WO2015137883A1 - Procédé de conjugaison de protéines - Google Patents

Procédé de conjugaison de protéines Download PDF

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Publication number
WO2015137883A1
WO2015137883A1 PCT/SG2015/050033 SG2015050033W WO2015137883A1 WO 2015137883 A1 WO2015137883 A1 WO 2015137883A1 SG 2015050033 W SG2015050033 W SG 2015050033W WO 2015137883 A1 WO2015137883 A1 WO 2015137883A1
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polypeptide
analog
lysine residue
optionally substituted
group
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PCT/SG2015/050033
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English (en)
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Chuan-Fa Liu
Renliang YANG
Xiaobao BI
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Nanyang Technological University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
    • C12Y601/01026—Pyrrolysine-tRNAPyl ligase (6.1.1.26)
    • 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
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention generally relates to the field of biochemistry.
  • it refers to a method of conjugating two or more polypeptides.
  • the method employs a protected lysine, or analog thereof, incorporated into a polypeptide during translation, as a temporary and orthogonal lysine precursor, or analog thereof, for the installation of a conjugatable moiety, which facilitates ligation between two polypeptides.
  • Polypeptide conjugation is generally used to generate custom-made polypeptides in order to study their regulatory role as hormones and inhibitors and their involvement in immunological recognition.
  • the significant biological role of polypeptides makes it important to understand their interactions with the receptors to which they bind. This role involves targeted delivery of therapeutic, diagnostic, and research agents to targeted cells in the patient in order to improve their efficacy and to minimize potentially adverse side effects.
  • therapeutic, diagnostic, and research agents, or their carriers are chemically conjugated to polypeptides that can selectively bind to targeted cells.
  • the resulting conjugates are structurally and functionally heterogeneous because they are formed randomly via chemical reactions with few of several available chemical groups, usually ⁇ -amino groups of lysine residues, in the targeting protein. Since random conjugation does not discriminate between functionally important and dispensable amino acid residues in the targeting protein, it would be desirable to custom-develop and optimize the reaction to increase the proportion of functionally active polypeptides.
  • ubiquitination is one of the most important protein post-translational modifications in eukaryotic cells. It is involved in almost all of the cellular processes, including protein degradation and the regulation of gene expression. There is mounting evidence that ubiquitination process is related to many human diseases like Alzheimer's and Parkinson's diseases. To study and understand the physiological roles of ubiquitination as well as the roles of ubiquitination in disease development, it is important to generate homogenously ubiquitinated proteins.
  • ubiquitination process is catalyzed through the consecutive actions of three enzymes, ubiquitin-activating enzymes (El), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3). Due to the difficulties in identifying or isolating the substrate-specific ligases, enzymatic ubiquitination in vitro often faces problems like reaction inefficiency, requirement of several enzymes and product heterogeneity. A chemical approach to protein conjugation, in particular ubiquitination could circumvent these problems.
  • polypeptide conjugations with non-native linkages including a disulfide bond, oxime, triazole, thioether, as well as isopeptide bond with the C-terminal Gly76 mutated to D-Cys or Ala, have also been reported. While these non-native polypeptide conjugates could be used for certain studies, they do not reflect all the physiological properties of their native counterparts.
  • a method of conjugating two or more polypeptides comprises incorporating during translation a target lysine residue, or analog thereof, protected by a first protecting group in a first polypeptide.
  • the method comprises deprotecting said target lysine residue, or analog thereof, and conjugating a conjugatable moiety group to the target lysine residue, or analog thereof.
  • the target lysine residue, or analog thereof is conjugated to a second polypeptide.
  • conjugated polypeptide produced according to the method as described herein.
  • lysine residue analog refers to an amino acid residue, which has one or two more, or one or two less -CH2- groups in its side chain when compared to a lysine residue.
  • the lysine residue analog may have a side chain comprising or consisting of an aliphatic linear or branched C2 -5 -alkyl or C 2 . 4-alkyl bearing a terminal nitrogen functionality. This functionality can include azides, amines and carbamates.
  • the lysine analog may be a valine residue.
