-succinimidyl ester (17mg, 0.05mmol), triethylamine (20 ⁇ L, 0.15 mmol), stirred at room temperature overnight, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound 30 was obtained by freeze-drying (yield 91%).
  • Step 2 Weigh compound 30 (1.33 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N- After reacting with hydroxysuccinimide (0.69 mg, 6 ⁇ mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) at room temperature overnight, compound 31 was obtained. Crude.
  • Step 3 Weigh compound ATS-8 (7 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L). Add it to the reaction solution obtained in step 2, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 1 hour, and monitor with LC-MS. After the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain a white powder, which is compound ATC-29 (yield 74%).
  • HRMS calculated: C 114 H 157 N 33 O 28 S 4 : [M+H] + 2565.0837, [M+3H] 3+ 855.6998, [M+4H] 4+ 642.0268, measured 855.7043, 642.0276.
  • Step 1 Weigh biotin (50mg, 0.2mmol) and dissolve it in DMF, add HATU (152mg, 0.4mmol), DIPEA (103 ⁇ L, 0.6mmol) and (2-aminoethoxy)carbamic acid tert-butyl ester (35mg , 0.2 mmol), reacted at room temperature for 2 h, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound 32 was obtained by freeze-drying (yield 85%).
  • HRMS calculated for C 17 H 30 N 4 O 5 S: [M+H] + 403.2015, measured 403.2114.
  • Step 2 Weigh compound 32 (20 mg, 0.05 mmol) and dissolve it in 500 ⁇ L dichloromethane. Under ice bath conditions, add 450 ⁇ L trifluoroacetic acid and 50 ⁇ L triisopropylsilane. After reacting at room temperature for 1 hour, blow dry with N 2 and set aside. .
  • Step 4 Weigh compound 33 (1.43 mg, 4 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add N-hydroxysuccinimide (0.69 mg, 6 ⁇ mol), 1-(3-dimethylaminopropyl)-3- After reacting with ethylcarbodiimide hydrochloride (1.15 mg, 6 ⁇ mol) at room temperature overnight, the crude compound 34 was obtained.
  • Step 5 Weigh compound ATS-8 (7 mg, 3.2 ⁇ mol) and dissolve it in DMF (50 ⁇ L), add it to the reaction solution obtained in step 2, then add PB 7.4 buffer (200 ⁇ L), stir at room temperature for 1 hour, and monitor with LC-MS After the reaction is complete, use semi-preparative isolation and purification, and freeze-dry to obtain a white powder, which is compound ATC-30 (yield 63%).
  • HRMS calculated: C 110 H 156 N 34 O 29 S 4 : [M+H] + 2546.0738, [M+3H] 3+ 849.3631, [M+4H] 4+ 637.2743, measured 849.3654, 637.2842.
  • Step 1 Weigh aminofluorescein (10.4mg, 0.03mmol) and dissolve it in DMF (500 ⁇ L). Add disuccinimide glutarate (DSG, 39mg, 0.12mmol) and triethylamine (12.5 ⁇ L, 0.09 mmol), react at room temperature for 2 hours in the dark, and LC-MS monitors that the reaction is complete. Compound 35 is obtained. Thioglycolic acid (6.3 ⁇ L, 0.09 mmol) and PB 7.4 buffer (500 ⁇ L) are added to the reaction solution, and the reaction is carried out at room temperature in the dark for 2 hours.
  • Step 2 Weigh compound 36 (5.1 mg, 9.51 ⁇ mol) and dissolve it in DMF (300 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). , reacted at room temperature for 2 hours, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound ATC-33 was obtained by freeze-drying.
  • HRMS calculated: C 120 H 154 N 32 O 30 S 3 : [M+H] + 2620.0749, [M+3H] 3+ 874.0302, [M+4H] 4+ 655.7746, measured 874.0562, 655.7789.
  • Step 1 Weigh aminofluorescein (17 mg, 49 ⁇ mol) and dissolve it in DMF (400 ⁇ L). Add HATU (28 mg, 73.5 ⁇ mol), DIPEA (18.9 ⁇ L, 110 ⁇ mol) and carboxylic acid-polyethylene glycol-carboxylic acid (17.4 mg, 98 ⁇ mol), the reaction was carried out at room temperature in the dark for 1 hour. After the reaction was completed by LC-MS, semi-preparative isolation and purification was used, and compound 37 was obtained by freeze-drying (yield 92%). HRMS, calculated: C 26 H 21 NO 10 : [M+H] + 508.1244, measured 508.1312.
  • Step 2 Weigh compound 37 (15.2 mg, 0.03 mmol) and dissolve it in DMF (300 ⁇ L). Add N-hydroxysuccinimide (NHS, 4.1 mg, 0.036 mmol) and EDCI (6.4 ⁇ L, 0.036 mmol) at room temperature. The reaction was carried out overnight in the dark. After the reaction was monitored by LC-MS, thioglycolic acid (6.3 ⁇ L, 0.09 mmol) and PB 7.4 buffer (300 ⁇ L) were added to the reaction solution. The reaction was carried out at room temperature for 2 hours in the dark. After the reaction was monitored by LC-MS, the semi-preparation process was completed. Isolate, purify, and freeze-dry to obtain yellow powder (yield 81%). HRMS, calculated for C 28 H 23 NO 11 S: [M+H] + 582.1070, measured 582.1235.
  • Step 3 Weigh compound 38 (5.5 mg, 9.51 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). , reacted at room temperature for 2 hours, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound ATC-34 was obtained by freeze-drying (yield 84%).
  • Step 1 Weigh 2-methyl-2-cyclopropenecarboxylic acid (20mg, 0.2mmol) and dissolve it in DMF (400 ⁇ L). Add NHS (27.6mg, 0.24mmol) and EDCI (42.4 ⁇ L, 0.24mmol) and react at room temperature. Overnight, 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid (53mg, 0.2mmol), triethylamine ( 83 ⁇ L, 0.6 mmol) After the reaction was monitored by LC-MS, semi-preparative isolation and purification was used, and compound 39 was obtained by freeze-drying (yield 78%). HRMS, calculated for C 16 H 27 NO 7 : [M+H] + 346.1866, measured 346.1902.
  • Step 2 Weigh compound 39 (10.4 mg, 0.03 mmol) and dissolve it in DMF (300 ⁇ L). Add N-hydroxysuccinimide (NHS, 4.1 mg, 0.036 mmol) and EDCI (6.4 ⁇ L, 0.036 mmol) at room temperature. After reacting overnight, thioglycolic acid (6.3 ⁇ L, 0.09 mmol) and PB 7.4 buffer (300 ⁇ L) were added to the reaction solution, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed after LC-MS monitoring, semi-preparation separation and purification was performed, and a white powder was obtained by freeze-drying (yield 84%). HRMS, calculated for C 18 H 29 NO 8 S: [M+H] + 420.1692, measured 420.1589.
  • Step 3 Weigh compound 40 (4 mg, 9.51 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). The reaction was carried out at room temperature for 2 hours. After the reaction was completed after monitoring by LC-MS, the compound was separated and purified using semi-preparative method and lyophilized to obtain compound ATC-35 (yield 90%).
  • Step 1 Weigh DBCO-NHS (40 mg, 0.1 mmol) and dissolve it in DMF (400 ⁇ L). Add NHS (13.8 mg, 0.12 mmol) and EDCI (21.2 ⁇ L, 0.12 mmol). React at room temperature overnight. Add 3 to the reaction solution. -[2-[2-[2-(2-Aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid (26.5mg, 0.1mmol), triethylamine (41.5 ⁇ L, 0.3mmol) After the completion of the reaction was monitored by LC-MS, semi-preparative separation and purification was used, and compound 41 was obtained by freeze-drying (yield 81%). HRMS, calculated for C 30 H 36 N 2 O 8 :[M+H] + 553.2550, measured 553.2548.
  • Step 2 Weigh compound 41 (16.5 mg, 0.03 mmol) and dissolve it in DMF (400 ⁇ L). Add N-hydroxysuccinimide (NHS, 4.1 mg, 0.036 mmol) and EDCI (6.4 ⁇ L, 0.036 mmol) at room temperature. After reacting overnight, thioglycolic acid (6.3 ⁇ L, 0.09 mmol) and PB 7.4 buffer (400 ⁇ L) were added to the reaction solution, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed after LC-MS monitoring, semi-preparative separation and purification was performed, and a white powder was obtained by freeze-drying, which is the compound 42 (yield 87%).
  • HRMS calculated for C 32 H 38 N 2 O 9 S: [M+H] + 627.2376, measured 627.2315.
  • Step 3 Weigh compound 42 (6 mg, 9.51 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). The reaction was carried out at room temperature for 2 hours. After the reaction was completed after monitoring by LC-MS, the compound was separated and purified using semi-preparative method and lyophilized to obtain compound ATC-37 (yield 92%).
  • Step 1 Weigh tert-butyl 2-aminoethoxycarbamate (17.6mg, 0.1mmol) and dissolve it in DMF (400 ⁇ L), add HATU (76mg, 0.2mmol), DIPEA (51.5 ⁇ L, 0.3mmol) and carboxylic acid Acid-polyethylene glycol-carboxylic acid (53.4 mg, 0.3 mmol) was reacted at room temperature for 2 h. After the reaction was completed after LC-MS monitoring, semi-preparative isolation and purification was used, and compound 43 was obtained by freeze-drying (yield 88%). HRMS, calculated for C 13 H 24 N 2 O 8 :[M+H] + 337.1611, measured 337.1587.
  • Step 2 Weigh compound 43 (20 mg, 0.06 mmol) and dissolve it in DMF (400 ⁇ L), add N-hydroxysuccinimide (NHS, 8.2 mg, 0.072 mmol), EDCI (12.8 ⁇ L, 0.072 mmol), and store at room temperature. The photoreaction was carried out overnight. After LC-MS monitored that the reaction was complete, thioglycolic acid (12.6 ⁇ L, 0.18 mmol) and PB 7.4 buffer (400 ⁇ L) were added to the reaction solution. The reaction was carried out at room temperature in the dark for 2 hours. After LC-MS monitored that the reaction was complete, semi-preparative separation was performed. Purify and freeze-dry to obtain a white powder, which is compound 44 (yield 83%). HRMS, calculated for C 16 H 28 N 2 O 9 S: [M+H] + 425.1594, measured 425.1608.
  • Step 3 Weigh compound 44 (4 mg, 9.51 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). React at room temperature for 2 hours. After LC-MS monitors that the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain compound 45 (product rate 90%). HRMS, calculated: C 108 H 159 N 33 O 30 S 3 : [M+H] + 2495.1171, [M+3H] 3+ 832.3776, [M+4H] 4+ 624.5351, measured 832.3810, 624.5305.
  • Step 4 Weigh compound 45 (9 mg, 3.6 ⁇ mol), add 200 ⁇ L of dichloromethane, 180 ⁇ L of trifluoroacetic acid, and 20 ⁇ L of isopropylsilane, react at 4°C for 30 min, and dry the solvent with N 2 to obtain compound ATC-38 (yield 95 %).
  • HRMS calculated: C 103 H 151 N 33 O 28 S 3 : [M+H] + 2395.0646, [M+3H] 3+ 799.0267, [M+4H] 4+ 599.5220, measured 799.0248, 599.5278.
  • Step 1 Weigh levulinic acid (23.2mg, 0.2mmol) and dissolve it in DMF (400 ⁇ L). Add N-hydroxysuccinimide (NHS, 27.6mg, 0.24mmol) and EDCI (42.4 ⁇ L, 0.24mmol). After reacting at room temperature overnight, 4-mercaptopropylacetic acid (67.2 mg, 0.4 mmol) and triethylamine (83 ⁇ L, 0.6 mmol) were added to the reaction solution. The reaction was carried out at room temperature for 2 hours. After the reaction was completed after LC-MS monitoring, the semi-preparative separation and purification was carried out and frozen. The white powder obtained after drying was compound 46 (yield 73%). HRMS, calculated for C 14 H 16 O 4 S: [M+H] + 281.0848, measured 281.0923.
  • HRMS calculated: C 101 H 145 N 31 O 25 S 3 : [M+H] + 2290.0346, [M+3H] 3+ 764.0167, [M+4H] 4+ 573.2645, measured 764.0205, 573.0618.
  • Step 1 Weigh diglycolic acid (26.8 mg, 0.2 mmol) and dissolve it in DMF (400 ⁇ L), add p-nitrophenol (66.7 mg, 0.48 mmol), EDCI (84.8 ⁇ L, 0.48 mmol), and react at room temperature overnight. , add diethyl ether to precipitate, and obtain product 47 (yield 69%).
  • Step 2 Dissolve compound 47 (11.3 mg, 30 ⁇ mol) in DMF (200 ⁇ L), add compound 17 (11.2 mg, 10 ⁇ mol), triethylamine (6.94 ⁇ L, 50 ⁇ mol), react at room temperature for 20 min, then add thioglycolic acid (2.1 ⁇ L , 30 ⁇ mol), PB7.4 buffer, reaction at room temperature for 1 h, semi-preparative isolation and purification, and compound 48 was obtained (yield 76%).
  • HRMS calculated: C 64 H 100 N 10 O 17 S: [M+H] + 1313.7067, measured 1313.7054.
  • Step 3 Weigh compound 48 (6.2 mg, 4.76 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (2.7 mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10 mg, 4.76 ⁇ mol). , reacted at room temperature for 2 hours, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound ATC-40 was obtained by freeze-drying (yield 91%).
  • Step 1 Weigh carboxylic acid-polyethylene glycol-carboxylic acid (17.8mg, 0.1mmol) and dissolve it in DMF (400 ⁇ L), add NHS (66.7mg, 0.24mmol), EDCI (42.4 ⁇ L, 0.24mmol), room temperature After reacting overnight, thioglycolic acid (20.8 ⁇ L, 0.3 mmol) and PB 7.4 buffer (400 ⁇ L) were added, and the reaction was carried out at room temperature for 1 h. Product 49 was obtained by semi-preparative isolation and purification (yield 79%). HRMS, calculated: C 10 H 14 O 8 S 2 :[M+H] + 327.0208, measured 327.0197.
  • Step 2 Weigh compound 49 (9.8 mg, 30 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add compound 17 (11.2 mg, 10 ⁇ mol) and triethylamine (125 ⁇ L, 0.9 mmol). After reacting at room temperature for 2 hours, monitor by LC-MS. The reaction was complete, and compound 50 was obtained by semi-preparative isolation and purification (yield 88%).
  • HRMS calculated value: C 66 H 104 N 10 O 18 S: [M+H] + 1357.7329, [M+2H] 2+ 679.3704, measured value 679.3759.
  • Step 3 Weigh compound 50 (6.5 mg, 4.76 ⁇ mol) Dissolve in DMF (200 ⁇ L), add HATU (2.7mg, 7.14 ⁇ mol), DIPEA (1.84 ⁇ L, 10.7 ⁇ mol) and AT-1 (10mg, 4.76 ⁇ mol), react at room temperature for 2h, monitor the reaction with LC-MS and use After semi-preparative isolation and purification, compound ATC-41 was obtained by freeze-drying (yield 85%).
  • HRMS calculated: C 159 H 237 N 41 O 39 S 3 : [M+H] + 3441.7062, [M+4H] 4+ 861.1824, [M+5H] 5+ 689.1475, measured 861.1859, 689.1512.
  • Step 1 Dissolve compound 47 (11.3 mg, 30 ⁇ mol) in DMF (200 ⁇ L), add compound 19 (23.9 mg, 10 ⁇ mol), triethylamine (6.94 ⁇ L, 50 ⁇ mol), react at room temperature for 20 min, then add thioglycolic acid (2.1 ⁇ L , 30 ⁇ mol), PB7.4 buffer, reaction at room temperature for 1 h, semi-preparative isolation and purification, and compound 51 was obtained (yield 81%).
  • HRMS calculated: C 122 H 214 N 12 O 44 S: [M+H] + 2584.4676, [M+3H] 3+ 862.1611, [M+4H] 4+ 646.8728, measured 862.1596, 646.8756.
  • Step 2 Weigh compound 51 (6.15 mg, 2.38 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (1.35 mg, 4.76 ⁇ mol), DIPEA (1.23 ⁇ L, 7.14 ⁇ mol) and AT-1 (5 mg, 2.38 ⁇ mol). , reacted at room temperature for 2 hours, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound ATC-42 was obtained by freeze-drying (yield 84%).
  • Step 1 Weigh compound 49 (4.9 mg, 15 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add compound 19 (12 mg, 5 ⁇ mol) and triethylamine (62.5 ⁇ L, 0.45 mmol). After reacting at room temperature for 3 hours, monitor by LC-MS. The reaction was complete, and compound 52 was obtained by semi-preparative isolation and purification (yield 85%).
  • HRMS calculated: C 124 H 218 N 12 O 45 S: [M+H] + 2628.4938, [M+3H] 3+ 876.8365, [M+4H] 4+ 657.8793, measured 876.8395, 657.8852.
  • Step 2 Weigh compound 52 (6.25 mg, 2.38 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (1.8 mg, 4.76 ⁇ mol), DIPEA (1.23 ⁇ L, 7.14 ⁇ mol) and AT-1 (5 mg, 2.38 ⁇ mol). , reacted at room temperature for 1 hour, and after LC-MS monitored that the reaction was complete, semi-preparative separation and purification was used, and compound ATC-43 was obtained by freeze-drying (yield 87%).
  • Step 2 Weigh compound 53 (8.74 mg, 8.9 ⁇ mol) and dissolve it in DMF, add HATU (6.8 mg, 17.8 ⁇ mol), DIPEA (4.6 ⁇ L, 27.6 ⁇ mol), compound 17 (10 mg, 8.9 ⁇ mol), and react at room temperature for 2 h. , after monitoring the reaction with LC-MS, add 20% piperidine, react at room temperature for 15 minutes, use semi-preparative separation and purification, and freeze-dry to obtain white powder 54 (yield 78%).
  • HRMS calculated for C 92 H 160 N 12 O 27 : [M+H] + 1866.1594, [M+3H] 3+ 622.7250, measured 622.7289.
  • Step 3 Weigh compound 54 (9.33 mg, 5 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add compound 49 (4.9 mg, 15 ⁇ mol) and triethylamine (20.8 ⁇ L, 0.15 mmol). After reacting at room temperature for 2 hours, LC-MS The reaction was monitored to be complete, and compound 55 was obtained by semi-preparative isolation and purification (yield 87%).
  • HRMS calculated: C 100 H 170 N 12 O 33 S: [M+H] + 2100.1792, [M+3H] 3+ 700.7316, [M+4H] 4+ 525.8007, measured 700.7359, 525.8095.
  • Step 4 Weigh compound 52 (5 mg, 2.38 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (1.8 mg, 4.76 ⁇ mol), DIPEA (1.23 ⁇ L, 7.14 ⁇ mol) and AT-1 (5 mg, 2.38 ⁇ mol). The reaction was carried out at room temperature for 1 hour. After the reaction was completed after monitoring by LC-MS, the compound was separated and purified using semi-preparative method and lyophilized to obtain compound ATC-44 (yield 86%).
  • Step 1 Weigh fluorenylmethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (20mg, 0.047mmol) and dissolve it in DMF, add HATU (35.8mg, 0.094mmol), DIPEA (24.2 ⁇ L, 0.14mmol), 3-Amino-1-propanesulfonic acid (6.5 mg, 0.047 mmol) was reacted at room temperature for 2 h. After the reaction was completed after LC-MS monitoring, it was separated and purified using semi-preparation and lyophilized to obtain white powder 56 (yield 85%). HRMS, calculated: C 27 H 34 N 2 O 8 S: [M+H]+547.2114, measured 547.2156.
  • Step 2 Weigh compound 56 (4.9 mg, 8.9 ⁇ mol), add 200 ⁇ L of dichloromethane, 180 ⁇ L of trifluoroacetic acid, and 20 ⁇ L of isopropylsilane. React at room temperature for 1 hour. Dry the solvent with N 2 to obtain compound 57. Dissolve in DMF. Add HATU (6.8 mg, 17.8 ⁇ mol), DIPEA (4.6 ⁇ L, 27.6 ⁇ mol), and compound 17 (10 mg, 8.9 ⁇ mol), and react at room temperature for 2 hours. After LC-MS monitors that the reaction is complete, add 20% piperidine, and react at room temperature for 15 minutes.
  • Step 3 Weigh compound 58 (7.2 mg, 10 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add compound 49 (9.8 mg, 30 ⁇ mol) and triethylamine (41.6 ⁇ L, 0.30 mmol). After reacting at room temperature for 2 hours, perform LC-MS The reaction was monitored to be complete, and compound 59 was obtained by semi-preparative isolation and purification (yield 82%). HRMS, calculated for C 74 H 118 N 12 O 23 S 2 :[M+H] + 1607.7952,[M+2H] 2+ 804.4015, measured 804.4110.
  • Step 4 Weigh compound 59 (3.8 mg, 2.38 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (1.8 mg, 4.76 ⁇ mol), DIPEA (1.23 ⁇ L, 7.14 ⁇ mol) and AT-1 (5 mg, 2.38 ⁇ mol). , react at room temperature for 1 hour, after LC-MS monitoring the reaction is complete, use semi-preparative isolation and purification, and lyophilize to obtain the compound ATC-45 (90% yield). HRMS, calculated: C 167 H 251 N 43 O 44 S 4 : [M+H] + 3691.7686, [M+5H] 5+ 739.1600, measured 739.1642.
  • Step 1 Weigh Fmoc-PEG 4 -COOH (8.8 mg, 17.8 ⁇ mol), add HATU (13.6 mg, 35.6 ⁇ mol), DIPEA (9.2 ⁇ L, 55.2 ⁇ mol), compound 17 (20 mg, 17.8 ⁇ mol), and react at room temperature for 2 hours. , after LC-MS monitors that the reaction is complete, add 20% piperidine, react at room temperature for 15 minutes, use semi-preparative separation and purification, and freeze-dry to obtain white powder 60 (yield 87%).
  • HRMS calculated: C 69 H 115 N 11 O 17 : [M+H] + 1370.8550, measured 1370.8547.
  • Step 2 Weigh 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)glutaric acid (10 mg, 27.1 ⁇ mol) Dissolve in DMF (300 ⁇ L), add NHS (3.8 mg, 32.5 ⁇ mol), DCC (6.7 mg, 32.5 ⁇ mol), and react at 4°C overnight to obtain crude compound 61. Take 80 ⁇ L of the reaction solution, add 80 ⁇ L of DMF solution of compound 60 (10 mg, 7.3 ⁇ mol), and triethylamine (3 ⁇ L, 21.9 ⁇ mol), and react at room temperature for 2 hours. After the reaction is complete after LC-MS monitoring, add 40 ⁇ L of piperidine, and react at room temperature for 15 min.
  • Step 3 Weigh compound 62 (7.12 mg, 2.5 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add compound 49 (1.7 mg, 5 ⁇ mol) and triethylamine (16.6 ⁇ L, 0.12 mmol). After reacting at room temperature for 5 hours, LC- MS monitored the reaction to be complete, and semi-preparative isolation and purification was performed to obtain compound 63 (yield 70%).
  • HRMS calculated for C 151 H 245 N 23 O 42 S: [M+H] + 3085.7541, [M+3H] 3+ 1029.2566, [M+4H] 4+ 772.1944, measured, 1029.1856,772.2015.
  • Step 4 Weigh compound 63 (5 mg, 1.62 ⁇ mol) and dissolve it in DMF (200 ⁇ L). Add HATU (1.2 mg, 3.24 ⁇ mol), DIPEA (0.84 ⁇ L, 4.86 ⁇ mol) and AT-1 (5 mg, 2.38 ⁇ mol). The reaction was carried out at room temperature for 2 hours. After the reaction was completed after monitoring by LC-MS, the compound was separated and purified using semi-preparative method and lyophilized to obtain compound ATC-46 (yield 88%). HRMS, calculated: C 244 H 378 N 54 O 63 S 3 :[M+H] + 5169.7275,[M+5H] 5+ 1034.7517, measured 1034.7451.
  • trastuzumab (5mg/mL) and compound ATC-2 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), place it at 37°C for reaction for 2h, and monitor with LC-MS Complete response. After protein A purification, ultrafiltration tube concentration, and then replacement with 1 ⁇ PBS buffer, biotinylated antibody Ab-1 was obtained. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two biotins was observed at 146355 ( Figure 1a).
  • Nano-HPLC EASY-nLC 1200 UHPLC system (ThermoFisher Scientific)
  • Mass spectrometer Q Exactive HF-X mass spectrometer (ThermoFisher Scientific)
  • Mobile phase A aqueous solution containing 0.1% formic acid
  • Mobile phase B acetonitrile solution containing 0.1% formic acid
  • Raw data were analyzed using the protein identification software Mascot 2.3.
  • the trastuzumab sequence was added to the peptide identification database provided to the search engine. Trypsin was set as the digestive enzyme (C-terminal cleavage to Lys and Arg residues), allowing up to two missing cleavages. Oxidation of methionine residues (+15.9949Da), novel modifications to lysine (+226.0776Da), and deamidation of asparagine (+0.9840Da) were set as variable modifications. Methyl carbamate on cysteine (+57.0215Da) was searched as a fixed modification. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.05 Da, respectively. All modified peptides were manually verified.
  • trastuzumab (5mg/mL) and compound ATC-1 (5eq, 0.168mM) into NaOAc buffer solution (50mM, pH 5.5, containing 20% DMF), incubate at 37°C for 30min, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain biotinylated antibody Ab-2. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two azidoacetic acids was observed at 146037 ( Figure 2a).
  • trastuzumab (5mg/mL) and compound ATC-34 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete reaction. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain FITC site-specific modified antibody Ab-3. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two fluoresceins was observed at 147150 ( Figure 3).
  • Example 50 Preparation and characterization of methylcyclopropene site-directed modified antibody Ab-4
  • trastuzumab (5mg/mL) and compound ATC-35 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response.
  • the solution was concentrated through an ultrafiltration tube and then replaced with 1 ⁇ PBS buffer to obtain the methylcyclopropene site-specific modified antibody Ab-4.
  • the mass was measured by ESI-TOFMS.
  • the peak of the raw material trastuzumab was observed at 145870.
  • the peak of the product coupled with two methylcyclopropenes was observed at 146524 ( Figure 4).
  • ADC-1 Dilute ADC-1 to 5 mg/mL with 1 ⁇ PBS and inject 5 ⁇ L.
  • the detection wavelength is 280nm.
  • the retention time of 9.391 min is considered to be an impurity peak introduced by the drug linker D1 that is not pure enough, and the retention time of 10.354 min is considered to be a compound that introduces two drug linkers D1 into trastuzumab ( Figure 7d).
  • ADC-1 was diluted to 1mg/mL with 1 ⁇ PBS, and heated in a PCR machine at 60°C for 48h, 0h, 6h, 12h, 24h, and 48h. Samples were taken for SEC analysis, and 15 ⁇ L was sampled at each time point.
  • the SEC column uses BioCore SEC-300column (7.8 ⁇ 300mm 5 ⁇ m).
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.907 min is considered to be the peak of monomer (Figure 7e).
  • ADC-2 The preparation conditions of ADC-2 are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the peak of the raw material trastuzumab was observed at 145870.
  • the peak of the product coupled with two drug linkers D2 was observed at 148636 ( Figure 8a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and product.
  • the heavy chain of the raw material had a peak at 49499.
  • a drug linker D2 was introduced into the heavy chain and a peak was observed at 50811 ( Figure 8b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and product.
  • the Fc peak of the raw material was observed at 24136.
  • a drug linker was introduced into the Fc region and a peak was observed at 25518 ( Figure 8c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 9.071 min is considered to be a compound that introduces one drug linker D2 into trastuzumab, and the retention time of 10.170 min is considered to be a compound that introduces two drug linkers D2 into trastuzumab ( Figure 8d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.923 min is considered to be the peak of monomer (Figure 8e).
  • ADC-3 The preparation conditions of ADC-3 are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the peak of the raw material trastuzumab was observed at 145870.
  • the peak of the product coupled with two drug linkers D3 was observed at 151180 ( Figure 9a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and product.
  • the heavy chain of the raw material had a peak at 49499.
  • a drug linker D3 was introduced into the heavy chain and a peak was observed at 52154 ( Figure 9b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and product.
  • the raw material Fc peak was observed at 24136.
  • a drug linker D3 was introduced into the Fc region and a peak was observed at 26790 (Figure 9c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 8.846 min is considered to be a compound that introduces one drug linker D3 into trastuzumab
  • the retention time of 9.611 min is considered to be a compound that introduces two drug linkers D3 into trastuzumab ( Figure 9d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.850 min is considered to be the peak of monomer (Figure 9e).
  • ADC-4 The preparation conditions of ADC-4 are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the peak of the raw material trastuzumab was observed at 145870.
  • the peak of the product coupled with two drug linkers D4 was observed at 148377 ( Figure 10).
  • trastuzumab (5mg/mL) and compound ATC-6 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 3h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-5. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two drug linkers D5 was observed at 148311 ( Figure 11a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and the product.
  • the peak of the heavy chain of the raw material was observed at 49499.
  • a drug linker D5 was introduced into the heavy chain and a peak was observed at 50718 ( Figure 11b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and product.
  • the Fc peak of the raw material was observed at 24136.
  • a drug linker D5 was introduced into the Fc region and a peak was observed at 25355 (Figure 11c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 8.687 min is considered to be a compound that introduces one drug linker D5 into trastuzumab, and the retention time of 9.676 min is considered to be a compound that introduces two drug linkers D5 into trastuzumab ( Figure 11d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.940 min is considered to be the peak of monomer (Figure 11e).
  • trastuzumab (5mg/mL) and compound ATC-7 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete reaction. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-6. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two drug linkers D6 was observed at 148139 ( Figure 12a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and product.
  • the heavy chain of the raw material had a peak at 49499.
  • a drug linker D6 was introduced into the heavy chain and a peak was observed at 50633 ( Figure 12b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and product.
  • the Fc peak of the raw material was observed at 24136.
  • a drug linker D6 was introduced into the Fc region and a peak was observed at 25270 ( Figure 12c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 8.623 min is considered to be a compound that introduces one drug linker D6 into trastuzumab, and the retention time of 9.565 min is considered to be a compound that introduces two drug linkers D6 into trastuzumab ( Figure 12d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.883 min is considered to be the peak of monomer (Figure 12e).
  • trastuzumab (5mg/mL) and compound ATC-8 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 10h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-7. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two drug linkers D7 was observed at 147938 ( Figure 13a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and the product.
  • the peak of the heavy chain of the raw material was observed at 49499.
  • a drug linker D7 was introduced into the heavy chain and a peak was observed at 50533 ( Figure 13b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and the product.
  • the Fc peak of the raw material was observed at 24136.
  • a drug linker D7 was introduced into the Fc region and a peak was observed at 25170 (Figure 13c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 8.623 min is considered to be a compound that introduces two drug linkers D7 into trastuzumab ( Figure 13d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.690 min is considered to be the peak of monomer (Figure 13e).
  • trastuzumab (5mg/mL) and compound ATC-9 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-8. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145870. Regarding the reaction product, the peak of the product coupled with two drug linkers D8 was observed at 148225 ( Figure 14a).
  • TCEP processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same light chain peak was observed for the raw material and product.
  • the heavy chain of the raw material had a peak at 49499.
  • a drug linker D8 was introduced into the heavy chain and a peak was observed at 50677 ( Figure 14b) .
  • IdeS protease processing conditions are the same as ADC-1.
  • the mass was measured by ESI-TOFMS.
  • the same Fab peak was observed for the raw material and product.
  • the Fc peak of the raw material was observed at 24136.
  • a drug linker D8 was introduced into the Fc region and a peak was observed at 25315 ( Figure 14c).
  • the HIC analysis operation is the same as ADC-1.
  • the retention time of 8.370 min is considered to be a compound that introduces one drug linker D8 into trastuzumab, and 9.063 min is considered to be a compound that introduces two drug linkers D8 into trastuzumab ( Figure 14d).
  • the aggregation stability analysis operation is the same as ADC-1.
  • the retention time of 5.4-8.0 min is considered to be the peak of aggregates, and the retention time of 8.623 min is considered to be the peak of monomer (Figure 14e).
  • Toripalimab (5mg/mL) and compound ATC-7 (10eq, 0.334mM) were added to PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), and incubated at 37°C for 2h, LC-MS Monitor response for completeness. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-12. The mass was measured by ESI-TOFMS. The peak of the raw material toripalimab was observed at 147729. Regarding the reaction product, the peak of the product coupled with two drug linkers D6 was observed at 149996 ( Figure 18).
  • panitumumab (2mg/mL) and compound ATC-7 (10eq, 0.134mM) into 1x PBS buffer solution (containing 20% DMF), and incubate at 37°C for 2 hours.
  • LC-MS monitors the reaction to be complete. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-13. The mass was measured by ESI-TOFMS. The peak of the raw material panitumumab was observed at 144723. Regarding the reaction product, the peak of the product coupled with two drug linkers D6 was observed at 146992 ( Figure 19).
  • LC-MS monitors the reaction to be complete. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-13. The mass was measured by ESI-TOFMS. The peak of the raw material nivolumab was observed at 144016. Regarding the reaction product, the peak of the product coupled with two drug linkers D6 was observed at 146313 ( Figure 20).
  • trastuzumab (5mg/mL) and compound ATC-40 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-15. The mass was measured by ESI-TOFMS, and the peak of the raw material trastuzumab was observed at 145860, and the peak of the reaction product was observed at 148299 ( Figure 21a).
  • the HIC analysis operation is the same as ADC-1 ( Figure 21b).
  • trastuzumab (5mg/mL) and compound ATC-41 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-16. via ESI-TOFMS The mass was measured and a peak was observed at 145860 for the raw material trastuzumab and at 148385 for the reaction product (Fig. 22a).
  • the HIC analysis operation is the same as ADC-1 ( Figure 22b).
  • trastuzumab (5mg/mL) and compound ATC-42 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete reaction. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-17. The mass was measured by ESI-TOFMS. A peak was observed at 145860 for the raw material trastuzumab, and a peak at 150841 for the reaction product ( Figure 23a).
  • the HIC analysis operation is the same as ADC-1 ( Figure 23b).
  • Example 70 Preparation and characterization of one-step ADC-18
  • trastuzumab (5mg/mL) and compound ATC-43 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-18. The mass was measured by ESI-TOFMS. A peak was observed at 145860 for the raw material trastuzumab, and a peak was observed at 150930 for the reaction product (Figure 24a).
  • the HIC analysis operation is the same as ADC-1 ( Figure 24b).
  • trastuzumab (5mg/mL) and compound ATC-44 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at 37°C for 2h, and monitor by LC-MS Complete response. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-19. The mass was measured by ESI-TOFMS, and the peak of the raw material trastuzumab was observed at 145860, and the peak of the reaction product was observed at 149875 ( Figure 25a).
  • the HIC analysis operation is the same as ADC-1 ( Figure 25b).
  • trastuzumab (5mg/mL) and compound ATC-45 (10eq, 0.334mM) into PIPES buffer solution (50mM, pH 7.4, containing 20% DMF), incubate at room temperature for 1 hour, and monitor the reaction with LC-MS completely. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-20. The mass was measured by ESI-TOFMS. The peak of the raw material trastuzumab was observed at 145860, and the peak of the reaction product was observed at 148882 ( Figure 26).
  • trastuzumab (5mg/mL) and compound ATC-46 (10eq, 0.334mM) into sodium acetate buffer solution (50mM, pH 5.5, containing 20% DMF), incubate at 37°C for 1 hour, and perform LC-MS Monitor response for completeness. Concentrate through an ultrafiltration tube and replace with 1 ⁇ PBS buffer to obtain ADC-21. The mass was measured by ESI-TOFMS. A peak was observed at 145860 for the raw material trastuzumab, and a peak was observed at 151842 for the reaction product (Figure 27).
  • MTT method was used to measure the cell activity and toxicity of some of the above ADCs. Trypsinize the cells, collect by centrifugation after termination, resuspend the cells in 1-3 mL of culture medium, mix and count the cells, and supplement the culture medium to adjust the concentration of the cell suspension. Use a 96-well plate for cell experiments. Add 90 ⁇ L of cell suspension to each well so that the density of cells to be tested is ⁇ 6000/well. Set up 3 duplicate wells. Fill the edge holes with 100 ⁇ L 1x PBS and set zero adjustment holes (90 ⁇ L culture medium).
  • control wells (90 ⁇ L cells, no drugs), incubate in 5% CO 2 in a 37°C incubator for 12 hours to allow cells to completely adhere to the wall.
  • concentration gradients for each sample, the highest concentration is 1 ⁇ M, 5-fold gradient dilution, add 10 ⁇ L of the corresponding concentration gradient ADC molecules to each well, and add 10 ⁇ L of culture medium to each of the 6 control wells.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了亲和片段导的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用。具体地，本发明提供了一种带有配体亲和导向基团的偶联物，所述偶联物如式I所示：AT-CL-R(I)；其中，AT为针对目标蛋白(TP)的亲和性部分；CL为可裂解片段，其具有自裂解反应性；R为需要被修饰到所述目标蛋白的基团。