  • lysine in the context of amino acids such as lysine (e.g. lysine residue) refers to an amino acid moiety bearing an aliphatic side chain (e.g. (CH 2 ) 4 NH 3 + in case of lysine side chain) associated with the amino acid backbone (NH 2 -CH-COOH). Attached to this side chain may be a functionality which may not be part of the natural occurring amino acid, and which is capable of chemical manipulation, such as an azide, amino or carbamate.
  • the term “residue” in context with the conjugatable moiety refers to parts of the conjugatable moiety which are attached to the conjugation site, but do not form part of the conjugated polypeptide.
  • conjugatable moiety refers to a chemical moiety, which is attached to an amine functionality and which is capable of influencing the selectivity of a particular chemical reaction.
  • the conjugatable moiety may be divided into 3 groups: one group may remain in the product to form an isopeptide bond, one group may be removed in the reaction of the peptide conjugation, and one group may be removed in a deprotection reaction.
  • the group which remains to form the isopeptide bond may comprise or consist of -C(0)CH 2 N-.
  • the group which may be removed in the peptide conjugation reaction may comprise an auxiliary group.
  • the group which may be removed in a deprotection reaction may comprise or consist of -C3 ⁇ 4-.
  • auxiliary group refers to any chemical moiety, which is temporarily incorporated into an organic synthesis for the purpose of altering the selectivity of a subsequent reaction. After it has served its purpose, it can be removed at a later stage in the synthesis.
  • isopeptide bond refers to an amide bond between a carboxyl group and an amino group, at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone.
  • An isopeptide bond may form within a single protein or may occur between two peptides or a peptide and a protein.
  • an isopeptide may form intramolecularly within a single protein or intermolecularly i.e. between two peptide/protein molecules.
  • isopeptide bonds are Gly- ⁇ -Lys isopeptide bond or Ala-s-Lys isopeptide bond.
  • Translation refers to the process by which the amino acid sequence of a polypeptide chain is derived from the nucleotide sequence of an mRNA molecule associated with a ribosome.
  • Polypeptide refers to a molecule or moiety containing two or more amino acids bound through a peptide linkage. Examples can include, but are not limited to proteins such as antibodies, enzymes, lectins and receptors; lipoproteins and lipopolypeptides; and glycoproteins and glycopolypeptides.
  • protecting group refers to a species which prevents a portion of a molecule from undergoing a specific chemical reaction, but which is removable from the molecule following completion of that reaction.
  • a “protecting group” is used in the conventional chemical sense as a group which reversibly renders unreactive a functional group under certain conditions of a desired reaction. After the desired reaction, protecting groups may be removed to deprotect the protected functional group. All protecting groups should be removable (and hence, labile) under conditions which do not degrade a substantial proportion of the molecules being synthesized.
  • the term “deprotect” or deprotection” refers to the removal of at least one protecting group from the polypeptide of interest.
  • Receptor refers to a polypeptide that binds (or ligates) a specific molecule (ligand) and, when expressed in a cell, may initiate a response in the cell. Receptors may specifically bind ligands without a signaling response.
  • RNA refers to a polynucleotide or oligonucleotide which comprises at least one ribonucleotide residue.
  • tRNA or "tRNA molecule” refers to a specialized RNA molecule, which acts as a template to direct the synthesis of the polypeptide.
  • tRNA synthetase refers to an aminoacyl-tRNA synthetase, which is an enzyme specifically linking a particular amino acid to a particular tRNA, thereby implementing the genetic code.
  • tRNA synthetase/fRNA pair refers to the pair of a tRNA molecule and a tRNA synthetase specific to that tRNA molecule.
  • ubiquitin-like moiety refers to molecules other than ubiquitin, which confer similar modes of functional protein modification and are henceforth called ubiquitin- like proteins, molecules, or modifiers (ULMs).
  • ULMs ubiquitin-like protein modifiers
  • the family heritage of ubiquitin-like protein modifiers (ULMs) is not so much by sequence homology but rather by a common 3D structure, the ubiquitin fold, and a C-terminal glycine residue, whose carboxyl group is the site of attachment to the lysine residue of substrates via isopeptide bond formation. Hence, they are conjugated to proteins and function in "ubiquitin-like" manner. At least 10 different ULMs exist in mammals.
  • Examples can include, but are not limited to Interferon-induced 17 kDa protein, ISG15 (UCRP), UniProt P05161 (2 ubiquitins); FUBl (MNSFp), UniProt P35544; NEDD8 (Rubl), UniProt Q15843; FAT 10 (2 ubiquitins), Ubiquitin D; Small ubiquitin-related modifier 1, SUMO-1 (SMT3C, GMP1, UBL1), UniProt P63165; Small ubiquitin-related modifier 2, SUMO-2 (SMT3B), UniProt P61956; Small ubiquitin-related modifier 3, SUMO-3 (SMT3A), UniProt P55854; Autophagy protein 8, Apg 8, LC3 Antibody; Autophagy protein 12, Apg 12; Ubiquitin-related modifier- 1, Urml ; Ubiquitin-like protein 5, UBL5 (Hubl), UniProt Q9BZL1 ; and Ubiquitin- fold modifier
  • Conjugation is defined as the process of linking, connecting, associating, bonding (covalently or non-covalently) or any combination thereof, two or more smaller entities to form a larger entity. This term is intended to encompass a covalent bond.
  • alkyl refers to monovalent (“alkyl”) and divalent (“alkylene”) straight chain or branched chain saturated aliphatic groups having from 1 to 24 carbon atoms, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
  • alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1,2-dimethylpropyl, 1,1- dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3- methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1- methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpent
  • alkenyl refers to a branched, unbranched or cyclic (e.g. in the case of C5 and C6) hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containing at least one double bond, such as ethenyl, vinyl, allyl, octenyl, decenyl, and the like.
  • alkynyl refers to a branched or unbranched hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containing at least one triple bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.
  • alkoxy or variants such as “alkoxide” as used herein refers to an -O-alkyl radical. Representative examples include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.
  • amino includes an amine group (i.e., -NH 2 ) or a substituted amine group.
  • heteroalkyl refers to a straight-or bra ched-chain alkyl group having from 2 to 12 atoms in the chain, one or more of which is a heteroatom selected from S,0, and N.
  • exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, and the like.
  • heterocycloalkenyl refers to a non-aromatic, cyclic moiety having at least one ring heteroatom and at least one double bond in the ring, such as pyranyl.
  • halogen and halo refer to a fluoro, chloro, bromo, or iodo moiety.
  • aromatic group refers to monovalent (“aryl”) and divalent (“arylene”) single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like.
  • heteroaryl refers to an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. "Heteroaryl” may also include a heteroaryl as defined above fused to an aryl as defined above.
  • Non- limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[l,2-a]pyridinyl, imidazo[2,l-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl
  • heteroaryl also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like. Heteroaryl groups may be optionally substituted.
  • carbocyclyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, e.g. .
  • a carbocyclyl comprises three to ten carbon atoms.
  • a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond.
  • Carbocyclyl may be saturated, (i.e., containing single C-C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds.)
  • a fully saturated carbocyclyl radical is also referred to as "cycloalkyl.”
  • monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • cycloalkenyl refers to a non-aromatic mono or multicyclic ring system comprising about 3 to about 10 carbon atoms which contains at least one carbon- carbon double bond.
  • suitable monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cyclohepta-1 ,3-dienyl, and the like.
  • Non-limiting example of a suitable multicyclic cycloalkenyl is norbornylenyl, as well as unsaturated moieties of the examples shown above for cycloalkyl. Cycloalkenyl groups may be optionally substituted.
  • OMe refers to an alkoxy group, wherein Me stands for CH 3 .
  • Such groups may be, for example, halogen, hydroxy, oxo, cyano, nitro, alkyl, alkoxy, haloalkyl, haloalkoxy, arylalkoxy, alkylthio, hydroxyalkyl, alkoxyalkyl, cycloalkyl, cycloalkylalkoxy, alkanoyl, alkoxycarbonyl, alkylsulfonyl, alkylsulfonyloxy, alkylsulfonylalkyl, arylsulfonyl, arylsulfonyloxy, arylsulfonylalkyl, alkylsulfonamido, alkylamido, alkylsulfonamidoalkyl, alkylamidoalkyl, arylsulfonamido, arylcarboxamido, arylsulfonamidoalkyl, arylcarboxamid
  • arylalkyl When compounded chemical names, e.g. "arylalkyl” and “arylimine” are used herein, they are understood to have a specific connectivity to the core of the chemical structure.