Description

亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用 技术领域
本发明属于药物化学领域，具体涉及一类亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用。
背景技术
抗体-药物偶联物是通过化学链接将具有生物活性的细胞毒药物分子连接到抗体上，通过抗体的靶向作用将细胞毒小分子运输到目标细胞发挥作用的一类靶向药物。由于随机偶联小分子药物不利于药代动力学药效学的研究，且临床评价难以得到均一数据，生产过程中不同批次质量也难以一致，因此发展了许多定点定量引入小分子药物的偶联策略，呈现出很好的应用前景。
抗体-药物偶联物中的抗体主要是IgG类抗体，拥有可变的Fab区和恒定可结晶的Fc结构域。在Fc结构域定点连接小分子药物不影响抗体-抗原的识别，同时表现出更好的药效和稳定性。因此，在Fc结构域定点偶联小分子药物是重要的研究方向。目前，针对抗体Fc结构域对抗体进行选择性修饰的技术方法有很多，其中基于Fc配体导向实现特定氨基酸选择性修饰的研究为定点ADC药物研究提供了很好的方法。然而，基于Fc配体导向的定点修饰方法，或不能脱除Fc配体，或脱除过程复杂，不利于抗体的稳定性和安全性。因此，发展一种高效的Fc配体导向的复合物，直接实现天然抗体的定点定量修饰，具有重要的研究意义。
发明内容
本发明的目的就是提供一种亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用。具体地，本发明提供了一类硫酯结构，利用该硫酯结构，制备得到基于硫酯的酰基转移试剂，该试剂在亲和片段导向下，可以实现抗体的定点定量的修饰，本发明涉及该片段的设计、合成以及它们的用途。
在本发明的第一方面，提供了一种带有配体亲和导向基团的偶联物，其特征在于，所述偶联物如式I所示：
AT-CL-R   (I)
其中，
AT为针对目标蛋白(TP)的亲和性部分；
CL为可裂解片段；并且所述CL中存在如下所示的二价片段
其中，A1各自独立地为任选取代的C1-10亚烷基(较佳地，C1-3亚烷基)、任选取代的C6-10芳基、任选取代的5至10元杂芳基：
R为需要被修饰到所述目标蛋白的基团(该基团可以是任意所需基团或含功能性物质的基团，在R基团中可以包含单一的所需基团，也可以包含2种以上的相同或不同所需基团或功能性物质的)；
除非特别说明，所述的取代是指基团中一个或多个H被选自下组的取代基取代：卤素(如F、Cl、Br、I)、C1-6烷基(较佳地，C1-4烷基，如甲基、乙基)、C1-6卤代烷基(较佳地，C1-4卤代烷基)。
在另一优选例中，所述可裂解片段具有自裂解反应性(例如在特定反应条件下/与特定反应物或反应基团接触时可裂解)。
在另一优选例中，所述的亲和性部分衍生自可与目标蛋白形成可逆共价键的小分子化合物，或者衍生自对目标蛋白具有亲和力的蛋白或多肽结构。
在另一优选例中，中，A1为靠近AT的一端，CO为靠近R的一端。
在另一优选例中，所述偶联物为用于目标蛋白(如抗体或Fc融合蛋白)定点修饰的酰基转移试剂。
在另一优选例中，所述针对蛋白的亲和性部分是指针对目标蛋白的具有亲和性的或能够可逆结合(能够可逆共价结合)的部分。
在另一优选例中，所述的目标蛋白是抗体或融合蛋白。在另一优选例中，所述抗体为含Fc结构域的抗体或者融合蛋白。
在另一优选例中，所述目标蛋白是抗体，且所述抗体为含Fc结构域的抗体；此时对应的AT为可以与抗体可逆共价结合的小分子化合物，或具有亲和力的蛋白或多肽结构。
在另一优选例中，所述抗体包括：单克隆抗体、双功能抗体、单克隆抗体、含有Fc片段的纳米抗体、Fc融合蛋白，或其组合。
在另一优选例中，所选抗体包括：曲妥珠单抗、帕妥珠单抗、利妥昔单抗、西妥昔单抗、莫罗单抗、吉妥珠单抗、阿昔单抗、达利珠单抗、阿达木单抗、帕利珠单抗、巴利昔单抗、贝伐珠单抗、帕尼单抗、尼妥珠单抗、德尼单抗、迪西妥单抗、雷莫尼单抗、耐昔妥珠单抗、易普利姆玛、达雷木单抗、本妥昔单抗、阿仑单抗、埃罗妥珠单抗、博纳吐单抗、纳武单抗、派姆单抗、阿特珠单抗、阿维鲁单抗、度伐鲁单抗、特瑞普利单抗、卡妥索单抗、贝林妥欧单抗、艾美赛珠单抗、埃万妥单抗(Rybrevant)，或其组合。
在另一优选例中，所述抗体包括：曲妥珠单抗(Trastuzumab)、利妥昔单抗(Rituximab)、帕妥珠单抗(Pertuzumab)、贝伐单抗(Bevacizumab)、特瑞普利单抗(Toripalimab)，纳武单抗(Nivolumab,IgG4)，帕尼单抗(Panituzumab, IgG2)或其组合。
在另一优选例中，AT为衍生自可与目标蛋白可逆结合的小分子化合物(较佳地，形成可逆共价结合的小分子化合物)的部分，或者衍生自对目标蛋白具有亲和力的蛋白或多肽的部分。
在另一优选例中，AT为衍生自可与抗体可逆结合的小分子化合物(较佳地，形成可逆共价结合的小分子化合物)的部分，或者衍生自对抗体具有亲和力的蛋白或多肽(较佳地，衍生自Fc结合肽；更佳地，衍生自来源于ProteinA或ProteinG的且与Fc(Fc段)具有结合能力的多肽片段)的部分。
在另一优选例中，AT衍生自蛋白A(与Fc亲和的ProteinA)中的序列。
在另一优选例中，AT衍生自与抗体Fab具有亲和力的结合肽。
在另一优选例中，所述多肽为环肽。
在另一优选例中，AT衍生自选自下组的肽：Fc-III肽(如DCAWHLGELVWCT(SEQ ID No.2))、Fc结合肽(如GPDCAYHRGELVWCTFH(SEQ ID No.3)、RGNCAYHRGQLVWCTYH(SEQ ID No.4)、CDCAWHLGELVWCTC(SEQ ID No.5)等)，或其组合。
在另一优选例中，AT为衍生自包含式II所示序列的Fc结合多肽的核心序列：
(Xs1)t1-Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Ys2)t2   (II)
其中，
X所在位置为多肽的N端，Y所在位置为多肽的C端；
s1＝0、1、2或3；
s2＝0、1、2或3；
X和Y各自独立地为氨基酸残基；
t1和t2各自独立为0-10的整数(较佳地，t1和t2各自独立为0、1、2或3，更佳地，1和t2各自独立为0或1)；
Cys为半胱氨酸残基；
Aa1、Aa2、Aa3、Aa5、Aa7、Aa8和Aa9各自独立地为氨基酸残基；
Aa4和Aa6各自独立地为氨基酸残基，并且Aa4和Aa6中的至少一个为侧链含NH2基团的氨基酸残基(如赖氨酸残基(K)等)或侧链含-COOH基团的氨基酸残基(如天冬氨酸残基(D)、谷氨酸残基(E)等)。
在另一优选例中，Aa1、Aa2、Aa3、Aa4、Aa5、Aa6、Aa7、Aa8和Aa9不为半胱氨酸残基。
在另一优选例中，所述的衍生是指Aa4或Aa6中侧链-NH2脱去一个H形成-NH-，或侧链-COOH与氨基形成的-CONH-(即AT通过Aa4或Aa6的侧链与CL连接)。
在另一优选例中，Aa6为侧链含NH2基团的氨基酸残基或侧链含-COOH基团的氨基酸残基。
在另一优选例中，所述的衍生是指Aa6中侧链-NH2脱去一个H形成-NH-，或侧链-COOH与氨基形成的-CONH-(即AT通过Aa6的侧链与CL连接)。
在另一优选例中，所述氨基酸残基为衍生自天然或非天然氨基酸的的氨基酸残基。
在另一优选例中，AT衍生自环肽。
在另一优选例中，AT衍生自式II所示序列中两个Cys之间形成-S-S-键从而形成的环肽。
在另一优选例中，Xs1和Ys2各自独立地为无或1-3个连续的相同的或不同的氨基酸残基；较佳地，Ys2和Ys2各自独立地为1-3个连续的相同的或不同的氨基酸残基(即s1＝1、2或3，s2＝1、2或3)。
在另一优选例中，
Aa1为丙氨酸残基(A)、丝氨酸残基(S)、或苏氨酸残基(T)；和/或
Aa2为酪氨酸残基(Y)、组氨酸残基(H)、或色氨酸残基(W)；和/或
Aa3为组氨酸残基(H)、苯丙氨酸残基(F)、酪氨酸残基(Y)、色氨酸残基(W)、精氨酸残基(R)或甘氨酸残基(G)；和/或
Aa5为甘氨酸残基(G)、丝氨酸残基(S)、天冬酰胺残基(N)、谷氨酰胺残基(Q)、天冬氨酸残基(D)、谷氨酸残基(E)、苯丙氨酸残基(F)、酪氨酸残基(Y)、色氨酸残基(W)、组氨酸残基(H)、苏氨酸残基(T)、亮氨酸残基(L)、丙氨酸残基(A)、缬氨酸残基(V)、异亮氨酸残基(I)、或精氨酸残基(R)；和/或
Aa7为亮氨酸残基(L)、异亮氨酸残基(I)、缬氨酸残基(V)、丙氨酸残基(A)、谷氨酰胺残基(Q)、或谷氨酸残基(E)；和/或
Aa8为缬氨酸(V)、异亮氨酸残基(I)、或亮氨酸残基(L)；和/或
Aa9为色氨酸残基(W)、或苯丙氨酸残基(F)。
在另一优选例中，所述侧链含-NH2基团的氨基酸残基为如下所示
其中，*是指与CL连接的部分；LA为取代或未取代的C1-8亚烷基；较佳地，为-(CH2)n1-且n1＝1、2、3、4、5或6；较佳地，n1＝3、4或5。
在另一优选例中，所述侧链含-COOH基团的氨基酸残基为如下所示
其中，*是指与-NH-CL连接的部分；LA为取代或未取代的C1-8亚烷基；较佳地，为-(CH2)n1-且n1＝1、2、3、4、5或6；较佳地，n1＝2、3、4或5。
在另一优选例中，所述侧链含-NH2基团的氨基酸残基为赖氨酸残基(K)。
在另一优选例中，所述侧链含-COOH基团的氨基酸残基为天冬氨酸残基(D)、谷氨酸残基(E)。
在另一优选例中，Aa4为侧链含-NH2基团的氨基酸残基，且Aa6为谷氨酰胺残基(Q)、谷氨酸残基(E)、组氨酸残基(H)、天冬酰胺残基(N)、脯氨酸残基(P)、天冬氨酸残基(D)、赖氨酸残基(K)、或甘氨酸残基(G)；或者，Aa6为侧链含-NH2基团的氨基酸残基，且Aa4为精氨酸残基(R)、亮氨酸残基(L)、赖氨酸残基(K)、天冬氨酸残基(D)、谷氨酸残基(E)、2-氨基辛二酸或二氨基丙酸。
在另一优选例中，Aa4为侧链含-COOH基团的氨基酸残基、2-氨基辛二酸或二氨基丙酸，且Aa6为谷氨酰胺残基(Q)、谷氨酸残基(E)、组氨酸残基(H)、天冬酰胺残基(N)、脯氨酸残基(P)、天冬氨酸残基(D)、赖氨酸残基(K)、或甘氨酸残基(G)；或者，Aa6为侧链含-COOH基团的氨基酸残基、2-氨基辛二酸或二氨基丙酸，且Aa4为精氨酸残基(R)、亮氨酸残基(L)、赖氨酸残基(K)、天冬氨酸残基(D)、谷氨酸残基(E)、2-氨基辛二酸或二氨基丙酸。
在另一优选例中，式II所示序列为HYTCWVLKGRHYACNGR(SEQ ID No.6)、HYTCWVLDGRHYACNGR(SEQ ID NO.7)、或HYTCWVLEGRHYACNGR(SEQ ID No.8)。
在另一优选例中，AT为与蛋白具有可逆共价相互作用的小分子片段，较佳地，AT选自下组：
在另一优选例中，CL如下所示
其中，
*代表与R连接的位置
W1选自下组：无(单键)、-NH-、-C(O)-、-C(O)-NH-和-NH-C(O)-；
W2为无(单键)、-NH-、或-C(O)-；
L1为无(单键)或二价连接基团；
A1如前定义。
在另一优选例中，L1为无(单键)或由选自下组的一个或多个(较佳地，1-10个；更佳地，1、2、3、4、或5个，最佳地，1、2或3个)单元构成的二价连接基团：氨基酸残基、任选取代的C1-4亚烷基、-(CH2-NH-CO)-、-(CH2-CH2-CO)-、-(CH2-CH2-O)-、-(CH2-O-CH2)-。
在另一优选例中，W2为-NH-或-C(O)-。
在另一优选例中，W1和L1为无。
在另一优选例中，W1为-C(O)-和L1为C1-10亚烷基(较佳地，C1-3亚烷基)。
在另一优选例中，CL选自组1，并且组1包括如下基团：
其中，n为1-10的整数；较佳地，n＝1、2、3、4或5。
在另一优选例中，所述的偶联物如式III所示，
其中，为-W2-L1-W1-A1-；W2、L1、W1、A1、AT和R如前定义，
在另一优选例中，为-CO-L1-W1-A1-。
在另一优选例中，R为包含一个或多个选自下组的活性基团的部分：可被进一步修饰的活性基团、具有生物活性和/或可检测的活性基团。
在另一优选例中，所述可被进一步修饰的活性基团是指可进行生物正交反应的活性基团(又称为生物正交基团)；优选地，所述可进行生物正交反应的活性基团包括：叠氮基(-N3)、醛基(-CHO)、硫醇基(-SH)、炔基(如C2-C6炔基、环张力炔基BCN、DBCO等)、烯基(如C2-C6烯基)、卤素(如F、Cl、Br和I)、四嗪基、硝酮基、羟胺基、腈基、肼基、酮基、硼酸残基、氰基苯并噻唑基、烯丙基基、膦基、马来酰亚胺基、二硫基、硫酯基、α-卤代羰基、异腈基、斯德酮基、硒残基、共轭二烯基、磷酸基、环炔基(如C3-C7环炔基)及环烯基(如C3-C7环烯基)中的一种或多种。
在另一优选例中，所述具有生物活性和/或可检测的活性基团为小分子药物、细胞毒、或其他功能性分子中的任意一种或多种。
在另一优选例中，所述小分子药物为放射性治疗物、或分子影像试剂。
在另一优选例中，所述具有生物活性和/或可检测的活性基团衍生自选自下组的化合物：美登素、DM-1、DM-4、MMAE(Monomethyl auristatin E)、MMAF、SN-38、Dxd、PBD及其类似物、鹅膏蕈碱、长春新碱、长春碱、长春瑞滨、VP-16、喜树碱、紫杉醇、多烯紫杉醇、埃博霉素A、埃博霉素B、诺考达唑、秋水仙碱、雌莫司汀、西马多丁、艾榴塞洛素、荧光试剂、单糖、二糖、寡糖、多聚乙二醇(PEG)、细胞毒、免疫激动剂，或者放射性治疗物、分子影像试剂。
在另一优选例中，R为-L2-(L3-B)o，其中，下标o为1-10的整数，L2和L3为各自 独立地为无或连接片段，B各自独立地为相同或不同的可被进一步修饰的活性基团(如生物正交基团)。
在另一优选例中，R为-L2-(L3-D)o，其中，下标o为1-10的整数，L2和L3各自独立地为无或连接片段，D各自独立地为相同或不同的具有生物活性和/或可检测的活性基团(如小分子化合物)。
在另一优选例中，o为1。
在另一优选例中，o为2、3、4、或5。
在另一优选例中，L2和L3各自独立地为无或由选自下组的一个或多个(如1-10个，较佳地，1、2、3、4、5或6个)单元结构组成的连接片段：CH2OCH2、C1-C4亚烷基、CO、NH、氨基酸残基。
在另一优选例中，L2和L3中不含由正交反应形成的连接基团(例如，如式上定义的B经正交反应形成的连接基团)。
在另一优选例中，所述的偶联物如式III-A1、III-A2或III-A3所示，
其中，
标注有1、2、3、4、5、7、8和9的圆圈分别如式II中Aa1、Aa2、Aa3、Aa4、Aa5、Aa7、Aa8、和Aa9的定义；
靠近标注有1的圆圈的末端为多肽N端，靠近标注有9的圆圈的末端为多肽C端；
Ac为N端保护基或不存在；
各个空白圆圈如式II中的X和Y定义；
为-W2-L1-W1-A1-；较佳地，为-CO-L1-W1-A1-；
圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
圆圈K为赖氨酸残基的主链部分(该氨基酸侧链已示出)；圆圈D为天冬氨酸残基(D)的主链部分(该氨基酸侧链已示出)、圆圈E为谷氨酸残基(E)的主链部分(该氨基酸侧链已示出)；
R如前定义。
在另一优选例中，
空白圆圈(如中的任一一个圆圈)各自独立地为无(不存在)或氨基 酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有数字的圆圈(即圆圈1、圆圈2、圆圈3、圆圈4、圆圈5、圆圈7、圆圈8、和圆圈9)各自独立地为氨基酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有C的圆圈(圆圈C)为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
标注有K的圆圈(圆圈K)为赖氨酸残基的主链部分；标注有D的圆圈(圆圈D)为天冬氨酸残基(D)的主链部分、标注有E的圆圈(圆圈E)为谷氨酸残基(E)的主链部分；
为-W2-L1-W1-A1-；较佳地-CO-L1-W1-A1-(即连接在上述式II多肽链的Aa6位点)；
R如上所述；
Ac为N端保护基或不存在(较佳地，为乙酰基)，连接在多肽的N端；和
-CONH2为酰胺基，为多肽的C端；
当CL连接于环肽Aa6位置时，结合优选的连接子结构，可以实现任意R基团或物质修饰到靶蛋白上(优选地，靶蛋白为抗体)。
在另一优选例中，所述偶联物所述偶联物如式III-A1-1、III-A2-1或III-A3-1所示，
其中，
圆圈R为多肽N端，圆圈H为多肽C端；
Ac为N端保护基或不存在；
为-W2-L1-W1-A1-(较佳地-CO-L1-W1-A1-)；
圆圈R为精氨酸残基、G为甘氨酸残基、N为天冬酰胺残基、A为丙氨酸残基、Y为酪氨酸残基、H为组氨酸残基、L为亮氨酸残基、V为缬氨酸残基、W为色氨酸残基、T为苏氨酸残基；
圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
圆圈K为赖氨酸残基的主链部分；圆圈D为天冬氨酸残基(D)的主链部分、 圆圈E为谷氨酸残基(E)的主链部分；
R如前定义。
在另一优选例中，式III-A1为
其中，除标注有C和K的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。
在另一优选例中，式III-A2为
其中，除标注有C和D的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。
在另一优选例中，式III-A3为
其中，除标注有C和E的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。
在另一优选例中，所述的偶联物如式III-B1、III-B2或III-B3所示，
其中，
标注有1、2、3、5、6、7、8和9的圆圈分别如式II中Aa1、Aa2、Aa3、Aa5、Aa6、Aa7、Aa8、和Aa9的定义；
靠近标注有1的圆圈的末端为多肽N端，靠近标注有9的圆圈的末端为多肽C端；
Ac为N端保护基或不存在；
各个空白圆圈如式II中的X和Y定义；
为-W2-L1-W1-A1-；较佳地-CO-L1-W1-A1-；
圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
圆圈K为赖氨酸残基的主链部分(该氨基酸侧链已示出)；圆圈D为天冬氨酸残基(D)的主链部分(该氨基酸侧链已示出)、圆圈E为谷氨酸残基(E)的主链部分(该氨基酸侧链已示出)；
R如前定义。
在另一优选例中，
空白圆圈各自独立地为无(不存在)或氨基酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有数字的圆圈(即圆圈1、圆圈2、圆圈3、圆圈5、圆圈6、圆圈7、圆圈8、和圆圈9)各自独立地为氨基酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有C的圆圈(圆圈C)为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
标注有K的圆圈(圆圈K)为赖氨酸残基的主链部分；标注有D的圆圈(圆圈D)为天冬氨酸残基(D)的主链部分；标注有E的圆圈(圆圈E)为谷氨酸残基(E)的主链部分；
为-W2-L1-W1-A1-；较佳地-CO-L1-W1-A1-(即连接在式II上述多肽链的Aa4位点)；
R如上所述；
Ac为N端保护基或不存在(较佳地为乙酰基)，连接在多肽的N端；和
-CONH2为酰胺基，为多肽的C端。
在另一优选例中，所述的偶联物如式III-C1、III-C2或III-C3所示，
其中，
标注有1、2、4、3、5、7、8和9的圆圈分别如式II中Aa1、Aa2、Aa3、Aa4、Aa5、Aa7、Aa8、和Aa9的定义；
靠近标注有1的圆圈的末端为多肽N端，靠近标注有9的圆圈的末端为多肽C端；
Ac为N端保护基或不存在；各个空白圆圈如式II中的X和Y定义；
为-W2-L1-W1-A1-；较佳地为-CO-L1-W1-A1；
圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
圆圈K为赖氨酸残基的主链部分；圆圈D为天冬氨酸残基(D)的主链部分、圆圈E为谷氨酸残基(E)的主链部分。
在另一优选例中，