  • the group listed farthest to the right e.g. alkyl in “arylalkyl”
  • alkyl in “arylalkyl” is the group that is directly connected to the core.
  • an "arylalkyl” group for example, is an alkyl group substituted with an aryl group (e.g. phenylmethyl (i.e., benzyl)) and the alkyl group is attached to the core.
  • An “alkylaryl” group is an aryl group substituted with an alkyl group (e.g., p- methylphenyl (i.e., p-tolyl)) and the aryl group is attached to the core.
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the present invention refers to a method of linking the C-terminus of a polypeptide to the lysine side chain or analog thereof of another polypeptide through an isopeptide bond.
  • the lysine residue, or analog thereof is incorporated or built in during translation and is protected by a first protection group in said first polypeptide (step a).
  • a polypeptide may be produced having a target lysine, or analog thereof, protected site-specifically by genetically encoded protection of that residue.
  • said first protection group is deprotected.
  • a conjugatable moiety group is subsequently conjugated to the deprotected target lysine residue, or analog thereof (step c).
  • Conjugation of the target lysine residue, or analog thereof, and a second protein gives the conjugated polypeptide (step d).
  • the method is depicted in a simplified and highly schematized manner in Figure 1. It consists of a translation step a), in which a protected lysine residue, or analog thereof (Lys), is being incorporated into a first polypeptide (1. PP), followed by step b), wherein said target lysine residue, or analog thereof, is being deprotected in order to cany out step c), the conjugation with a conjugatable moiety (CM) to the target lysine residue, or analog thereof, and step d), the conjugation of a second polypeptide (2. PP) to this target lysine, or analog thereof, to form the conjugated polypeptide.
  • CM conjugatable moiety
  • the first step (step a) entails the provision of a nucleic acid which is encoding the first polypeptide.
  • said genetic incorporation preferably uses an orthogonal or expanded genetic code, in which one or more specific orthogonal codons have been allocated to encode the specific lysine residue, or analog thereof, with the lysine side chain protected so that it can be incorporated by using an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA synthetase/tRNA pair can in principle be any such pair capable of charging the tRNA with the protected lysine or analog thereof and capable of incorporating that protected lysine into the polypeptide chain in response to the orthogonal codon.
  • An "orthogonal tRNA synthetase/tRNA pair” is hereby understood in its biochemical meaning as the requirement for molecular recognition between host and guest, hereby exhibiting molecular complementarity. It should not be confused with the term Orthogonal protecting group', which is commonly used in organic chemistry and is explained further below.
  • the herein provided nucleic acid has an orthogonal codon encoding the incorporated lysine or analog thereof.
  • Step a) can be performed in the following manner:
  • the method details the translation of a nucleic acid in the presence of an orthogonal tRNA synthetase and tRNA pair, which is capable of recognizing said orthogonal codon and incorporating said target lysine residue, or analog thereof, protected by a first protecting group into the first polypeptide.
  • the protected lysine residue, or analog thereof may be genetically incorporated through a variety of synthetases, with the only requirement that it provides the same tRNA charging function employed by the presented method.
  • the tRNA synthetase may include, but is not limited to natural occurring synthetases or engineered synthetases.
  • the tRNA synthetase may be from any species such as from archea, for example from Methanosarcina barkeri MS, Methanosarcina barkeri str.and Fusaroor Methanosarcina mazei Gol.
  • the tRNA synthetase may be from bacteria, for example from Desulfitobacterium hafniense PCP1 or Desulfotomaculum acetoxidans DSM 771.
  • the tRNA synthetase may be from eukaryotes, such as yeast.
  • the process may be conducted using engineered methioninyl- tRNA synthetase, or MetRS, in Met-auxotrophic E. Coli cells. It may also be conducted using pyrrolysine tRNA synthetases, with the species being from Methanosarcina barkeri MS, Methanosarcina barkeri str. or Fusaroor Methanosarcina mazei Gol .
  • the pyrrolysine tRNA synthetase is especially useful in introducing protecting groups such as a CBz or Boc groups on the lysine residue, or analog thereof.
  • the process may be performed when the tRNA synthetase is MetRS with LI 3 A mutation (MetRSL13A).
  • MetRSL13A is especially useful for introducing an azide as a protection group on the lysine residue, or analog thereof.
  • the MetRSL13A system is useful to incorporate an azidonorleucin or azidonorvaline into the first polypeptide.