空白圆圈各自独立地为无(不存在)或氨基酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有数字的圆圈(即圆圈1、圆圈2、圆圈3、圆圈4、圆圈5、圆圈7、圆圈8、和圆圈9)各自独立地为氨基酸残基(较佳地，所述氨基酸残基不为赖氨酸残基和半胱氨酸残基)；
标注有C的圆圈为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
标注有K的圆圈为赖氨酸残基的主链部分；标注有D的圆圈为天冬氨酸残基(D)的主链部分；标注有E的圆圈为谷氨酸残基(E)的主链部分；
为-W2-L1-W1-A1-；较佳地为-CO-L1-W1-A1(即连接在式II上述多肽链的Aa6位点)；
L2、L3、下标o、和R如前定义；
Ac为N端保护基或不存在(较佳地为乙酰基)，连接在多肽的N端；和
-CONH2为酰胺基，为多肽的C端；
当CL连接于环肽Aa6位置时，结合优选的连接子结构，可以实现任意L2-(L3-D)o基团或物质修饰到靶蛋白上(优选地，靶蛋白为抗体)。
在另一优选例中，式III-C1为
其中，除标注有C和K的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。
在另一优选例中，式III-C2为
其中，除标注有C和D的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。
在另一优选例中，式III-C3为
其中，除标注有C和E的圆圈外，标注有字母的圆圈分别代表有该字母表示的氨基酸残基。在另一优选例中，上述各式中，标注有1、2、3、4、5、6、7、8和9的圆圈分别如式II中Aa1、Aa2、Aa3、Aa4、Aa5、Aa6、Aa7、Aa8和Aa9的定义，各个空白圆圈如式II中的X和Y定义。
在另一优选例中，所述的偶联物选自表A1和表A2。
在本发明的第二方面，提供了一种中间体I，所述中间体如式I-A所示
其中，Ra为H或保护基团(如三苯甲基(Trt))；L1、W1和A1如前定义。
在本发明的第三方面提供了一种中间体II，所述中间体如式I-B所示
其中，Ra为H或保护基团(如三苯甲基(Trt))(较佳地，Ra为H)；AT、L1、W1和A1如前定义。
在另一优选例中，AT为衍生自包含如II所示序列的Fc结合多肽的核心序列。
在本发明的第四方面提供了一种如第一方面所述的偶联物的制备方法，包括步骤：
(1)使式I-A中间体与包含如II所示序列的Fc结合多肽的核心序列反应从而形成式I-B中间体；以及
(2)使式I-B中间体与R-COOH酯化物(羧酸化合物的活性酯)(如)形式反应，从而得到如式I所示的偶联物。
在本发明的第五方面，提供了一种蛋白位点选择性修饰的方法，包括步骤：
(1)提供本发明第一方面所述的偶联物；
(2)使需要修饰的目标蛋白与所述的偶联物接触，并使得所述偶联物与蛋白上侧链氨基和/或末端的氨基进行反应，从而得到经修饰的目标蛋白。
在另一优选例中，所述的经修饰的目标蛋白被一个或多个所述的R基团修饰。
在另一优选例中，所述的修饰为位点选择性修饰。
在另一优选例中，所述的氨基包括赖氨酸(K)的侧链氨基。
在另一优选例中，所述的修饰通过一步反应即可得到得到经修饰的目标蛋白。
在另一优选例中，所述经修饰的目标蛋白是修饰有一个或多个选自下组的活性基团的蛋白：具有生物活性和/或可检测的活性基团、可被进一步修饰的活性基团。
在另一优选例中，所述经修饰的目标蛋白如式IV所示
TP-(NHCO-R)p   (IV)
其中，R如前定义，TP为目标蛋白部分，下标p为1-8。
在另一优选例中，p＝2-4。
在另一优选例中，TP为目标蛋白中侧链含NH2基团的氨基酸残基(较佳地，赖氨酸残基；更佳地，位于Fc区域的赖氨酸残基；最佳地，Fc区域中的第246位-248位的赖氨酸残基)中NH2的与所述的偶联物中-CO-R连接形成-NHCO-R后其余的部分。
在另一优选例中，所述目标蛋白为抗体或Fc融合蛋白。
在另一优选例中，所述目标蛋白为抗体，且所述经修饰的目标蛋白如式V所示
Ab-(NHCO-R)p   (V)。
其中，R和下标p如前定义，Ab为抗体。
在另一优选例中，Ab为抗体中p个侧链含NH2基团的氨基酸残基(较佳地，赖氨酸残基；更加地，位于Fc区域的赖氨酸残基；最佳地，Fc区域中的第246位-248位的赖氨酸残基)中NH2的与所述的偶联物中-CO-R连接形成-NHCO-R后其余的部分。
在另一优选例中，所述目标蛋白具有与式I所示复合物的亲和的片段或序列。
在另一优选例中，所述抗体或Fc结构为人IgG来源。
在另一优选例中，所述目标蛋白被修饰的位置为Fc区域。
在另一优选例中，所述目标蛋白被修饰的位置为包含人IgG的Fc 80％同源序列区域。
在另一优选例中，人IgG的Fc原始序列如SEQ ID No.1所示：
在另一优选例中，所述目标蛋白被修饰的位置为人IgG的Fc区域中第246位- 第248位的氨基酸残基所形成的靶区域。
在另一优选例中，与式I偶联物相互作用的共价修饰区域为由连续1-10个氨基酸残基组成的区域，并且该区域包括由人IgG Fc区域中的第246位-248位的氨基酸残基形成的靶区域。
在另一优选例中，所述靶区由人IgG Fc区域中的第246位或第248位为赖氨酸残基。
在另一优选例中，所述方法的区域的选择性为≥90％。
在另一优选例中，所述修饰位点为人IgG Fc区域中的第248位赖氨酸残基。
在本发明的第六方面，提供了一种如式IV所示的经位点选择性修饰的蛋白，
TP-(NHCO-R)p   (IV)
其中，TP、R和下标p如前定义。
在另一优选例中，R为-L2-(L3-D)o；其中，L2、L3、D、下标o如前定义。
在另一优选例中，R为-L2-(L3-B)o；其中，L2、L3、B、下标o如前定义。
在另一优选例中，R为-L2-(L3-B'-L4-D)o；其中，L2、L3、B、下标o如前定义；B'为B与L4'-D进行正交反应形成的连接基团，L4为连接片段，L4'为带有可与B进行正交反应的活性基团并在反应后可形成L4的基团。
在另一优选例中，所述的经位点选择性修饰的蛋白为抗体药物偶联物，即TP为抗体且R为-L2-(L3-D)o或R为-L2-(L3-B'-L4-D)o。
在另一优选例中，TP为抗体，且所述的经位点选择性修饰的蛋白如式V所示；
Ab-(NHCO-R)p   (V)
其中，Ab为抗体，R和下标p如前定义。
在另一优选例中，所述的经位点选择性修饰的蛋白如式IV-A所示；
TP-(NHCO-L2-(L3-D)o)p   (IV-A)
其中，TP、L2、L3、D、下标o和下标p如前定义。
在另一优选例中，所述的经位点选择性修饰的蛋白如式V-A所示；
Ab-(NHCO-L2-(L3-D)o)p   (V-A)
其中，Ab、L2、L3、D、下标o和下标p如前定义。
在另一优选例中，所述抗体药物偶联物选自：ADC-1、ADC-2、ADC-3、ADC-4、ADC-5、ADC-6、ADC-7、ADC-8、ADC-9、ADC-10、ADC-11、ADC-12、ADC13、ADC-14、ADC-15、ADC-16、ADC-17、ADC-18、ADC-19、ADC-20和ADC-21。
在本发明的第七方面，提供了一种本发明第一方面所述的偶联物的用途，用于位点选择性修饰蛋白。
在本发明的第八方面，提供了一种本发明第一方面所述的偶联物的用途，用于制备抗体药物偶联物(ADC)。
在另一优选例中，所述用途不依赖于生物正交反应，所述偶联物可经一步反应直接将需要被修饰到目标蛋白的药物分子，以位点选择性方式修饰至抗体上。
在本发明的第九方面，提供了一种如式V所示的经位点选择性修饰抗体，
Ab-(NHCO-R)p   (V)
其中，Ab为抗体，R和下标p如前定义。
在本发明的第十方面，提供了一种抗体药物偶联物(ADC)，所述抗体药物偶联物如式V-A所示：
Ab-(NHCO-L2-(L3-D)o)p   (V-A)
其中，Ab、L2、L3、D、下标o和下标p如前定义。
在另一优选例中，所述抗体药物偶联物是不依赖于生物正交反应、一步制备得到的，且抗体被定点修饰的定点抗体药物偶联物。
在另一优选例中，Ab为抗体，下标p为1～8(较佳地，p＝2-4)，o为1-10的整数，L2和L3为连接片段，D为小分子药物；当o为2-10时，D可以为同一个小分子药物，也可以独立为不同的小分子药物。
在另一优选例中，所述定点抗体药物偶联物，D(如小分子药物)定点偶联在抗体恒定区(Fc区域)。
在另一优选例中，所述定点抗体药物偶联物，D(如小分子药物)定点偶联在人IgG Fc区域中的由246-248位氨基酸残基构成的区域。
在另一优选例中，所述抗体药物偶联物选自：ADC-1、ADC-2、ADC-3、ADC-4、ADC-5、ADC-6、ADC-7、ADC-8、ADC-9、ADC-10、ADC-11、ADC-12、ADC13、ADC-14、ADC-15、ADC-16、ADC-17、ADC-18、ADC-19、ADC-20和ADC-21。在另一优选例中，所述抗体药物偶联物选自：ADC-5、ADC-6、ADC-7、ADC-8、ADC-9、ADC-10、ADC-11、ADC-12、ADC13、ADC-14、ADC-15、ADC-16、ADC-17、ADC-18、ADC-19、ADC-20和ADC-21。
在另一优选例中，，上述定点ADC制备方法为：将天然抗体与物式(III、III-A1-3、III-B1-3、III-C1-3)所示亲和片段-可裂解连接片段-功能性片段复合物孵育后，功能性片段上含有的羰基直接与抗体Fc区域的赖氨酸残基共价形成酰胺键，一步反应制备定点ADC化合物，不依赖生物正交反应。
在本发明的第十一方面，提供了一种如式V-B所示的位点特异性的，双价或多价生物正交基团修饰的抗体，其特征在于，
Ab-(NHCO-L2-(L3-B)o)p   (V-B)
其中，Ab、L2、L3、B、下标o和下标p如前定义。
在另一优选例中，Ab为抗体，下标p为1～8(较佳地，p＝2-4)，o为2-10的整数，L2和L3为连接片段；
B为生物正交基团，多个B可以相同也可以独立不同，优选自：叠氮残基、醛残基、硫醇残基、炔烃残基、烯烃残基、卤素残基、四嗪残基、硝酮残基、羟胺残基、腈残基、肼残基、酮残基、硼酸残基、氰基苯并噻唑残基、烯丙基残基、膦残基、马来酰亚胺残基、二硫残基、硫酯残基、α-卤代羰基残基、异腈残基、斯德酮残基、硒残基、共轭二烯残基、磷酸残基、环炔残基及环烯残基。
上述生物正交基团定点偶联在抗体恒定区(Fc区域)；
上述生物正交基团定点偶联在人IgG Fc区域中的由246-248位氨基酸残基构成的区域；
在本发明的第十二方面，提供了一种如式V-C所示的位点特异性的，双价或多价药物修饰的抗体，其特征在于，
Ab-(NHCO-L2-(L3-B’-L4-D)o)p   (V-C)
其中，L2、L3、B、下标o如前定义；且B’为B与L4’-D进行正交反应形成的连接基团，L4为连接片段，L4’为带有可与B进行正交反应的活性基团并在反应后可形成L4的基团。
在另一优选例中，Ab为抗体，下标p为1～8(较佳地，p＝2-4，更佳地p＝2)，o为2-10的整数，L2和L3为连接片段，B’为包含药物片段与上述生物正交基团B之间反应生成的连接片段，B’相同或各自不同，L4为连接B’与药物D的连接子，D如上所述，D在此的结构相同或各自不同。
上述生物正交基团定点偶联在抗体恒定区(Fc区域)；
上述生物正交基团定点偶联在人IgG Fc区域中的由246-248位氨基酸残基构成的区域。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a-e显示了实施例47制备得到的生物素化曲妥珠单抗Ab-1的各表征结果。其中，图1a、1b和1c显示了显示ESI-TOFMS测定结果，图1d显示了曲妥珠单抗经胰蛋白酶消化的包含对赖氨酸的修饰部位的由33个氨基酸残基形成的肽 (THTCPPCPAPELLGGPSVFLFPPKPK(C10H14N2O2S)DTLMISR)的肽片段的MS谱图，图1e显示了经Mascot 2.3软件分析，确定修饰高选择性地发生在第251位赖氨酸残基。
图2a-c显示了实施例48制备得到的偶联叠氮基团的曲妥珠单抗Ab-2的各表征结果。其中，图2a、2b和2c显示了显示ESI-TOFMS测定结果。
图3显示了实施例49偶联生物荧光素FITC前后的曲妥珠单抗Ab-3的ESI-TOFMS测定结果。
图4显示了实施例50偶联正交基团环丙烯前后的曲妥珠单抗Ab-4的ESI-TOFMS测定结果。
图5显示了实施例51偶联正交基团DBCO前后的利妥昔单抗Ab-5的ESI-TOFMS测定结果。
图6显示了实施例52偶联正交基团DBCO前后的利妥昔单抗Ab-6的ESI-TOFMS测定结果。
图7a-e显示了实施例53制备得到的ADC-1的各表征结果。其中，图7a、7b和7c显示了ESI-TOFMS测定结果，图7d显示了疏水色谱分析结果，图7e显示了分子排阻色谱分析结果。
图8a-e显示了实施例54制备得到的ADC-2的各表征结果。其中，图8a、8b和8c显示了ESI-TOFMS测定结果，图8d显示了疏水色谱分析结果，图8e显示了分子排阻色谱分析结果。
图9a-e显示了实施例55制备得到的ADC-3的各表征结果。其中，图9a、9b和9c显示了ESI-TOFMS测定结果，图9d显示了疏水色谱分析结果，图9e显示了分子排阻色谱分析结果。
图10显示了实施例56制备得到的ADC-4的ESI-TOFMS测定结果。
图11a-e显示了实施例57制备得到的ADC-5的各表征结果。其中，图11a、11b和11c显示了ESI-TOFMS测定结果，图11d显示了疏水色谱分析结果，图11e显示了分子排阻色谱分析结果。
图12a-e显示了实施例58制备得到的ADC-12的各表征结果。其中，图12a、12b和12c显示了ESI-TOFMS测定结果，图12d显示了疏水色谱分析结果，图12e显示了分子排阻色谱分析结果。
图13a-e显示了实施例59制备得到的ADC-7的各表征结果。其中，图13a、13b和13c显示了ESI-TOFMS测定结果，图13d显示了疏水色谱分析结果，图13e显示了分子排阻色谱分析结果。
图14a-e显示了实施例60制备得到的ADC-8的各表征结果。其中，图14a、14b和14c显示了ESI-TOFMS测定结果，图14d显示了疏水色谱分析结果，图14e显示了分子排阻色谱分析结果。
图15显示了实施例61偶联药物连接子D6前后的利妥昔单抗的ESI-TOFMS测定结果。
图16显示了实施例62偶联药物连接子D6前后的帕妥珠单抗的ESI-TOFMS测定结果。
图17显示了实施例63偶联药物连接子D6前后的贝伐单抗的ESI-TOFMS测定结果。
图18显示了实施例64偶联药物连接子D6前后的特瑞普利单抗的ADC-12的ESI-TOFMS测定结果。
图19显示了实施例65偶联药物连接子D6前后的帕尼单抗的ESI-TOFMS测定结果。
图20显示了实施例66偶联药物连接子D6前后的纳武单抗的ESI-TOFMS测定结果。
图21a显示了实施例67中原料曲妥珠单抗及制备得到的ADC-15的ESI-TOFMS测定结果，图21b显示了疏水色谱分析结果。
图22a显示了实施例68中原料曲妥珠单抗及制备得到的ADC-16的ESI-TOFMS测定结果,图22b显示了疏水色谱分析结果。
图23a显示了实施例69中原料曲妥珠单抗及制备得到的ADC-17的ESI-TOFMS测定结果,图23b显示了疏水色谱分析结果。
图24a显示了实施例70中原料曲妥珠单抗及制备得到的ADC-18的ESI-TOFMS测定结果,图24b显示了疏水色谱分析结果。
图25a显示了实施例71中原料曲妥珠单抗及制备得到的ADC-19的ESI-TOFMS测定结果,图25b显示了疏水色谱分析结果。
图26显示了实施例72中原料曲妥珠单抗及制备得到的ADC-20的ESI-TOFMS测定结果。
图27显示了实施例73中原料曲妥珠单抗及制备得到的ADC-21的ESI-TOFMS测定结果。
图28显示了本发明实施例制备得的ADC的细胞活性及细胞毒性结果。
具体实施方式
经过广泛而深入地研究，本发明人意外地发现一种结构独特的偶联物，它具有所示的二价片段的可裂解片段和亲和性部分(AT)，本发明的偶联物十分适合对蛋白进行直接修饰，尤其是抗体或Fc融合蛋白的Fc段)。采用本发明的偶联物，无需在修饰前对蛋白中的侧链上的活性基团预先修饰，可直接反应(如仅一步直接反应)而得到被R基团修饰的所需产物(如ADC等)。尤其是，采用特定序列的环肽作为亲和性部分且可裂解片段连接于该环肽的特定位置上时， 本发明偶联物可以极高的效率进行位点选择性修饰。在此基础上，发明人完成了本发明。
术语
除非另有说明，在本文中所有的缩写具有本领域技术人员所熟知的含义。
如本文所用，“卤素”指F、Cl、Br、和I。更佳地，卤原子选自F、Cl和Br。
除非另有说明，术语“烷基”，本身或作为另一取代基的一部分，是指具有指定碳原子数的直链或支链烃基(即，C1-6表示1-6个碳)。烷基的例子包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、正戊基、正己基、正庚基、正辛基等。
如本文所用，术语“亚烷基”，其本身或作为另一取代基的一部分，是指衍生自烷烃的二价基团，例如-CH2CH2CH2CH2-。烷基(或亚烷基)通常具有1-10个碳原子(即C1-10亚烷基)。亚烷基的例子包括但不限于：亚甲基、亚乙基。
除非另有说明，术语“芳基”表示多不饱和的(通常芳香性)的烃基，其可以是单环或稠合在一起或共价连接的多环(最多三环)。一般地，芳基具有6-10个环原子。术语"杂芳基"是指含有1至5个选自N、O、和S的杂原子的芳基(或环)，其中氮和硫原子任选被氧化，氮原子任选被季铵化。一般地，杂芳基具有5-10个环原子即5-10元杂芳基，优选地，具有5-6个环原子即5-6元杂芳基，并含有1、2、3或4个杂原子。杂芳基可通过杂原子连接于分子的其余部分。芳基的非限制性例子包括苯基、萘基和联苯基，而杂芳基的非限制性例子包括吡啶基等。
在本文中，以虚线表示的键代表基团(或片段或部分)与其余部分连接的位点。
在本文中，除非另有定义，所述取代是指基团中一个或多个H被选自下组的取代基取代：卤素、C1-6烷基、C1-6卤代烷基。
如本文所用，术语“生物正交基团”是指含可用于生物正交反应的官能团的基团，例如含叠氮基(-N3)、环辛炔、醛基、酮羰基、四嗪、反式环辛烯、硫醇、卤素、肼、羟胺等基团。
如本文所用，术语“氨基酸残基”是指氨基酸的N端-NH2脱去一个H，C端的-COOH脱去-OH所形成的基团。一般地，将氨基酸(残基)中包括N端和C段的链段称为主链，而决定氨基酸具体种类的部分称为侧链。除非另有定义，在本文中，氨基酸包括天然氨基酸或非天然氨基酸，包括D型和/或L型氨基酸。氨基酸的例子包括但不限于Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp (W)、Tyr(Y)、Val(V)。优选地，在本文中，氨基酸为选自下组的氨基酸：L-甘氨酸(L-Gly)，L-丙氨酸(L-Ala)，β-丙氨酸(β-Ala)，L-谷氨酸(L-Glu)，L-天冬氨酸(L-Asp)，L-组氨酸(L-His)，L-精氨酸(L-Arg)，L-赖氨酸(L-Lys)，L-缬氨酸(L-Val)，L-丝氨酸(L-Ser)，L-苏氨酸(L-Thr)；此外，当氨基酸存在2个或以上的氨基和/或者2个或以上的羧基时，该术语还包括不在同一个碳原子上的-NH2脱去一个H和-COOH脱去-OH所形成的基团，例如由谷氨酸的-NH2和非α位-COOH分别脱去一个H后形成的二价基团-C(O)-(CH2)2-C(COOH)-NH-。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
多肽
在本发明中，术语“Fc结合多肽”指具有可逆结合目标蛋白，尤其是抗体的Fc区域能力的肽；较佳地，其具有如式II所示的核心序列(即(Xs1)t1-Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Ys2)t2)。此外，所述术语还包括所述Fc结合多肽的变异形式。这些变异形式包括(但并不限于)：1-5个(通常为1-4个，较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为5个以内，较佳地为3个以内，更佳地为2个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1

发明还提供本发明多肽的类似物。这些类似物与天然本发明多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
亲和片段导向的可裂解片段
在本发明的一个方面，提供了一种亲和片段导向的可裂解片段(也可称为本发明的偶联物)或其衍生物(如其盐)，所述由以下式I表示：
AT-CL-R   (I)
该复合物作为酰基转移试剂，可以直接将R基团偶联到蛋白上，与特定位点氨基酸形成新的共价键。
在一些实施方案中，AT(affinity tag)为针对蛋白的亲和性物质；CL(cleavable linker)为可裂解片段，其具有自水解反应性的切割片段；R(reagent)为化合物。
在另一些优选实施方案中，AT为蛋白的亲和片段，该片段既可以是与蛋白形成可逆共价结合的小分子化合物，也可以是具有亲和力的蛋白或多肽结构。特别地，在修饰抗体方面，AT既可以是与抗体形成可逆共价结合的小分子化合物，也可以是与抗体具有亲和力的蛋白或多肽结构如Fc结合肽，来源于ProteinA或ProteinG的与Fc具有结合能力的多肽片段结构等。
在另一些优选实施方案中，AT可以是例如Fc-III肽(DCAWHLGELVWCT(SEQ ID No.2))、其他Fc结合肽(GPDCAYHRGELVWCTFH(SEQ ID No.3)、RGNCAYHRGQLVWCTYH(SEQ No.4)、CDCAWHLGELVWCTC(SEQ ID No.5))等，其核心结构序列如下(II)所示：
(X0-3)t1-Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Y0-3)t2   (II)
其中，C为半胱氨酸残基，X0-3和Y0-3为除了赖氨酸和半胱氨酸以外的任意氨基酸，可为1-3个连续的相同的或不同的氨基酸残基，m和n分别独立为0-10的数字。Aa1-Aa3和Aa5以及Aa7-Aa9为除了半胱氨酸和赖氨酸的任意氨基酸，Aa4或Aa6分 别独立为赖氨酸残基，即当Aa4为赖氨酸时，Aa6为除了半胱氨酸和赖氨酸的其他任意氨基酸，当Aa6为赖氨酸时，Aa4为除了半胱氨酸和赖氨酸的其他任意氨基酸。
优选地，所述的亲和性片段(AT)为或衍生自可以与抗体Fc具有亲和力的环多肽片段，优选结构为：
其中，无标记或以数字标记的圆圈代表除半胱氨酸和赖氨酸的任意氨基酸，无标记的圆圈也可以不存在，C代表半胱氨酸且-S-S-代表有该两个半胱氨酸中-SH基团形成的-S-S-键，以及K代表赖氨酸主链，并描绘出其侧链结构。
更优选地，所述的亲和性片段(AT)及可裂解片段(CL)的一部分(即AT-W2-L1-W1-A1-S-)衍生自含有巯基的Fc结合肽，其优选修饰结构如下任一所述：
在另一些优选实施方案中，AT也可以为与抗体Fab具有亲和力的结合肽；例如曲妥珠单抗Fab结合肽等。
在一些实施方案中，AT也可以为与蛋白具有可逆共价相互作用的小分子片段，例如，如下所示的邻羟基苯甲醛、临乙酰基苯硼酸等，
在一些实施方案中，CL为可裂解片段，该片段可以在亲核试剂的进攻下形成新的化学键，将R片段共价连接到蛋白特定位点，同时该片段发生断裂，释放出亲和性片段AT。优选地，CL如下所示，CL片段可为硫酯片段、马来酰亚胺酯片段、酯片段等酰基转移试剂：
其中，R1和R2分别独立为氢原子、甲基、乙基等脂肪族或芳香族片段，或R1与R2形成环状烷烃或烯烃结构。
更优选地，CL如下任一所示
其中，可裂解片段的羰基连接R片段，可裂解片段的S或O部分偶联亲和性片段。
在一些优选实施方案中，CL片段是以硫酯为核心的酰基转移试剂，其结构如下式(III)所示：
其中，连接子(linker)的结构为不同长度的亚甲基结构、氨基酸、多聚甘氨酸、多聚乙醇(PEG)结构等；更优选地，连接子的结构如下所示：
其中，n为1-10的整数。
在一些实施方案中，R衍生自将要修饰到蛋白上的化合物，其包括可被进一步修饰的活性基团如生物正交基团等或者含或衍生自生物素、荧光试剂、小分子药物、mRNA、多肽化合物及蛋白等。R的来源化合物本身包含羧酸片段或可衍生获得羧酸结构，通过羧酸与巯基、马来酰亚胺、苯酚等形成CL可裂解片段中的-CO-结构，即酰基转移试剂。
在另一优选例中，-CO-R选自组2(其中-CO-为CL中的-CO-)，并且组2包括如下所示的基团：
其中，m为0或任意正整数；优选地为1～20的正整数。
在另一优选例中，L3-D选自下组：
其中，
p、q和r为0或任意正整数；优选地为0或1～20的正整数；更优选地，p和r各自独立地为0、1、2或3，且q为1、2、3、4、5或6；
MMAE/MMAF是指MMAE或MMAF，并且MMAE和MMAF的结构如下：
在一些实施方案中，式III所示的亲和片段导向的可裂解片段如下式任一所示
其中，R和linker的定义如前定义，圆圈1、2、3、4、5、6、7、8和9分别指如前定义的Aa1、Aa2、Aa3、Aa4、Aa5、Aa6、Aa7、Aa8、和Aa9，圆圈C是指Cys，圆圈K是指赖氨酸残基的主链部分(即赖氨酸残基的-NH-C-CO-部分)。
在一些实施方案中，AT、CL和R如第一方面中定义。
在一些实施方案中，AT、CL和R各自独立地为表A1和表A2所示的偶联物或实施例中各具体化合物中对应的基团。
在一些具体实施方式中，所述的偶联物(或称亲和片段导向的可裂解片段)选自表A1和表A2所示的化合物(环肽中K的侧链结构(即-(CH2)4-NH-)已示出)：
表A1

其中，Ac为N端保护基或不存在(较佳地为乙酰基)。
表A2



其中，Ac为N端保护基或不存在(较佳地为乙酰基)。
优选地，所述的亲和片段导向的可裂解片段(酰基转移试剂)，可以直接将R基团转移到天然抗体(Ab)上，实现抗体的氨基酸选择性修饰，形成抗体的官能团化修饰、荧光修饰、药物偶联，多肽偶联、mRNA偶联等，形成如下式(V)所示的抗体修饰结构：
Ab-NHCO-R   (V)
其中，Ab为抗体，-NHCO-为酰胺键结构，R为修饰到抗体上的修饰片段。
在本发明的另一个方面，提供了一种如上所述的亲和片段导向的可裂解片段的制备方法，其如下任一反应式所示：

其中，linker 1为-L1-W1-A1-；PG为硫醇以外任意巯基保护基团，如Trt-、Acm-、Mob等；AE为任意活性酯形式，如NHS酯、酰氯、酰基叠氮、硫酯、对硝基苯酚酯等；L1、W1、A1、圆圈1-9、圆圈K、圆圈C、和R如前定义。
在另一个实施方案中，所述方法包括以下步骤：
步骤1：亲和片段的合成：通过固相合成获得多肽后，将其溶解于DMSO中使终浓度为5mM，加入过氧化氢(10mM)、氨水(100mM)，室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干得亲和片段。
步骤2：带保护的亲和片段-巯基衍生物的合成
步骤3：亲和片段-巯基衍生物的合成：通过相应的脱保护策略得亲和片段-巯基衍生物粗品。
步骤4：亲和片段导向的可裂解片段的制备：将上述亲和片段-巯基衍生物粗品与羧酸化合物的活性酯形式混合LC-MS监测反应完全后，使用半制备分离纯化，冻干即得亲和片段导向的可裂解片段。
在另一个实施方案中，所述方法包括以下步骤：
步骤1：亲和片段的合成：通过固相合成获得多肽后，将其溶解于DMSO中使终浓度为5mM，加入过氧化氢(10mM)、氨水(100mM)，室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干得亲和片段。
步骤2：可裂解片段的合成：利用羧酸化合物的活性酯形式与巯基衍生物反应得到可裂解片段——硫酯化合物。
步骤3：亲和片段导向的可裂解片段的制备：亲和片段与可裂解片段通过缩合、还原胺化等反应获得亲和片段导向的可裂解片段。
在本发明的一个方面，提供一种基于上述亲和片段导向的可裂解片段(即导向基-硫酯-载体分子复合物)的定点抗体药物偶联物的制备方法(或抗体或Fc融 合蛋白定点修饰方法)，如以下反应式所示：
其中，R为偶联到抗体上的修饰结构，R1和R2为生物正交基团，R3为R1与R2反应形成的接头结构。
优选地，所述制备方法或定点修饰方法包括式(I)所示复合物(如前所述)，与抗体或Fc融合蛋白孵育，实现抗体或Fc融合蛋白Fc区域特定位点赖氨酸的定点修饰。
优选地，所述抗体或Fc融合蛋白具有与式(I)所示复合物的亲和片段或序列。
优选地，抗体或Fc结构为人IgG来源
优选地，与(I)所示复合物相互作用形成的共价修饰区域，由连续1-10个氨基酸残基组成的区域，其中所述靶区由人IgG Fc区域中的第246位-248位的氨基酸残基形成的区域。
优选地，其中所述靶区由人IgG Fc区域中的第246位或第248位为赖氨酸残基。
优选地，所述的抗体或Fc融合蛋白，其中，所述区域的选择性为90％以上。
优选地，抗体药物偶联物或者定点修饰后的抗体或Fc融合蛋白
本发明的主要优点包括
(a)本发明提供的偶联物用于修饰蛋白如抗体时修饰效率高(>95％)。
(b)本发明提供的偶联物可直接修饰在蛋白需被修饰部位的氨基酸残基的侧链氨基上而无需预先对该侧链氨基先进行修饰。
(c)本发明提供的偶联物对抗体活性的影响小。
(d)本发明提供的偶联物对抗体修饰操作简便，对pH，浓度无严苛的要求(pH 5.5-8.0，抗体浓度0.5mg/mL-10mg/mL)，便于技术推广。
(e)本发明构建的方法用途广泛，可用于构建抗体快速修饰平台，即抗体的多样化修饰与抗体药物偶联物(ADC)的制备。
(f)本发明构建的方法首次实现了抗体的一步定点无痕修饰。
(g)本发明构建的方法可用于一步制备定点修饰的ADC，有望构建大容量ADC库，用于ADC的药物连接子的系统筛选与优化。
(h)本发明提供的硫酯片段作为酰基转移试剂，与Fc结合肽的特定位点连接，可以实现小分子药物的“一步”高效定点偶联，且兼容多样性的药物连接子结构，底物适用性广泛。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另有说明，各实施例中的检测方法和条件如下：
ESI-TOFMS
流动相A：0.1％FA的水溶液，流动相B：0.1％FA的乙腈溶液。
方法A：用于小分子化合物的质谱分析。使用C18色谱柱(ACQUITY UPLC BEH C18,1.7μm,2.1x50mm)，其液相程序为0-0.2-1.5-1.8-2.0-2.5分钟时对应流动相B为10-10-70-70-10-10％，检测波长为214nm，柱温：35℃。
方法B：用于抗体及ADC分子的质谱分析。使用C4柱(ACQUITY UPLC Protein BEH C4,1.7μm,2.1mm x 50mm)，其液相程序为0-2.0-6.0-7.2-7.3-7.6-7.7-8.0-8.1-10.0分钟时对应流动相B为5-5-90-90-5-90-5-90-5-5％，检测波长为280nm，柱温：80℃。
疏水色谱分析
将待测ADC分子使用1x PBS稀释至5mg/mL，进样5μL。流动相A：0.1M磷酸钠，1.5M硫酸铵缓冲液，pH＝7.0；流动相B：0.1M磷酸钠缓冲液，pH＝7.0，流速1mL/min，0-15min，线性梯度0-100％流动相B。检测波长为280nm。
分子排阻色谱分析
将待测ADC分子使用1x PBS稀释至1mg/mL，60℃ PCR仪加热48h，0h，6h，12h，24h，48h分别取点进行SEC分析，每个时间点取样15μL。SEC分析使用流动相为150mM磷酸钠，pH＝7.4，流速为1mL/min，等度洗脱15min，检测波长为280nm。
I：亲和片段的制备
制备例1：化合物AT-1的合成
化合物AT-1的结构以及合成方法如下：

称取化合物1(200mg,0.095mmol)溶解于DMSO中使终浓度为5mM，加入过氧化氢(10mM)、氨水(100mM)，室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干后得到白色粉末即化合物AT-1(产率91％)。HRMS，计算值C93H135N31O22S2:[M+H]+2102.9918,[M+2H]2+1051.9998,[M+3H]3+701.6691,[M+4H]4+526.5038测量值701.6684,526.5048.
制备例2：化合物AT-2的合成
化合物AT-2的结构以及合成方法如下：
称取化合物2(200mg,0.096mmol)溶解于DMSO中使终浓度为5mM，加入过氧化氢(10mM)、氨水(100mM)，室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干后得到白色粉末即化合物AT-2(产率90％)。HRMS，计算值C92H131N29O23S2:[M+H]+2074.9491,[M+2H]2+1037.9785,[M+3H]3+692.3216,测量值1037.9715,692.3156.
Ⅱ：亲和片段-巯基衍生物的制备
制备例3：化合物ATS-1的合成
化合物ATS-1的结构以及合成方法如下：
步骤1：称取2-(三苯甲硫基)乙酸(15.7mg,0.047mmol)溶解于DMF中，依 次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-1(20mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物3(产率92％)。HRMS，计算值C114H151N31O23S3:[M+H]+2419.0839,[M+3H]3+807.0332,[M+4H]4+605.5269,测量值807.0342,605.5232.
步骤2：称取化合物3(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-1。HRMS，计算值C95H137N31O23S3:[M+H]+2176.9744,[M+3H]3+726.3300,[M+4H]4+544.9995,测量值726.3278,545.0035.
制备例4：化合物ATS-2的合成
化合物ATS-2的结构以及合成方法如下：
步骤1：称取3-(三苯甲硫基)丙酸(16.36mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-1(20mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物4(产率89％)。HRMS，计算值C115H153N31O23S3:[M+H]+2433.0996,[M+3H]3+811.7051,[M+4H]4+609.0308,测量值811.7012,609.0299.
步骤2：称取化合物4(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-2。HRMS，计算值C96H139N31O23S3:[M+H]+2190.9900,[M+3H]3+731.0019,[M+4H]4+548.5034,测量值731.0054,548.5102.
制备例5：化合物ATS-3的合成
化合物ATS-3的结构以及合成方法如下：

步骤1：称取NaH(34.5mg,1.44mmol)溶解于DMF(5mL)中，在N2保护，0℃，冰浴条件下，加入三苯基甲硫醇(200mg,0.72mmol)，冰浴搅拌30min后，加入4-溴丁酸(60mg,0.36mmol)，待温度缓慢升至室温后，搅拌过夜反应，硅胶柱分离纯化，得化合物5(产率76％)。1H NMR(600MHz,DMSO-d6)δ12.02(s,1H),7.33–7.15(m,15H),2.12(t,J＝7.3Hz,2H),2.08(t,J＝7.4Hz,2H),1.48(p,J＝7.3Hz,2H).13C NMR(151MHz,DMSO)δ173.70,144.50,129.08,128.02,126.70,66.02,32.72,30.72,23.56.
步骤2：称取化合物5(17mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-1(20mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物6(产率91％)。HRMS，计算值C116H155N31O23S3:[M+H]+2447.1152,[M+3H]3+816.3770,[M+4H]4+612.5347,测量值816.3715,612.5382.
步骤3：称取化合物6(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-3。HRMS，计算值C97H141N31O23S3:[M+H]+2205.0057,[M+3H]3+735.6738,[M+4H]4+552.0073,测量值735.6742,552.0153.
制备例6：化合物ATS-4的合成
化合物ATS-4的结构以及合成方法如下：
步骤1：称取2-(三苯甲硫基)乙酸(334mg,1mmol)溶解于5mL DMF中，加入N-羟基丁二酰亚胺(138mg,1.2mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺 盐酸盐(230mg,1.2mmol)，室温搅拌过夜。然后加入甘氨酸(75mg,1mmol)，室温反应2h。1H NMR(500MHz,DMSO-d6)δ8.24(t,J＝5.8Hz,1H),7.38–7.23(m,15H),3.67(d,J＝5.8Hz,2H),2.84(s,2H).13C NMR(126MHz,DMSO)δ170.93,167.63,144.03,129.10,128.11,126.83,66.03,40.89,35.77.
步骤2：称取化合物7(18.4mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-1(20mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物8(产率87％)。HRMS，计算值C116H154N32O24S3:[M+H]+2476.1054,[M+3H]3+826.0404,[M+4H]4+619.7822,测量值826.0475,619.1098.
步骤3：称取化合物8(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-4。HRMS，计算值C97H140N32O24S3:[M+H]+2233.9955,[M+3H]3+745.3372,[M+4H]4+559.2548,测量值745.3356,559.2573.
制备例7：化合物ATS-5的合成
化合物ATS-5的结构以及合成方法如下：
步骤1：称取2-(三苯甲硫基)乙酸(15.7mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物9(产率87％)。HRMS，计算值C113H147N29O24S3:[M+H]+2391.0414,[M+3H]3+797.6857,[M+4H]4+598.5162,测量值797.685,598.5136.
步骤2：称取化合物9(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-5。HRMS，计算值C94H133N29O24S3:[M+H]+2148.9319,[M+3H]3+716.9825,[M+4H]4+537.9888,测量值716.9867,537.9872.
制备例8：化合物ATS-6的合成
化合物ATS-6的结构以及合成方法如下：
步骤1：称取3-(三苯甲硫基)丙酸(16.36mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物10(产率89％)。HRMS，计算值C114H149N29O24S3:[M+H]+2405.0571,[M+3H]3+802.3576,[M+4H]4+602.0201,测量值802.3512,602.0258.
步骤2：称取化合物10(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-6。HRMS，计算值C95H134N29O24S3:[M+H]+2162.9475,[M+3H]3+721.6544,[M+4H]4+541.4928,测量值721.6523,514.4953.
制备例9：化合物ATS-7的合成
化合物ATS-7的结构以及合成方法如下：
步骤1：称取化合物5(17mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物11(产率92％)。HRMS，计算值C115H151N29O24S3:[M+H]+ 2419.0727,[M+3H]3+807.0294,[M+4H]4+605.5240,测量值807.0287,605.5198.
步骤2：称取化合物11(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-7。HRMS，计算值C96H137N29O24S3:[M+H]+2176.9632,[M+3H]3+726.3263,[M+4H]4+544.9967,测量值726.3248,544.9913.
制备例10：化合物ATS-8的合成
化合物ATS-8的结构以及合成方法如下：
步骤1：称取化合物7(18.4mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物12(产率87％)。HRMS，计算值C115H150N30O25S3:[M+H]+2448.0619,[M+3H]3+816.6928,[M+4H]4+612.7716,测量值816.6915,612.7742.
步骤2：称取化合物12(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-8。HRMS，计算值C96H136N30O25S3:[M+H]+2205.9533,[M+3H]3+735.9897,[M+4H]4+552.2442,测量值735.9910,552.2418.
制备例11：化合物ATS-9的合成
化合物ATS-9的结构以及合成方法如下：
步骤1：称取NaH(34.5mg,1.44mmol)溶解于DMF(5mL)中，在N2保护，0℃，冰浴条件下，加入三苯基甲硫醇(200mg,0.72mmol)，冰浴搅拌30min后，加入6-溴己酸(70mg,0.36mmol)，待温度缓慢升至室温后，搅拌过夜反应，硅胶柱分离纯化，得化合物13(产率82％)。
步骤2：称取化合物13(18.3mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物14(产率87％)。HRMS，计算值C117H155N29O24S3:[M+H]+2447.1040,[M+3H]3+816.3732,[M+4H]4+612.5319,测量值816.3798,612.5326.
步骤3：称取化合物14(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-9。HRMS，计算值C98H141N29O24S3:[M+H]+2204.9945,[M+3H]3+735.6700,[M+4H]4+552.0044,测量值735.6712,552.0019.
制备例12：化合物ATS-10的合成
化合物ATS-10的结构以及合成方法如下：

步骤1：称取2-(三苯甲硫基)乙酸(334mg,1mmol)溶解于5mL DMF中，加入N-羟基丁二酰亚胺(138mg,1.2mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(230mg,1.2mmol)，室温搅拌过夜。然后加入4-氨基丁酸(103mg,1mmol)，室温反应2h，得化合物15(产率78％)。HRMS，计算值C25H25NO3S:[M+H]+420.1633,测量值420.1649.
步骤2：称取化合物15(19.7mg,0.047mmol)溶解于DMF中，依次加入HATU(7.14mg,0.0188mmol)，DIPEA(4.84μL,0.028mmol)以及化合物AT-2(19.5mg,0.0094mmol)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末即化合物16(产率82％)。HRMS，计算值C117H154N30O25S3:[M+H]+2476.0942,[M+3H]3+826.0366,[M+4H]4+619.7794,测量值826.0342,619.7758.
步骤3：称取化合物16(15mg)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干，得化合物ATS-10。HRMS，计算值C98H140N30O25S3:[M+H]+2233.9846,[M+3H]3+745.3334,[M+4H]4+559.2520,测量值745.3316,559.2561.
Ⅲ：药物连接子的合成
制备例13：化合物D-1的合成
化合物D-1的结构以及合成方法如下：

称取化合物17(10mg,8.9μmol)溶解在200mL DMF中，加入DBCO-CONHS(4.3mg,10.7μmol)，三乙胺(3.7mL,26.7μmol)，室温搅拌2h，LC-MS监测反应完全，使用半制备分离纯化，冻干得白色粉末即为化合物D1(产率81％)。HRMS，计算值:C77H107N11O14:[M+H]+1410.8077,测量值1410.8052.
制备例14：化合物D-2的合成
化合物D-2的结构以及合成方法如下：
称取化合物17(10mg,8.9μmol)溶解在200μL DMF中，加入BCN-O-PNP(3.5mg,10.7μmol)，三乙胺(3.7μL,26.7μmol)，室温搅拌3h，LC-MS监测反应完全，使用半制备分离纯化，冻干得化合物D2(产率76％)。HRMS，计算值:C69H106N10O14:[M+H]+1299.7968,测量值1299.8016.
制备例15：化合物D-3的合成
化合物D-3的结构以及合成方法如下：
步骤1：称取CH3O-PEG24-COOH(58mg,0.05mmol)溶解于乙腈中，加入NHS(6.8mg,0.06mmol)，EDC(7.9μL,0.06mmol)，室温反应过夜后，加入Fmoc-Lys(NH2)-OH(18.4mg,0.05mmol)的乙腈溶液，室温搅拌2h，LC-MS监测反应完全，使用半制备分离纯化，冻干得白色粉末，得化合物18(产率78％)。HRMS，计算值:C73H126N2O30:[M+H]+756.4275,测量值756.4218.
步骤2：称取化合物18(13.5mg,8.9μmol)溶解于DMF中，加入HATU(6.8mg,17.8μmol)，DIPEA(4.6μL,27.6μmol)，化合物17(10mg,8.9μmol)，室温反应2h，LC-MS监测反应完全，使用半制备分离纯化，冻干得白色粉末即化合物19(产率86％)。HRMS，计算值:C116H208N12O39:[M+H]+2394.4741,[M+3H]3+798.8299,测量值798.8312.
步骤3：称取化合物19(10mg,4.2μmol)溶解于DMF中，加入BCN-O-PNP(1.63mg,5μmol)，三乙胺(1.7μL,12.6μmol)，室温搅拌2h，LC-MS监测反应完全，使用半制备分离纯化，冻干得化合物D3(产率76％)。HRMS，计算值:C127H220N12O41:[M+H]+2570.5577,[M+3H]3+857.5244,测量值857.5267.
制备例16：化合物D-4的合成
化合物D-4的结构以及合成方法如下：

步骤1：H2N-PEG4-COOH(26.5mg,0.1mmol)溶于DMF中，加入9-芴甲基-N-琥珀酰亚胺基碳酸酯(67.4mg,0.2mmol)，三乙胺(41.6μL,0.3mmol)，室温反应3h后，LC-MS监测反应体系，显示反应完全，半制备分离纯化后冻干得到化合物20。HRMS，计算值：C26H33NO8:[M+H]+488.2284，测量值488.2243。
步骤2：称取化合物20(17mg,0.035mmol)溶于DMF中，加入对硝基苯酚(9.7mg,0.07mmol)，EDC(12.7μL,0.07mmol)，室温反应1h后，LC-MS监测反应体系，显示反应完全，半制备分离纯化后冻干得到化合物21。HRMS，计算值：C32H36N2O10:[M+H]+609.2448，测量值609.2252。
步骤3：称取化合物21(10mg,0.016mmol)溶于DMF中，加入MMAF(12mg,0.02mmol)，HOBt(0.54mg,3.2μmol)，吡啶(36μL,0.45mmol)，室温反应1h，LC-MS监测基本反应完全后，加入20％哌啶反应15min，半制备分离纯化后冻干得到化合物22。HRMS，计算值：C50H86N6O13:[M+H]+979.6331，测量值979.6328。
步骤4：称取化合物22(10mg,0.01mmol)溶于DMF中，加入BCN-O-PNB(9.45mg,0.03mmol)，三乙胺(8.3μL,0.06mmol)，室温反应4h，LC-MS监测基本反应完全，半制备分离纯化后冻干得到化合物D4。HRMS，计算值：C61H98N6O15:[M+H]+1155.7168，[M+2H]2+578.3623，测量值578.3619。
制备例17：化合物D-5的合成
化合物D-5的结构以及合成方法如下：

称取化合物17(10mg,9.8μmol)溶解于DMF(500μL)中，加入双琥珀酰亚胺戊二酸酯(DSG,15.6mg,0.048mmol)，三乙胺(4μL,0.029mmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末D5(产率91％)。HRMS，计算值:C67H103N11O17:[M+H]+1334.7612,测量值1334.7655.
制备例18：化合物D-6的合成
化合物D-6的结构以及合成方法如下：
称取化合物23(10.1mg,9.8μmol)溶解于DMF(500μL)中，加入双琥珀酰亚胺戊二酸酯(DSG,15.6mg,0.048mmol)，三乙胺(4μL,0.029mmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末D6(产率85％)。HRMS，计算值:C64H97N9O16:[M+H]+1248.7132,测量值1248.7154.
制备例19：化合物D-7的合成
化合物D-7的结构以及合成方法如下：

称取HOOC-PEG5-COOH(23mg,0.068mmol)溶解于DMF(500μL)中，加入HATU(26mg,0.068mmol)，DIPEA(23.5μL,0.136mmol)，药物毒素MMAF(10mg,0.0136mmol)，室温反应1h后，使用半制备分离纯化，冻干得白色粉末D7(产率82％)。HRMS，计算值:C53H89N5O16:[M+H]+1052.6383,测量值1052.6354.
制备例20：化合物D-8的合成
化合物D-8的结构以及合成方法如下：
步骤1：称取HOOC-PEG4-NH2(26.5mg,0.1mmol)溶解于DMF(400μL)中，加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(30.8mg,0.1mmol)，三乙胺(41.6μL,0.3mmol)，室温搅拌1h，LC-MS监测反应完全，使用半制备分离纯化，冻干得白色粉末，即为化合物24(产率86％)。HRMS，计算值:C20H32N2O10:[M+H]+461.2135,测量值461.2169.
步骤2：称取化合物24(10mg,0.022mmol)溶解于PB 7.4(500μL)，加入细胞毒素DM1(16mg,0.022mmol)，室温搅拌1h，LC-MS监测反应完全，使用半制备分离纯化，冻干得化合物D8(产率87％)。HRMS，计算值:C55H80ClN5O20S:[M+H]+1198.4884,测量值1198.4798.
Ⅳ：亲和片段导向的可裂解片段的制备
实施例1：化合物ATC-1的合成
化合物ATC-1的结构以及合成方法如下：
步骤1：称取叠氮乙酸(250mg，2.5mmol)溶解于THF(5mL)中，加入N-羟基琥珀酰亚胺(288mg，2.5mmol)，0℃搅拌10min后，加入二环己基碳二亚胺(516mg,2.5mmol)，并将反应液于0℃搅拌4h。过滤除去DCU后，收集滤液，加入乙醚(20mL)重结晶，并在4℃下静置过夜。过滤，滤饼用THF(40mL)洗涤并真空干燥，得到白色粉末状即为化合物25(产率82％)。1H NMR(600MHz,DMSO-d6)δ4.71(s,2H),2.84(s,4H).13C NMR(151MHz,DMSO)δ169.91,165.51,47.50,25.50.
步骤2：称取化合物ATS-1(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入化合物25(0.77mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-1(产率71％)。HRMS，计算值:C97H138N34O24S3:[M+H]+2259.9861,[M+3H]3+754.0006,[M+4H]4+565.7524,测量值753.9815,565.7080.
实施例2：化合物ATC-2的合成
化合物ATC-2的结构以及合成方法如下：
称取化合物ATS-1(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-2(产率77％)。HRMS，计算值:C105H151N33O25S4:[M+H]+2403.0520,[M+3H]3+801.6892,[M+4H]4+601.5188,测量值801.6801,601.5139.
实施例3：化合物ATC-3的合成
化合物ATC-3的结构以及合成方法如下：
步骤1：称取2-甲基-2-环丙烯甲酸(0.4mg，4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物26粗品。
步骤2：称取化合物ATS-1(7mg,3.2μmol)溶于DMF中(50μL)中，加至步骤1所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-3(产率67％)。HRMS，计算值:C100H141N31O24S3:[M+H]+2257.0006,[M+3H]3+753.0054,[M+4H]4+565.0060,测量值753.0013,565.0059.
实施例4：化合物ATC-4的合成
化合物ATC-4的结构以及合成方法如下：
步骤1：称取戊炔酸(0.4mg，4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物27粗品。
步骤2：称取化合物ATS-1(7mg,3.2μmol)溶于DMF中(50μL)中，加至步骤1所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-4(产率59％)。HRMS，计算值:C100H141N31O24S3:[M+H]+2257.0006,[M+3H]3+753.0054,[M+4H]4+565.0060,测量值753.0051,565.0086.
实施例5：化合物ATC-5的合成
化合物ATC-5的结构以及合成方法如下：
称取化合物ATS-1(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入DBCO-NHS(1.55mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-6(产率68％)。HRMS，计算值:C114H150N32O25S3:[M+H]+2464.0690,[M+3H]3+822.0282,[M+4H]4+616.7731,测量值822.0264,616.7715.
实施例6：化合物ATC-6的合成
化合物ATC-6的结构以及合成方法如下：
称取化合物D5(13.3mg,0.01mmol)溶解于DMF/PB7.4＝1/1混合溶液中，加入化合物ATS-1，室温反应30min后，使用半制备分离纯化，冻干得白色粉末即为化合物ATC-6(产率76％)。HRMS，计算值:C158H235N41O37S3:[M+H]+3395.7007,[M+3H]3+1132.5721,[M+4H]4+849.6810,测量值1133.5587,849.6596.
实施例7：化合物ATC-7的合成
化合物ATC-7的结构以及合成方法如下：
称取化合物D6(12.5mg,0.01mmol)溶解于DMF/PB7.4＝1/1混合溶液中，加入化合物ATS-1，室温反应30min后，使用半制备分离纯化，冻干得化合物ATC-7(产率81％)。HRMS，计算值:C155H229N39O36S3:[M+H]+3309.6526,[M+3H]3+1103.8894,[M+4H]4+828.1690,测量值1103.8900,828.1674.
实施例8：化合物ATC-8的合成
化合物ATC-8的结构以及合成方法如下：
称取D7(10.5mg,0.01mmol)溶解于DMF中，加入NHS(1.4mg,0.012mmol)，EDC(2.1μL,0.012mmol)，室温反应过夜。反应液中加入化合物ATS-1的DMF溶液，同时加入等体积的0.2M PB 7.4缓冲液，室温反应2h后，使用半制备分离纯化，冻干得化合物ATC-8(产率77％)。HRMS，计算值:C148H224N36O38S3:[M+H]+3210.5941,[M+3H]3+1070.8699,[M+4H]4+803.4044,测量值1070.4196,803.9044.
实施例9：化合物ATC-9的合成
化合物ATC-9的结构以及合成方法如下：
称取D8(12mg,0.01mmol)溶解于DMF中，加入NHS(1.4mg,0.012mmol)，EDC(2.1μL,0.012mmol)，室温反应过夜后，加入ATS-1的DMF溶液，再加入等体积的0.2M PB 7.4缓冲液，室温反应2h后，使用半制备分离纯化，冻干得白色粉末即化合物ATC-9(产率72％)。HRMS，计算值:C150H215ClN36O42S4:[M+H]+3356.4445,[M+3H]3+1119.4867,[M+4H]4+839.8669,测量值1119.4882,839.8637.
实施例10：化合物ATC-10的合成
化合物ATC-10的结构以及合成方法如下：
称取化合物ATS-2(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-10(产率79％)。HRMS， 计算值:C106H153N33O25S4:[M+H]+2417.0676,[M+3H]3+806.3611,[M+4H]4+605.0228,测量值806.3648,605.0256.
实施例11：化合物ATC-11的合成
化合物ATC-11的结构以及合成方法如下：
称取化合物ATS-3(7.05mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-11(产率67％)。HRMS，计算值:C107H155N33O25S4:[M+H]+2431.0833,[M+3H]3+811.0330,[M+4H]4+608.5267,测量值811.0325,608.5286.
实施例12：化合物ATC-12的合成
化合物ATC-12的结构以及合成方法如下：
称取化合物ATS-4(7.05mg,3.2μmol)溶于PB 7.0/DMF＝1/1混合溶液中，加入化合物25(0.77mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-12(产率89％)。HRMS，计算值:C99H141N35O25S3:[M+H]+2317.0078,[M+3H]3+773.0078,[M+4H]4+580.0078,测量值773.0123,580.0071.
实施例13：化合物ATC-13的合成
化合物ATC-13的结构以及合成方法如下：

称取化合物ATS-4(7.05mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-13(产率77％)。HRMS，计算值:C107H154N34O26S4:[M+H]+2460.0734,[M+3H]3+820.6963,[M+4H]4+615.7742,测量值820.7012,615.7726.
实施例14：化合物ATC-14的合成
化合物ATC-14的结构以及合成方法如下：
步骤1：称取2-甲基-2-环丙烯甲酸(0.4mg，4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物26粗品。
步骤2：称取化合物ATS-4(7.05mg,3.2μmol)溶于DMF中(50μL)中，加至步骤1所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-14(产率59％)。HRMS，计算值:C102H144N32O25S3:[M+H]+2314.0221,[M+3H]3+772.0126,[M+4H]4+579.2614,测量值772.0148,579.2658.
实施例15：化合物ATC-15的合成
化合物ATC-15的结构以及合成方法如下：
步骤1：称取戊炔酸(0.4mg，4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐 (1.15mg,6μmol)，室温反应过夜后，得化合物27粗品。
步骤2：称取化合物ATS-4(7.05mg,3.2μmol)溶于DMF中(50μL)中，加至步骤1所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-4(产率66％)。HRMS，计算值:C102H144N32O25S3:[M+H]+2314.0221,[M+3H]3+772.0126,[M+4H]4+579.2614,测量值772.0148,579.2594.
实施例16：化合物ATC-16的合成
化合物ATC-16的结构以及合成方法如下：
称取化合物ATS-4(7.05mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入DBCO-NHS(1.55mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-16(产率68％)。HRMS，计算值:C116H153N33O26S3:[M+H]+2521.0905,[M+3H]3+841.0354,[M+4H]4+631.0285,测量值841.0389,631.0268.
实施例17：化合物ATC-17的合成
化合物ATC-17的结构以及合成方法如下：
称取化合物D5(13.3mg,0.01mmol)溶解于DMF/PB7.4＝1/1混合溶液中，加入化合物ATS-4，室温反应30min后，使用半制备分离纯化，冻干得白色粉末即为化合物ATC-17(产率73％)。HRMS，计算值:C159H236N42O38S3:[M+H]+3438.7066,[M+3H]3+1146.9074,[M+4H]4+860.4325,测量值860.4365.
实施例18：化合物ATC-18的合成
化合物ATC-18的结构以及合成方法如下：
称取化合物ATS-5(6.9mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌20min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-2(产率77％)。HRMS，计算值:C104H147N31O26S4:[M+H]+2375.0094,[M+3H]3+792.3417,[M+4H]4+594.5082,测量值792.3395,594.5079.
实施例19：化合物ATC-19的合成
化合物ATC-19的结构以及合成方法如下：
称取化合物ATS-6(6.9mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-19(产率76％)。HRMS，计算值:C105H149N31O26S4:[M+H]+2389.0251,[M+3H]3+797.0136,[M+4H]4+598.0121,测量值797.0178,598.0135.
实施例20：化合物ATC-20的合成
化合物ATC-20的结构以及合成方法如下：
步骤1：称取巯基丙酸(26.5mg,0.25mmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(17mg,0.05mmol)，室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物28(产率53％)。
步骤2：称取化合物28(1.33mg,4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物29粗品。
步骤3：称取化合物ATS-6(6.9mg,3.2μmol)溶于DMF中(50μL)中，加至步骤2所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-20(产率75％)。HRMS，计算值:C108H153N31O27S5:[M+H]+2477.0234,[M+3H]3+826.3463,[M+4H]4+620.0117,测量值826.3457,619.9915.
实施例21：化合物ATC-21的合成
化合物ATC-21的结构以及合成方法如下：
称取化合物ATS-7(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-21(产率74％)。HRMS，计算值:C106H151N31O26S4:[M+H]+2403.0407,[M+3H]3+801.6854,[M+4H]4+601.5160,测量值801.6794,601.5172.
实施例22：化合物ATC-22的合成
化合物ATC-22的结构以及合成方法如下：
称取化合物ATS-8(7mg,3.2μmol)溶于PB 7.0/DMF＝1/1混合溶液中，加入化合物25(0.77mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-22(产率91％)。HRMS，计算值:C98H137N33O26S3:[M+H]+2288.9653,[M+3H]3+763.6603,[M+4H]4+572.9971,测量值763.6584,571.9829.
实施例23：化合物ATC-23的合成
化合物ATC-23的结构以及合成方法如下：
称取化合物ATS-8(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-23(产率81％)。HRMS，计算值:C106H150N32O27S4:[M+H]+2432.0309,[M+3H]3+811.3488,[M+4H]4+608.7636,测量值811.2514,608.7618.
实施例24：化合物ATC-24的合成
化合物ATC-24的结构以及合成方法如下：

步骤1：称取2-甲基-2-环丙烯甲酸(0.4mg，4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物26粗品。
步骤2：称取化合物ATS-8(7mg,3.2μmol)溶于DMF中(50μL)中，加至步骤1所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-24(产率64％)。HRMS，计算值:C101H140N30O26S3:[M+H]+2285.9795,[M+3H]3+762.6650,[M+4H]4+572.2507,测量值762.6612,572.2547.
实施例25：化合物ATC-25的合成
化合物ATC-25的结构以及合成方法如下：
称取化合物ATS-8(7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入DBCO-NHS(1.55mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-25(产率65％)。HRMS，计算值:C115H149N31O27S3:[M+H]+2493.0479,[M+3H]3+831.6878,[M+4H]4+624.0178,测量值831.6894,624.0189.
实施例26：化合物ATC-26的合成
化合物ATC-26的结构以及合成方法如下：

称取化合物D5(13.3mg,0.01mmol)溶解于DMF/PB7.4＝1/1混合溶液中，加入化合物ATS-8，室温反应30min后，使用半制备分离纯化，冻干得白色粉末即为化合物ATC-26(产率83％)。HRMS，计算值:C159H234N40O39S3:[M+H]+3424.6797,[M+3H]3+1142.2318,[M+4H]4+856.9258,测量值856.9314.
实施例27：化合物ATC-27的合成
化合物ATC-27的结构以及合成方法如下：
称取D7(10.5mg,0.01mmol)溶解于DMF中，加入NHS(1.4mg,0.012mmol)，EDC(2.1μL,0.012mmol)，室温反应过夜。反应液中加入化合物ATS-8的DMF溶液，同时加入等体积的0.2M PB 7.4缓冲液，室温反应2h后，使用半制备分离纯化，冻干得化合物ATC-27(产率70％)。HRMS，计算值:C149H223N35O40S3:[M+H]+3239.5732,[M+3H]3+1080.5296,[M+4H]4+810.6492,测量值810.6554.
实施例28：化合物ATC-28的合成
化合物ATC-28的结构以及合成方法如下：

称取D8(12mg,0.01mmol)溶解于DMF中，加入NHS(1.4mg,0.012mmol)，EDC(2.1μL,0.012mmol)，室温反应过夜后，加入ATS-8的DMF溶液，再加入等体积的0.2M PB 7.4缓冲液，室温反应2h后，使用半制备分离纯化，冻干得白色粉末即化合物ATC-28(产率73％)。HRMS，计算值:C149H210ClN35O43S4:[M+H]+3341.3971,[M+3H]3+1114.4709,[M+4H]4+836.1051,测量值836.1095.
实施例29：化合物ATC-29的合成
化合物ATC-29的结构以及合成方法如下：
步骤1：称取4-(氨甲基)苯甲酸(9mg,0.06mmol)溶于DMF中，加入生物素-N-琥珀酰亚胺基酯(17mg,0.05mmol)，三乙胺(20μL,0.15mmol),室温搅拌过夜，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物30(产率91％)。
步骤2：称取化合物30(1.33mg,4μmol)溶解于DMF(50μL)中，加入N- 羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物31粗品。
步骤3：称取化合物ATS-8(7mg,3.2μmol)溶于DMF中(50μL)中，加至步骤2所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-29(产率74％)。HRMS，计算值:C114H157N33O28S4:[M+H]+2565.0837,[M+3H]3+855.6998,[M+4H]4+642.0268,测量值855.7043,642.0276.
实施例30：化合物ATC-30的合成
化合物ATC-30的结构以及合成方法如下：
步骤1：称取生物素(50mg,0.2mmol)溶于DMF中，加入HATU(152mg,0.4mmol)，DIPEA(103μL,0.6mmol)以及(2-氨基乙氧基)氨基甲酸叔丁酯(35mg,0.2mmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物32(产率85％)。HRMS，计算值:C17H30N4O5S:[M+H]+403.2015,测量值403.2114.
步骤2：称取化合物32(20mg,0.05mmol)溶于500μL二氯甲烷中，冰浴条件下，加入450μL三氟乙酸、50μL三异丙基硅烷，室温反应1h后，N2吹干待用。
步骤3：在步骤2所得粗品中加入乙腈/水/醋酸＝9/9/2的混合溶液500μL，并加入乙醛酸(2.8μL,0.05mmol)，室温反应15min，半制备分离纯化，冻干得化 合物33(产率92％)。HRMS，计算值:C14H22N4O5S:[M+H]+359.1389,测量值359.1425.
步骤4：称取化合物33(1.43mg,4μmol)溶解于DMF(50μL)中，加入N-羟基琥珀酰亚胺(0.69mg，6μmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.15mg,6μmol)，室温反应过夜后，得化合物34粗品。
步骤5：称取化合物ATS-8(7mg,3.2μmol)溶于DMF中(50μL)中，加至步骤2所得反应液，再加入PB 7.4缓冲液(200μL)，室温搅拌1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末，即为化合物ATC-30(产率63％)。HRMS，计算值:C110H156N34O29S4:[M+H]+2546.0738,[M+3H]3+849.3631,[M+4H]4+637.2743,测量值849.3654,637.2842.
实施例31：化合物ATC-31的合成
化合物ATC-31的结构以及合成方法如下：
称取化合物ATS-9(7.05mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-31(产率78％)。HRMS，计算值:C112H160N32O28S5:[M+H]+2562.0761,[M+3H]3+854.6972,[M+4H]4+641.2749,测量值854.7015,641.2796.
实施例32：化合物ATC-32的合成
化合物ATC-32的结构以及合成方法如下：
称取化合物ATS-10(7.2mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入生物素-N-琥珀酰亚胺基酯(1.32mg,3.86μmol)，室温搅拌15min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-32(产率78％)。HRMS，计算值:C112H159N33O29S5:[M+H]+2591.0663,[M+3H]3+864.3606,[M+4H]4+648.5224,测量值864.3587,648.5246.
实施例33：化合物ATC-33的合成
化合物ATC-33的结构以及合成方法如下：
步骤1：称取氨基荧光素(10.4mg,0.03mmol)溶于DMF(500μL)中，加入双琥珀酰亚胺戊二酸酯(DSG,39mg,0.12mmol)，三乙胺(12.5μL,0.09mmol)，室温避光反应2h，LC-MS监测反应完全后，得化合物35，反应液加入巯基乙酸(6.3μL,0.09mmol)，PB 7.4缓冲液(500μL)，室温避光反应2h，LC-MS监测反应完全后，半制备分离纯化，冻干得黄色粉末(产率84％)。HRMS，计算值:C27H22NO9S:[M+H]+536.1015,测量值536.1028.
步骤2：称取化合物36(5.1mg,9.51μmol)溶于DMF(300μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-33。HRMS，计算值:C120H154N32O30S3:[M+H]+2620.0749,[M+3H]3+874.0302,[M+4H]4+655.7746,测量值874.0562,655.7789.
实施例34：化合物ATC-34的合成
化合物ATC-34的结构以及合成方法如下：
步骤1：称取氨基荧光素(17mg,49μmol)溶于DMF(400μL)中，加入HATU(28mg,73.5μmol)，DIPEA(18.9μL,110μmol)以及羧酸-聚乙二醇-羧酸(17.4mg,98μmol)，室温避光反应1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物37(产率92％)。HRMS，计算值:C26H21NO10:[M+H]+508.1244,测量值508.1312.
步骤2：称取化合物37(15.2mg,0.03mmol)溶于DMF(300μL)中，加入N-羟基琥珀酰亚胺(NHS,4.1mg,0.036mmol)，EDCI(6.4μL,0.036mmol)，室温避光反应过夜，LC-MS监测反应完全后，反应液加入巯基乙酸(6.3μL,0.09mmol)，PB 7.4缓冲液(300μL)，室温避光反应2h，LC-MS监测反应完全后，半制备分离纯化，冻干得黄色粉末(产率81％)。HRMS，计算值:C28H23NO11S:[M+H]+582.1070,测量值582.1235.
步骤3：称取化合物38(5.5mg,9.51μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-34(产率84％)。HRMS，计算值:C121H156N32O32S3:[M+H]+2666.0804,[M+3H]3+889.3653,[M+4H]4+667.2760,测量值889.3815,667.2715.
实施例35：化合物ATC-35的合成
化合物ATC-35的结构以及合成方法如下：
步骤1：称取2-甲基-2-环丙烯甲酸(20mg,0.2mmol)溶于DMF(400μL)中，加入NHS(27.6mg,0.24mmol)，EDCI(42.4μL,0.24mmol)，室温反应过夜，反应液中加入3-[2-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]乙氧基]丙酸(53mg,0.2mmol)，三乙胺(83μL,0.6mmol)LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物39(产率78％)。HRMS，计算值:C16H27NO7:[M+H]+346.1866,测量值346.1902.
步骤2：称取化合物39(10.4mg,0.03mmol)溶于DMF(300μL)中，加入N-羟基琥珀酰亚胺(NHS,4.1mg,0.036mmol)，EDCI(6.4μL,0.036mmol)，室温反应过夜后，反应液加入巯基乙酸(6.3μL,0.09mmol)，PB 7.4缓冲液(300μL)，室温反应1h，LC-MS监测反应完全后，半制备分离纯化，冻干得白色粉末(产率84％)。HRMS，计算值:C18H29NO8S:[M+H]+420.1692,测量值420.1589.
步骤3：称取化合物40(4mg,9.51μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-35(产率90％)。HRMS，计算值:C111H162N32O29S3:[M+H]+2531.1426,[M+3H]3+844.3861,[M+4H]4+633.5415,测量值844.3845,633.5436.
实施例36：化合物ATC-36的合成
化合物ATC-36的结构以及合成方法如下：

称取化合物ATS-1(6.7mg,3.2μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入DBCO-NHS(1.55mg,3.86μmol)，室温搅拌30min，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-36(产率62％)。HRMS，计算值:C114H150N32O25S3:[M+H]+2464.0690,[M+3H]3+822.0282,[M+4H]4+616.7731,测量值822.0315,616.7712.
实施例37：化合物ATC-37的合成
化合物ATC-37的结构以及合成方法如下：
步骤1：称取DBCO-NHS(40mg,0.1mmol)溶于DMF(400μL)中，加入NHS(13.8mg,0.12mmol)，EDCI(21.2μL,0.12mmol)，室温反应过夜，反应液中加入3-[2-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]乙氧基]丙酸(26.5mg,0.1mmol)，三乙胺(41.5μL,0.3mmol)LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物41(产率81％)。HRMS，计算值:C30H36N2O8:[M+H]+553.2550,测量值553.2548.
步骤2：称取化合物41(16.5mg,0.03mmol)溶于DMF(400μL)中，加入N-羟基琥珀酰亚胺(NHS,4.1mg,0.036mmol)，EDCI(6.4μL,0.036mmol)，室温反应过夜后，反应液加入巯基乙酸(6.3μL,0.09mmol)，PB 7.4缓冲液(400μL)，室温反应2h，LC-MS监测反应完全后，半制备分离纯化，冻干得白色粉末，为化合物42(产率87％)。HRMS，计算值:C32H38N2O9S:[M+H]+627.2376,测量值627.2315.
步骤3：称取化合物42(6mg,9.51μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)， 室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-37(产率92％)。HRMS，计算值:C125H171N33O30S3:[M+H]+2711.2110,[M+3H]3+904.4089,[M+4H]4+678.5586,测量值904.4123,678.5591.
实施例38：化合物ATC-38的合成
化合物ATC-38的结构以及合成方法如下：
步骤1：称取2-氨基乙氧基氨基甲酸叔丁酯(17.6mg,0.1mmol)溶于DMF(400μL)中，加入HATU(76mg,0.2mmol)，DIPEA(51.5μL,0.3mmol)以及羧酸-聚乙二醇-羧酸(53.4mg,0.3mmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物43(产率88％)。HRMS，计算值:C13H24N2O8:[M+H]+337.1611,测量值337.1587.
步骤2：称取化合物43(20mg,0.06mmol)溶于DMF(400μL)中，加入N-羟基琥珀酰亚胺(NHS,8.2mg,0.072mmol)，EDCI(12.8μL,0.072mmol)，室温避光反应过夜，LC-MS监测反应完全后，反应液加入巯基乙酸(12.6μL,0.18mmol)，PB 7.4缓冲液(400μL)，室温避光反应2h，LC-MS监测反应完全后，半制备分离纯化，冻干得白色粉末即为化合物44(产率83％)。HRMS，计算值:C16H28N2O9S:[M+H]+425.1594,测量值425.1608.
步骤3：称取化合物44(4mg,9.51μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物45(产 率90％)。HRMS，计算值:C108H159N33O30S3:[M+H]+2495.1171,[M+3H]3+832.3776,[M+4H]4+624.5351,测量值832.3810,624.5305.
步骤4：称取化合物45(9mg,3.6μmol)加入二氯甲烷200μL，三氟乙酸180μL，异丙基硅烷20μL，4℃反应30min，N2吹干溶剂，得化合物ATC-38(产率95％)。HRMS，计算值:C103H151N33O28S3:[M+H]+2395.0646,[M+3H]3+799.0267,[M+4H]4+599.5220,测量值799.0248,599.5278.
实施例39：化合物ATC-39的合成
化合物ATC-39的结构以及合成方法如下：
步骤1：称取乙酰丙酸(23.2mg,0.2mmol)溶于DMF(400μL)中，加入N-羟基琥珀酰亚胺(NHS,27.6mg,0.24mmol)，EDCI(42.4μL,0.24mmol)，室温反应过夜后，反应液加入4-巯基丙基乙酸(67.2mg,0.4mmol)，三乙胺(83μL,0.6mmol)，室温反应2h，LC-MS监测反应完全后，半制备分离纯化，冻干得白色粉末即为化合物46(产率73％)。HRMS，计算值:C14H16O4S:[M+H]+281.0848,测量值281.0923.
步骤2：称取化合物ATS1(6.7mg,3μmol)溶于PB 7.4/DMF＝1/1混合溶液中，加入化合物46(2.7mg,9.6μmol)，室温搅拌4h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-39(产率74％)。HRMS，计算值:C101H145N31O25S3:[M+H]+2290.0346,[M+3H]3+764.0167,[M+4H]4+573.2645,测量值764.0205,573.0618.
实施例40：化合物ATC-40的合成
化合物ATC-40的结构以及合成方法如下：
步骤1：称取二甘醇酸(26.8mg,0.2mmol)溶于DMF(400μL)中，加入对硝基苯酚(66.7mg,0.48mmol)，EDCI(84.8μL,0.48mmol)，室温反应过夜后，加入乙醚沉淀，得产物47(产率69％)。
步骤2：化合物47(11.3mg,30μmol)溶于DMF(200μL)中，加入化合物17(11.2mg,10μmol)，三乙胺(6.94μL,50μmol)，室温反应20min后，加入巯基乙酸(2.1μL,30μmol)，PB7.4缓冲液，室温反应1h，半制备分离纯化，得化合物48(产率76％)。HRMS，计算值:C64H100N10O17S:[M+H]+1313.7067,测量值1313.7054.
步骤3：称取化合物48(6.2mg,4.76μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-40(产率91％)。HRMS，计算值:C157H233N41O38S3:[M+H]+3397.6800,[M+3H]3+1133.2319,[M+4H]4+850.1759,测量值1133.2291,850.1782.
实施例41：化合物ATC-41的合成
化合物ATC-41的结构以及合成方法如下：

步骤1：称取羧酸-聚乙二醇-羧酸(17.8mg,0.1mmol)溶于DMF(400μL)中，加入NHS(66.7mg,0.24mmol)，EDCI(42.4μL,0.24mmol)，室温反应过夜后，加入巯基乙酸(20.8μL,0.3mmol)，PB 7.4缓冲液(400μL)，室温反应1h，半制备分离纯化得产物49(产率79％)。HRMS，计算值:C10H14O8S2:[M+H]+327.0208,测量值327.0197.
步骤2：称取化合物49(9.8mg,30μmol)溶于DMF(200μL)中，加入化合物17(11.2mg,10μmol)，三乙胺(125μL,0.9mmol)，室温反应2h后，LC-MS监测反应完全，半制备分离纯化，得化合物50(产率88％)。HRMS，计算值:C66H104N10O18S:[M+H]+1357.7329,[M+2H]2+679.3704,测量值679.3759.步骤3：称取化合物50(6.5mg,4.76μmol)溶于DMF(200μL)中，加入HATU(2.7mg,7.14μmol)，DIPEA(1.84μL,10.7μmol)以及AT-1(10mg,4.76μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-41(产率85％)。HRMS，计算值:C159H237N41O39S3:[M+H]+3441.7062,[M+4H]4+861.1824,[M+5H]5+689.1475,测量值861.1859,689.1512.
实施例42：化合物ATC-42的合成
化合物ATC-42的结构以及合成方法如下：

步骤1：化合物47(11.3mg,30μmol)溶于DMF(200μL)中，加入化合物19(23.9mg,10μmol)，三乙胺(6.94μL,50μmol)，室温反应20min后，加入巯基乙酸(2.1μL,30μmol)，PB7.4缓冲液，室温反应1h，半制备分离纯化，得化合物51(产率81％)。HRMS，计算值:C122H214N12O44S:[M+H]+2584.4676,[M+3H]3+862.1611,[M+4H]4+646.8728,测量值862.1596,646.8756.
步骤2：称取化合物51(6.15mg,2.38μmol)溶于DMF(200μL)中，加入HATU(1.35mg,4.76μmol)，DIPEA(1.23μL,7.14μmol)以及AT-1(5mg,2.38μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-42(产率84％)。HRMS，计算值:C215H347N43O65S3:[M+H]+4668.4409,[M+4H]4+1167.8661,[M+5H]5+934.4944,[M+6H]6+778.9133测量值934.5015,778.9215.
实施例43：化合物ATC-43的合成
化合物ATC-43的结构以及合成方法如下：

步骤1：称取化合物49(4.9mg,15μmol)溶于DMF(200μL)中，加入化合物19(12mg,5μmol)，三乙胺(62.5μL,0.45mmol)，室温反应3h后，LC-MS监测反应完全，半制备分离纯化，得化合物52(产率85％)。HRMS，计算值:C124H218N12O45S:[M+H]+2628.4938,[M+3H]3+876.8365,[M+4H]4+657.8793,测量值876.8395,657.8852.
步骤2：称取化合物52(6.25mg,2.38μmol)溶于DMF(200μL)中，加入HATU(1.8mg,4.76μmol)，DIPEA(1.23μL,7.14μmol)以及AT-1(5mg,2.38μmol)，室温反应1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-43(产率87％)。HRMS，计算值:C217H351N43O66S3:[M+H]+4712.4671,[M+5H]5+943.2997,[M+6H]6+786.2510测量值943.3005,786.2519.
实施例44：化合物ATC-44的合成
化合物ATC-44的结构以及合成方法如下：

步骤1：称取CH3O-PEG12-COOH(17mg,0.027mmol)溶解于乙腈中，加入NHS(3.71mg,0.032mmol)，EDCI(5.7μL,0.032mmol)，室温反应过夜后，加入Fmoc-Lys(NH2)-OH(10mg,0.027mmol)的乙腈/水＝1/1混合溶液，室温搅拌2h，LC-MS监测反应完全，使用半制备分离纯化，冻干得白色粉末53(产率78％)。HRMS，计算值:C49H78N2O18:[M+H]+983.5328,测量值983.5318.
步骤2：称取化合物53(8.74mg,8.9μmol)溶解于DMF中，加入HATU(6.8mg,17.8μmol)，DIPEA(4.6μL,27.6μmol)，化合物17(10mg,8.9μmol)，室温反应2h，LC-MS监测反应完全后，加入20％哌啶，室温反应15min后，使用半制备分离纯化，冻干得白色粉末54(产率78％)。HRMS，计算值:C92H160N12O27:[M+H]+1866.1594,[M+3H]3+622.7250,测量值622.7289.
步骤3：称取化合物54(9.33mg,5μmol)溶于DMF(200μL)中，加入化合物49(4.9mg,15μmol)，三乙胺(20.8μL,0.15mmol)，室温反应2h后，LC-MS监测反应完全，半制备分离纯化，得化合物55(产率87％)。HRMS，计算值:C100H170N12O33S:[M+H]+2100.1792,[M+3H]3+700.7316,[M+4H]4+525.8007,测量值700.7359,525.8095.
步骤4：称取化合物52(5mg,2.38μmol)溶于DMF(200μL)中，加入HATU(1.8mg,4.76μmol)，DIPEA(1.23μL,7.14μmol)以及AT-1(5mg,2.38μmol)，室温反应1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-44(产率86％)。HRMS，计算值:C193H303N43O54S3:[M+H]+4184.1526,[M+5H]5+837.6368,[M+6H]6+698.1986,测量值837.6389,698.2015.
实施例45：化合物ATC-45的合成
化合物ATC-45的结构以及合成方法如下：
步骤1：称取芴甲氧羰基-L-谷氨酸1-叔丁酯(20mg,0.047mmol)溶解于DMF中，加入HATU(35.8mg,0.094mmol)，DIPEA(24.2μL,0.14mmol)，3-氨基-1-丙烷磺酸(6.5mg,0.047mmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得白色粉末56(产率85％)。HRMS，计算值:C27H34N2O8S:[M+H]+547.2114,测量值547.2156.
步骤2：称取化合物56(4.9mg,8.9μmol)加入二氯甲烷200μL，三氟乙酸180μL，异丙基硅烷20μL，室温反应1h，N2吹干溶剂，得化合物57，溶于DMF中，加入HATU(6.8mg,17.8μmol)，DIPEA(4.6μL,27.6μmol)，化合物17(10mg,8.9μmol)，室温反应2h，LC-MS监测反应完全后，加入20％哌啶，室温反应15min后，使用半制备分离纯化，冻干得白色粉末58(产率81％)。HRMS，计算值:C66H108N12O17S:[M+H]+1373.7754,[M+2H]2+687.3916,测量值687.3988.
步骤3：称取化合物58(7.2mg,10μmol)溶于DMF(200μL)中，加入化合物49(9.8mg,30μmol)，三乙胺(41.6μL,0.30mmol)，室温反应2h后，LC-MS监测反应完全，半制备分离纯化，得化合物59(产率82％)。HRMS，计算值:C74H118N12O23S2:[M+H]+1607.7952,[M+2H]2+804.4015,测量值804.4110.
步骤4：称取化合物59(3.8mg,2.38μmol)溶于DMF(200μL)中，加入HATU(1.8mg,4.76μmol)，DIPEA(1.23μL,7.14μmol)以及AT-1(5mg,2.38μmol)，室温反应1h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物 ATC-45(产率90％)。HRMS，计算值:C167H251N43O44S4:[M+H]+3691.7686,[M+5H]5+739.1600,测量值739.1642.
实施例46：化合物ATC-46的合成
化合物ATC-46的结构以及合成方法如下：
步骤1：称取Fmoc-PEG4-COOH(8.8mg,17.8μmol)，加入HATU(13.6mg,35.6μmol)，DIPEA(9.2μL,55.2μmol)，化合物17(20mg,17.8μmol)，室温反应2h，LC-MS监测反应完全后，加入20％哌啶，室温反应15min后，使用半制备分离纯化，冻干得白色粉末60(产率87％)。HRMS，计算值:C69H115N11O17:[M+H]+1370.8550,测量值1370.8547.
步骤2：称取3-((((9H-芴-9-基)甲氧基)羰基)氨基)戊二酸(10mg,27.1μmol) 溶于DMF(300μL)，加入NHS(3.8mg,32.5μmol)，DCC(6.7mg,32.5μmol),4℃反应过夜，得化合物61粗品。取反应液80μL，加入化合物60(10mg,7.3μmol)的DMF溶液80μL,三乙胺(3μL,21.9μmol)，室温反应2h，LC-MS监测反应完全后，加入哌啶40μL，室温反应15min后，使用半制备分离纯化，冻干得化合物62(产率90％)。HRMS，计算值:C143H235N23O36:[M+H]+2851.7343,[M+3H]3+951.2500,测量值951.2495.
步骤3：称取化合物62(7.12mg,2.5μmol)溶于DMF(200μL)中，加入化合物49(1.7mg,5μmol)，三乙胺(16.6μL,0.12mmol)，室温反应5h后，LC-MS监测反应完全，半制备分离纯化，得化合物63(产率70％)。HRMS，计算值:C151H245N23O42S:[M+H]+3085.7541,[M+3H]3+1029.2566,[M+4H]4+772.1944，测量,1029.1856,772.2015.
步骤4：称取化合物63(5mg,1.62μmol)溶于DMF(200μL)中，加入HATU(1.2mg,3.24μmol)，DIPEA(0.84μL,4.86μmol)以及AT-1(5mg,2.38μmol)，室温反应2h，LC-MS监测反应完全后，使用半制备分离纯化，冻干得化合物ATC-46(产率88％)。HRMS，计算值:C244H378N54O63S3:[M+H]+5169.7275,[M+5H]5+1034.7517,测量值1034.7451.
Ⅴ：亲和片段导向的可裂解片段的应用实施例
实施例47：生物素化抗体Ab-1的制备及表征
47-1.抗体Ab-1的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-2(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃反应2h，LC-MS监测反应完全。经protein A纯化、超滤管浓缩，进而置换为1×PBS缓冲液后得到生物素化抗体Ab-1。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个生物素的产物在146355观测到峰(图1a)。
47-2.抗体Ab-1的重链选择性确认：
将抗体Ab-1用50mM PB 7.4缓冲液稀释至5mg/mL，加入TCEP(20eq，终浓度：0.668mM)，37℃孵育20min。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入了生物素标签在49725观测到峰(图1b)。
47-3抗体Ab-1的Fc选择性确认：
将抗体Ab-1用50mM PB 7.0缓冲液稀释至5mg/mL，加入IdeS protease(终浓度：0.5U/μL)，37℃孵育过夜。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入了生物素标签在24362观测到峰(图1c)。
47-4.抗体Ab-1的位点确证：
47-4-1.样品制备
取抗体Ab-1(10μg)用50mM碳酸氢铵缓冲液稀释至90μL。加入1μL的PNGase F酶，37℃孵育4h。加入DTT(5mM)37℃孵育30min，然后加入碘乙酰胺(11mM)室温避光反应30min。随后用0.5μg胰酶在37℃下消化过夜。用2％的三氟乙酸终止消化，对消化后的蛋白质脱盐、干燥。随后进行进一步的LC-MS/MS分析。
47-4-2.LC-MS/MS装置及分析条件
纳米HPLC：EASY-nLC 1200 UHPLC系统(ThermoFisher Scientific)
质谱仪：Q Exactive HF-X质谱仪(ThermoFisher Scientific)
分析柱：C18柱(75μm×25cm,1.9μm)(Dr.Maisch GmbH)
流动相A：含0.1％甲酸的水溶液
流动相B：含0.1％甲酸的乙腈溶液
梯度条件(B％)：5％-90％(0-30min)
使用纳米喷雾源进行分析。在Orbitrap中以60,000的分辨率检测到完整的肽。MS扫描范围为350-2000m/z，自动增益控制值为1e6，最大进样时间为45ms。使用28的归一化碰撞能量设置为MS/MS选择肽段；获得了质量分辨率为30,000的串联质谱。在每次MS扫描中选择具有2-5个电荷的前12个母离子用于随后的MS/MS扫描，自动增益控制值为5e4，最大进样时间为100ms。动态排除设置为10s。
47-4-3.数据获取
使用蛋白质鉴定软件Mascot 2.3分析原始数据。将曲妥珠单抗序列添加到提供给搜索引擎的肽识别数据库中。将胰蛋白酶设定为消化酶(C端裂解为Lys和Arg残基)，最多允许有两个缺失的裂解。将甲硫氨酸残基的氧化(+15.9949Da)、对赖氨酸的新修饰(+226.0776Da)以及天冬酰胺的脱酰胺(+0.9840Da)设置为可变修饰。对半胱氨酸(+57.0215Da)上的氨基甲酸甲酯作为固定修饰进行搜索。母离子和碎片离子的质量容差分别设置为10ppm和0.05Da。所有修饰的肽都经过人工验证。
47-4-4.结果分析
从LC-MS/MS分析结果中可观测到曲妥珠单抗的采用胰蛋白酶消化的包含对赖氨酸的修饰部位的由33个氨基酸残基形成的肽 (THTCPPCPAPELLGGPSVFLFPPKPK(C10H14N2O2S)DTLMISR)的肽片段的MS谱(图1d)，根据CID谱确认了重链上第251位的赖氨酸残基的修饰。此外通过Mascot 2.3软件进一步分析，表明了高选择性地发生了对第251位赖氨酸残基的修饰(图1e)。
实施例48：叠氮化抗体Ab-2的制备及表征
48-1.抗体Ab-2的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-1(5eq,0.168mM)加入NaOAc缓冲溶液中(50mM,pH 5.5,含20％DMF)，置于37℃孵育30min，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到生物素化抗体Ab-2。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个叠氮乙酸的产物在146037观测到峰(图2a)。
48-2.抗体Ab-2的重链选择性确认：
将抗体Ab-2用50mM PB 7.4缓冲液稀释至5mg/mL，加入TCEP(20eq，终浓度：0.668mM)，37℃孵育20min。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入了生物素标签在49583观测到峰(图2b)。
48-3抗体Ab-2的Fc选择性确认：
将抗体Ab-2用50mM PB 7.0缓冲液稀释至5mg/mL，加入IdeS protease(终浓度：0.5U/μL)，37℃孵育过夜。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入了叠氮基团在24219观测到峰(图2c)。
实施例49：FITC定点修饰抗体Ab-3的制备及表征
抗体Ab-3
将曲妥珠单抗(5mg/mL)，化合物ATC-34(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到FITC定点修饰抗体Ab-3。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个荧光素的产物在147150观测到峰(图3)。
实施例50：甲基环丙烯定点修饰抗体Ab-4的制备及表征
将曲妥珠单抗(5mg/mL)，化合物ATC-35(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到甲基环丙烯定点修饰抗体Ab-4。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个甲基环丙烯的产物在146524观测到峰(图4)。
实施例51：DBCO定点修饰抗体Ab-5的制备及表征
将利妥昔单抗(5mg/mL)，化合物ATC-36(10eq,0.334mM)，加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到DBCO定点修饰抗体Ab-5。经ESI-TOFMS测定质量，原料曲妥珠单抗在144891观测到峰，关于反应产物，偶联2个DBCO的产物在145465观测到峰(图5)。
实施例52：DBCO定点修饰抗体Ab-6的制备及表征
将利妥昔单抗(5mg/mL)，化合物ATC-37(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反 应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到DBCO定点修饰抗体Ab-6。经ESI-TOFMS测定质量，原料曲妥珠单抗在144891观测到峰，关于反应产物，偶联2个DBCO的产物在145947观测到峰(图6)。
实施例53：两步法ADC-1的制备及表征
53-1.ADC-1的制备：
将叠氮化抗体Ab-2(5mg/mL)，药物连接子D1(15eq,0.5mM)分别加入PB缓冲溶液(50mM,pH 7.4,含20％DMF)中，置于37℃孵育过夜，LC-MS监测反应完全，使用ProteinA纯化、超滤管浓缩进而置换为1×PBS缓冲液后得到ADC-1。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D1的产物在148859观测到峰(图7a)。
53-2.ADC-1的重链选择性确认：
将ADC-1用50mM PB 7.4缓冲液稀释至5mg/mL，加入TCEP(20eq，终浓度：0.668mM)，37℃孵育20min。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D1在50992观测到峰(图7b)。
53-3.ADC-1的Fc选择性确认：
将ADC-1用50mM PB 7.0缓冲液稀释至5mg/mL，加入IdeS protease(终浓度：0.5U/μL)，37℃孵育过夜。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子在25629观测到峰(图7c)。
53-4.ADC-1的HIC分析(疏水色谱分析)
将ADC-1使用1×PBS稀释至5mg/mL，进样5μL。HIC柱使用TSKgel,(4.6×10cm,2.5μm)；流动相A：0.1M磷酸钠，1.5M硫酸铵缓冲液，pH＝7.0；流动相B：0.1M磷酸钠缓冲液，pH＝7.0，流速1mL/min，0-15min，线性梯度0-100％流动相B。检测波长为280nm。保留时间9.391min被认为是药物连接子D1纯度不够以引入的杂峰，10.354min被认为是在曲妥珠单抗中引入2个药物连接子D1的化合物(图7d)。
53-5.ADC-1的聚集稳定性分析(分子排阻色谱分析)
将ADC-1使用1×PBS稀释至1mg/mL，60℃ PCR仪加热48h，0h，6h，12h，24h，48h分别取点进行SEC分析，每个时间点取样15μL。SEC柱使用BioCore SEC-300column(7.8×300mm 5μm)，SEC分析使用流动相为150mM磷酸钠，pH＝7.4，流速为1mL/min，等度洗脱15min，检测波长为280nm。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.907min被认为是单体的峰(图7e)。
实施例54：两步法ADC-2的制备及表征
54-1.ADC-2的制备：
ADC-2的制备条件同ADC-1。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D2的产物在148636观测到峰(图8a)。
54-2.ADC-2的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D2在50811观测到峰(图8b)。
54-3.ADC-2的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子在25518观测到峰(图8c)。
54-4.ADC-2的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间9.071min被认为是在曲妥珠单抗中引入1个药物连接子D2的化合物，10.170min被认为是在曲妥珠单抗中引入2个药物连接子D2的化合物(图8d)。
54-5.ADC-2的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.923min被认为是单体的峰(图8e)。
实施例55：两步法ADC-3的制备及表征
55-1.ADC-3的制备：
ADC-3的制备条件同ADC-1。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D3的产物在151180观测到峰(图9a)。
55-2.ADC-3的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D3在52154观测到峰(图9b)。
55-3.ADC-3的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子D3在26790观测到峰(图9c)。
55-4.ADC-3的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间8.846min被认为是在曲妥珠单抗中引入1个药物连接子D3的化合物，9.611min被认为是在曲妥珠单抗中引入2个药物连接子D3的化合物(图9d)。
55-5.ADC-3的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.850min被认为是单体的峰(图9e)。
实施例56：两步法ADC-4的制备及表征
ADC-4的制备：
ADC-4的制备条件同ADC-1。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D4的产物在148377观测到峰(图10)。
实施例57：一步法ADC-5的制备及表征
57-1.ADC-5的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-6(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育3h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-5。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D5的产物在148311观测到峰(图11a)。
57-2.ADC-5的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D5在50718观测到峰(图11b)。
57-3.ADC-5的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子D5在25355观测到峰(图11c)。
57-4.ADC-5的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间8.687min被认为是在曲妥珠单抗中引入1个药物连接子D5的化合物，9.676min被认为是在曲妥珠单抗中引入2个药物连接子D5的化合物(图11d)。
57-5.ADC-5的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.940min被认为是单体的峰(图11e)。
实施例58：一步法ADC-6的制备及表征
58-1.ADC-6的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-7(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-6。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D6的产物在148139观测到峰(图12a)。
58-2.ADC-6的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D6在50633观测到峰(图12b)。
58-3.ADC-6的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子D6在25270观测到峰(图12c)。
58-4.ADC-6的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间8.623min被认为是在曲妥珠单抗中引入1个药物连接子D6的化合物，9.565min被认为是在曲妥珠单抗中引入2个药物连接子D6的化合物(图12d)。
58-5.ADC-6的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.883min被认为是单体的峰(图12e)。
实施例59：一步法ADC-7的制备及表征
59-1.ADC-7的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-8(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育10h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-7。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D7的产物在147938观测到峰(图13a)。
59-2.ADC-7的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D7在50533观测到峰(图13b)。
59-3.ADC-7的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子D7在25170观测到峰(图13c)。
59-4.ADC-7的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间8.623min被认为是在曲妥珠单抗中引入2个药物连接子D7的化合物(图13d)。
59-5.ADC-7的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.690min被认为是单体的峰(图13e)。
实施例60：一步法ADC-8的制备及表征
60-1.ADC-8的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-9(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-8。经ESI-TOFMS测定质量，原料曲妥珠单抗在145870观测到峰，关于反应产物，偶联2个药物连接子D8的产物在148225观测到峰(图14a)。
60-2.ADC-8的重链选择性确认：
TCEP处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的轻链峰，原料重链在49499观测到峰，关于产物，在重链中引入1个药物连接子D8在50677观测到峰(图14b)。
60-3.ADC-8的Fc选择性确认：
IdeS protease处理条件同ADC-1。经ESI-TOFMS测定质量，原料、产物观测到相同的Fab峰，原料Fc在24136观测到峰，关于产物，在Fc区域中引入1个药物连接子D8在25315观测到峰(图14c)。
60-4.ADC-8的HIC分析(疏水色谱分析)
HIC分析操作同ADC-1。保留时间8.370min被认为是在曲妥珠单抗中引入1个药物连接子D8的化合物，9.063min被认为是在曲妥珠单抗中引入2个药物连接子D8的化合物(图14d)。
60-5.ADC-8的聚集稳定性分析(分子排阻色谱分析)
聚集稳定性分析操作同ADC-1。保留时间5.4-8.0min被认为是聚集体的峰，保留时间8.623min被认为是单体的峰(图14e)。
实施例61：一步法ADC-9的制备及表征
ADC-9的制备：
将利妥昔单抗(5mg/mL)，化合物ATC-7(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-9。经ESI-TOFMS测定质量，原料利妥昔单抗在144898观测到峰，关于反应产物，偶联2个药物连接子D6的产物在147136观测到峰(图15)。
实施例62：一步法ADC-10的制备及表征
ADC-10的制备：
将帕妥珠单抗(5mg/mL)，化合物ATC-7(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-10。经ESI-TOFMS测定质量，原料帕妥珠单抗在145909观测到峰，关于反应产物，偶联2个药物连接子D6的产物在148175观测到峰(图16)。
实施例63：一步法ADC-11的制备及表征
ADC-11的制备：
将贝伐单抗(5mg/mL)，化合物ATC-7(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-11。经ESI-TOFMS测定质量，原料贝伐单抗在147020观测到峰，关于反应产物，偶联2个药物连接子D6的产物在149286观测到峰(图17)。
实施例64：一步法ADC-12的制备及表征
ADC-12的制备：
将特瑞普利单抗(5mg/mL)，化合物ATC-7(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-12。经ESI-TOFMS测定质量，原料特瑞普利单抗在147729观测到峰，关于反应产物，偶联2个药物连接子D6的产物在149996观测到峰(图18)。
实施例65：一步法ADC-13的制备及表征
ADC-13的制备：
将帕尼单抗(2mg/mL)，化合物ATC-7(10eq,0.134mM)加入1x PBS缓冲溶液中(含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-13。经ESI-TOFMS测定质量，原料帕尼单抗在144723观测到峰，关于反应产物，偶联2个药物连接子D6的产物在146992观测到峰(图19)。
实施例66：一步法ADC-14的制备及表征
ADC-14的制备：
将纳武单抗(2mg/mL)，化合物ATC-7(10eq,0.134mM)加入1x PBS缓冲溶液中(含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-13。经ESI-TOFMS测定质量，原料纳武单抗在144016观测到峰，关于反应产物，偶联2个药物连接子D6的产物在146313观测到峰(图20)。
实施例67：一步法ADC-15的制备及表征
ADC-15的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-40(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-15。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在148299观测到峰(图21a)。
HIC分析操作同ADC-1(图21b)。
实施例68：一步法ADC-16的制备及表征
ADC-16的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-41(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-16。经ESI-TOFMS 测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在148385观测到峰(图22a)。
HIC分析操作同ADC-1(图22b)。
实施例69：一步法ADC-17的制备及表征
ADC-17的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-42(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-17。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在150841观测到峰(图23a)。
HIC分析操作同ADC-1(图23b)。
实施例70：一步法ADC-18的制备及表征
ADC-18的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-43(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-18。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在150930观测到峰(图24a)。
HIC分析操作同ADC-1(图24b)。
实施例71：一步法ADC-19的制备及表征
ADC-19的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-44(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于37℃孵育2h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-19。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在149875观测到峰(图25a)。
HIC分析操作同ADC-1(图25b)。
实施例72：一步法ADC-20的制备及表征
ADC-20的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-45(10eq,0.334mM)加入PIPES缓冲溶液中(50mM,pH 7.4,含20％DMF)，置于室温孵育1h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-20。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在148882观测到峰(图26)。
实施例73：一步法DAR4ADC-21的制备及表征
ADC-21的制备：
将曲妥珠单抗(5mg/mL)，化合物ATC-46(10eq,0.334mM)加入醋酸钠缓冲溶液中(50mM,pH 5.5,含20％DMF)，置于37℃孵育1h，LC-MS监测反应完全。经超滤管浓缩，进而置换为1×PBS缓冲液后得到ADC-21。经ESI-TOFMS测定质量，原料曲妥珠单抗在145860观测到峰，关于反应产物在151842观测到峰(图27)。
测试例1
活性数据实验过程及结果分析
采用MTT法对上述部分ADC进行细胞活性及毒性测定。胰酶消化细胞，终止后离心收集，使用1-3mL培养基重悬细胞，混匀后进行细胞计数，补充培养基调整细胞悬液浓度。使用96孔板进行细胞实验，每孔加入细胞悬液90μL，使得待测细胞密度为～6000/孔，设置3个复孔，边缘孔加入100μL 1x PBS填充，同时设置调零孔(90μL培养基)以及对照孔(90μL细胞，不加药物)，5％CO2，37℃培养箱孵育12h，使细胞完全贴壁。每个样品设置9个浓度梯度，最高浓度为1μM，5倍梯度稀释，每孔中加入相应浓度梯度ADC分子10μL，6个对照孔中也分别加入10μL培养基。5％CO2，37℃孵育72h后，每孔加入10μL MTT溶液(5mg/mL)，继续培养4h，随后每孔中加入90μL SDS裂解液，37℃孵育7小时后，使用酶联免疫检测仪测定各孔570nm的吸收值，使用GraphPad Prism 8软件进行数据处理(图28)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。





Claims (16)

  1. 一种带有配体亲和导向基团的偶联物，其特征在于，所述偶联物如式I所示：
    AT-CL-R   (I)
    其中，
    AT为针对目标蛋白(TP)的亲和性部分；
    CL为可裂解片段；并且所述CL中存在如下所示的二价片段
    其中，A1各自独立地为任选取代的C1-10亚烷基、任选取代的C6-10芳基、任选取代的5至10元杂芳基：
    R为需要被修饰到所述目标蛋白的基团；
    除非特别说明，所述的取代是指基团中一个或多个H被选自下组的取代基取代：卤素、C1-6烷基、C1-6卤代烷基。
  2. 如权利要求1所述的偶联物，其特征在于，所述的目标蛋白为具有Fc结构域的抗体或融合蛋白；和/或AT为具有和抗体Fc具有结合能力的多肽。
  3. 如权利要求1所述的偶联物，其特征在于，AT为衍生自包含如式II所示序列的Fc结合多肽的核心序列：
    (Xs1)t1-Cys-Aa1-Aa2-Aa3-Aa4-Aa5-Aa6-Aa7-Aa8-Aa9-Cys-(Ys2)t2   (II)
    其中，X所在位置为多肽的N端，Y所在位置为多肽的C端；
    s1＝0、1、2或3；
    s2＝0、1、2或3；
    X和Y各自独立地为氨基酸残基；
    t1和t2各自独立为0-10的整数；
    Cys为半胱氨酸残基；
    Aa1、Aa2、Aa3、Aa5、Aa7、Aa8和Aa9各自独立地为氨基酸残基；
    Aa4和Aa6各自独立地为氨基酸残基并且Aa4和Aa6中的至少一个为侧链含NH2基团的氨基酸残基或侧链含-COOH基团的氨基酸残基；
    和/或
    CL如下所示
    其中，
    *代表与R连接的位置
    W1选自下组：无(单键)、-NH-、-C(O)-、-C(O)-NH-和-NH-C(O)-；
    W2为无(单键)、-NH-、或-C(O)-；
    L1为无(单键)或二价连接基团；
    A1如权利要求1中定义；
    和/或
    R为包含一个或多个选自下组的活性基团的部分：可被进一步修饰的活性基团、具有生物活性和/或可检测的活性基团。
  4. 如权利要求3所述的偶联物，其特征在于，
    所述可被进一步修饰的活性基团是指可进行生物正交反应的活性基团；较佳地，所述可进行生物正交反应的活性基团选自：叠氮基、醛基、硫醇基、炔基、烯基、卤素、四嗪基、硝酮基、羟胺基、腈基、肼基、酮基、硼酸基、氰基苯并噻唑基、烯丙基基、膦基、马来酰亚胺基、二硫基、硫酯基、α-卤代羰基基、异腈基、斯德酮基、硒基、共轭二烯基、磷酸基、环炔基及环烯基，或其组合；和/或
    所述具有生物活性和/或可检测的活性基团选自：美登素、DM-1、DM-4、MMAE、MMAF、SN-38、Dxd、PBD及其类似物、鹅膏蕈碱、长春新碱、长春碱、长春瑞滨、VP-16、喜树碱、紫杉醇、多烯紫杉醇、埃博霉素A、埃博霉素B、诺考达唑、秋水仙碱、雌莫司汀、西马多丁、艾榴塞洛素、，荧光试剂、单糖、二糖、寡糖、多聚乙二醇(PEG)、免疫激动剂、细胞毒、放射性治疗物、分子影像试剂，或其组合。
  5. 如权利要求1所述的偶联物，其特征在于，所述的偶联物如式III-A1、III-A2或III-A3所示，
    其中，
    标注有1、2、3、4、5、7、8和9的圆圈分别如权利要求3中式II中Aa1、Aa2、Aa3、Aa4、Aa5、Aa7、Aa8、和Aa9的定义；
    靠近标注有1的圆圈的末端为多肽N端，靠近标注有9的圆圈的末端为多肽C端；
    Ac为N端保护基或不存在；
    各个空白圆圈如式II中的X和Y定义；
    为-W2-L1-W1-A1-；
    圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
    圆圈K为赖氨酸残基的主链部分；圆圈D为天冬氨酸残基(D)的主链部分、圆圈E为谷氨酸残基(E)的主链部分；
    R如权利要求1或3中定义。
  6. 如权利要求1所述的偶联物，其特征在于，所述的偶联物如式III-B1、III-B2或III-B3所示，
    其中，
    标注有1、2、3、5、6、7、8和9的圆圈分别如权利要求3中式II中Aa1、Aa2、Aa3、Aa5、Aa6、Aa7、Aa8和Aa9的定义；
    靠近标注有1的圆圈的末端为多肽N端，靠近标注有9的圆圈的末端为多肽C端；
    Ac为N端保护基或不存在；
    各个空白圆圈如式II中的X和Y定义；
    为-W2-L1-W1-A1-；
    圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
    圆圈K为赖氨酸残基的主链部分；圆圈D为天冬氨酸残基(D)的主链部分、圆圈E为谷氨酸残基(E)的主链部分；
    R如权利要求1或3中定义。
  7. 如权利要求1所述的偶联物，其特征在于，所述偶联物如式III-A1-1、 III-A2-1或III-A3-1所示，
    其中，
    圆圈R为多肽N端，圆圈H为多肽C端；
    Ac为N端保护基或不存在；
    为-W2-L1-W1-A1-；
    圆圈R为精氨酸残基、圆圈G为甘氨酸残基、圆圈N为天冬酰胺残基、圆圈A为丙氨酸残基、圆圈Y为酪氨酸残基、圆圈H为组氨酸残基、圆圈L为亮氨酸残基、圆圈V为缬氨酸残基、圆圈W为色氨酸残基、圆圈T为苏氨酸残基；
    圆圈C为半胱氨酸残基，-S-S-代表由半胱氨酸残基的侧链-SH形成的二硫键；
    圆圈K为赖氨酸残基的主链部分；圆圈D为天冬氨酸残基的主链部分、圆圈E为谷氨酸残基的主链部分；
    R如权利要求1或3中定义。
  8. 如权利要求1-7任一所述的偶联物，其特征在于，所述的偶联物选自表A1和表A2。
  9. 一种对蛋白进行修饰的方法，其特征在于，包括步骤：
    (1)提供如权利要求1-8任一所述的偶联物；
    (2)使需要修饰的目标蛋白与所述的偶联物接触，并使得所述偶联物与蛋白上侧链氨基和/或末端的氨基进行反应，从而得到经修饰的目标蛋白。
  10. 如权利要求9所述的方法，其特征在于，所述的修饰通过一步反应即可得到得到经修饰的目标蛋白。
  11. 一种如式IV所示的经位点选择性修饰的蛋白，其特征在于，
    TP-(NHCO-R)p  (IV)
    其中，TP为目标蛋白部分、R如权利要求1或3中定义，下标p为1-8的整 数。
  12. 一种抗体药物偶联物，其特征在于，所述抗体药物偶联物如式V-A所示：
    Ab-(NHCO-L2-(L3-D)o)p  (V-A)
    其中，Ab为抗体，下标o为1-10的整数，下标p为1-8的整数，L2和L3各自独立地为无或连接片段，D各自独立地为相同或不同的具有生物活性和/或可检测的活性基团。
  13. 如权利要求12所述的抗体药物偶联物，其特征在于，基团D定点偶联在抗体恒定区(Fc区域)；较佳地，基团D定点偶联在人IgG Fc区域中的由246-248位氨基酸残基构成的区域。
  14. 如权利要求12所述的蛋白，其特征在于，所述抗体药物偶联物选自下组：ADC-1、ADC-2、ADC-3、ADC-4、ADC-5、ADC-6、ADC-7、ADC-8、ADC-9、ADC-10、ADC-11、ADC-12、ADC13、ADC-14、ADC-15、ADC-16、ADC-17、ADC-18、ADC-19、ADC-20和ADC-21；较佳地，所述抗体药物偶联物选自下组：ADC-5、ADC-6、ADC-7、ADC-8、ADC-9、ADC-10、ADC-11、ADC-12、ADC13、ADC-14、ADC-15、ADC-16、ADC-17、ADC-18、ADC-19、ADC-20和ADC-21。
  15. 一种如权利要求1所述的偶联物的用途，其特征在于，用于位点选择性修饰蛋白。
  16. 一种如权利要求1所述的偶联物的用途，其特征在于，用于制备抗体药物偶联物(ADC)，较佳地，所述用途不依赖于生物正交反应，所述偶联物可经一步反应直接将需要被修饰到目标蛋白的药物分子，以位点选择性方式修饰至所述抗体上。
PCT/CN2023/083443 2022-03-25 2023-03-23 亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用 WO2023179723A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210307276.9A CN116836235A (zh) 2022-03-25 2022-03-25 亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用
CN202210307276.9 2022-03-25

Publications (1)

Publication Number Publication Date
WO2023179723A1 true WO2023179723A1 (zh) 2023-09-28

Family

ID=88100066

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/083443 WO2023179723A1 (zh) 2022-03-25 2023-03-23 亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用

Country Status (2)

Country Link
CN (1) CN116836235A (zh)
WO (1) WO2023179723A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010078376A2 (en) * 2008-12-30 2010-07-08 Ventana Medical Systems, Inc. Fc-specific polymer-conjugated antibodies and their diagnostic use
CN110637036A (zh) * 2017-04-28 2019-12-31 味之素株式会社 具有针对可溶性蛋白质的亲和性物质、切割性部分及反应性基团的化合物或其盐
CN112261954A (zh) * 2018-06-14 2021-01-22 味之素株式会社 具有针对抗体的亲和性物质和生物正交性官能团的化合物或其盐
CN112262152A (zh) * 2018-06-14 2021-01-22 味之素株式会社 具有针对抗体的亲和性物质、切割性部分及反应性基团的化合物或其盐
CN113227124A (zh) * 2018-10-31 2021-08-06 味之素株式会社 具有针对抗体的亲和性物质、切割性部分及反应性基团的化合物或其盐
AU2020210506A1 (en) * 2019-01-23 2021-09-16 AbTis Co., Ltd. Compound for preparation of antibody-payload conjugate and use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010078376A2 (en) * 2008-12-30 2010-07-08 Ventana Medical Systems, Inc. Fc-specific polymer-conjugated antibodies and their diagnostic use
CN110637036A (zh) * 2017-04-28 2019-12-31 味之素株式会社 具有针对可溶性蛋白质的亲和性物质、切割性部分及反应性基团的化合物或其盐
CN112261954A (zh) * 2018-06-14 2021-01-22 味之素株式会社 具有针对抗体的亲和性物质和生物正交性官能团的化合物或其盐
CN112262152A (zh) * 2018-06-14 2021-01-22 味之素株式会社 具有针对抗体的亲和性物质、切割性部分及反应性基团的化合物或其盐
CN113227124A (zh) * 2018-10-31 2021-08-06 味之素株式会社 具有针对抗体的亲和性物质、切割性部分及反应性基团的化合物或其盐
AU2020210506A1 (en) * 2019-01-23 2021-09-16 AbTis Co., Ltd. Compound for preparation of antibody-payload conjugate and use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Methods Mol Biol", vol. 2033, 23 July 2019, HUMANA PRESS, Totowa, NJ, ISBN: 978-1-62703-562-0, article TANG FENG, SHI WEI, HUANG WEI: "Homogeneous Antibody–Drug Conjugates via Glycoengineering", pages: 221 - 238, XP009543195, DOI: 10.1007/978-1-4939-9654-4_15 *
WANG, YUJIE: "Antibody-drug Conjugates: Design and Clinical Progress", ACTA PHARMACEUTICA SINICA, vol. 51, no. 8, 12 August 2016 (2016-08-12), XP055497140 *

Also Published As

Publication number Publication date
CN116836235A (zh) 2023-10-03

Similar Documents

Publication Publication Date Title
JP7417432B2 (ja) 新規リンカー、その製造方法およびその応用
CN108452321B (zh) 一种带酸性自稳定接头的抗体-药物偶联物
JP7387440B2 (ja) Ｍｔ１－ｍｍｐに結合するためのペプチドリガンド
TWI680765B (zh) 自行穩定之接合劑共軛物
EP2925368B1 (en) Methods for making conjugates from disulfide-containing proteins
US20060199770A1 (en) Functionalized carbon nanotubes, a process for preparing the same and their use in medicinal chemistry
CN105764530B (zh) 用于对酮修饰的多肽类进行肟缀合的方法
CN109689112A (zh) 化学选择性的巯基与烯基或炔基磷酰胺的偶联
CN108602878B (zh) C端赖氨酸缀合免疫球蛋白
CN107043406B (zh) 化合物、连接子-药物、及配体-药物耦合体
AU2017340314B2 (en) Cysteine modified antibody-drug conjugate and preparation method thereof
WO2023179723A1 (zh) 亲和片段导向的可裂解片段，其设计、合成及在制备定点药物偶联物中的应用
CN109125736B (zh) 非天然鹅膏毒肽类抗体偶联物
Hudecz 13 Synthesis of Peptide Bioconjugates
WO2018233572A1 (zh) 半胱氨酸改造的抗体-毒素偶联物(tdc)定点偶联位点筛选
WO2023171809A1 (ja) Ｆｃ含有分子修飾試薬の製造方法
WO2022080385A1 (ja) ペプチド、およびペプチドを用いる抗体の修飾
WO2024067754A1 (zh) 一种带有高稳定性亲水连接单元的奥瑞他汀类药物及其偶联物
RU2793125C2 (ru) Конъюгат антитело-лекарственное средство, имеющий кислотный самостабилизирующийся участок соединения
WO2023004687A1 (zh) 一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物
CN117298291A (zh) Fc结合肽偶联物、靶向TROP2的定点偶联ADC及其制备方法和应用
CN115192732A (zh) 一种dna毒性二聚体化合物及其偶联物
KR20140127224A (ko) 펩타이드 접합체 및 연결체의 개선된 제조 방법
CN116710138A (zh) 肽交联剂以及通过该交联剂交联的交联肽

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23773968

Country of ref document: EP

Kind code of ref document: A1