  • amino acid residues may be introduced after the target lysine residue, or analog thereof, within the polypeptide.
  • These amino acid residues can include, but are not limited to, all naturally occurring amino acids. In one example, they can include, but are not limited to, those bearing non-polar or polar residual moieties. In another example, such amino acids may be alanine or serine.
  • the introduction of additional amino acids after incorporation of the protected lysine residue, or analog thereof, is useful to facilitate removal of the initiator Met.
  • the target lysine, or analog thereof, (NPG 1 ) thus incorporated into the polypeptide is protected by a protecting group (e.g. step a of the method, Figure 1 and 2).
  • Said protecting group is different from the protecting group used to protect the further lysine(s) (NPG 2 ).
  • the method of deprotection used to selectively remove the protecting group from the target lysine or analog thereof in step b) of the method is in one example performed so as not to deprotect the further lysines at the same time.
  • This applied protecting groups may be orthogonal to each other.
  • the term "orthogonal protecting group” hereby refers to at least two different protecting groups, one of which can be removed under conditions that do not affect any others.
  • Chemical protecting agents for lysine side chains, or analogs thereof, are varied and can be chosen by the person skilled in the art depending on the type of deprotection methods to be used. However, said protecting agent would be chosen so as to allow the lysine residue, or analog thereof, to be incorporated genetically and thus allow it to be incorporated by an orthogonal tRNA synthetase/tRNA pair in a cell. Protection groups to be incorporated during step a) can include, but are not limited to Carboxybenzyl, ier/-Butyloxyearbonyl and an azide.
  • the target (or protected) lysine residue, or analog thereof, thus incorporated may refer to a target (or protected) amino acid residue, wherein the side chain consists of an aliphatic linear or branched C 2 -5-alkyl chain bearing a terminal nitrogen functionality.
  • the side chain may be an amino acid side chain, in which the aliphatic side chain has 2 to 5 carbon atoms.
  • the target amino acid residue may be selected from target amino acid residues such as a target lysine or target valine residue.
  • the target (or protected) lysine residue, or analog thereof, incorporated by this method may be azidonorleucine (Anl) or azidonorvaline (Anv).
  • the protecting group of the amine would be an azide and the amino acid targeted would be lysine ( Figure 3) or valine, which can be later selectively reduced to an amine, thereby revealing the free amine for further manipulation.
  • the polypeptide with its site-specific protected lysine, or analog thereof can then be chemically treated to protect each of the remaining (unprotected) lysines with a second protecting group ( Figure 2, step aa).
  • Figure 2, step aa This enables the target lysine, or analog thereof, to be selectively deprotected, leaving the other protecting groups on the additional lysines intact.
  • the target lysine, or analog thereof is site-specifically deprotected and can therefore be modified whilst the other lysines remain unaffected.
  • the first polypeptide after step a) may be treated with any amine protecting group, and may be orthogonal to the first protecting group incorporated under step a).
  • the first protecting group is an azide
  • the second protecting group may be any group which is stable under reducing reaction conditions as known to the person skilled in the art, such as treatment with a reducing agent including, but not limited to, tm(2-carboxyethyl)phosphine, Platinum oxide, boron trifluoride diethearate and indium trichloride.
  • the following protecting agents and ensuing protecting groups are contemplated: fluorenylmethyloxycarbonyl chloride, resulting in a fluorenylmethyloxycarbonyl (FMoc) protecting group, di(feri-butyloxy) anhydride, resulting in a tert-butyloxy (Boc) protecting group and benzyl chloroformate or N- benzyloxycarbonyloxy)succinimide, resulting in a carboxybenzyl (CBz) protecting group.
  • the second protecting group may be introduced with di(feri-butyloxy) anhydride as a reagent.
  • a base for this protection method may be selected from a variety of bases known to the person skilled in the art.
  • the base may be chosen from inorganic bases such as sodium hydroxide or potassium hydroxide. It may also be a Lewis base, in particular it may be a nitrogen base such as pyridine, triethyl amine, quinuclidine and N- ethyldiisopropylamine (DIEA). In one example, N-ethyldiisopropylamine (DIEA) was used in the protection reaction, which was added in a small amount to avoid side products.
  • This protection process may occur before step b) in this method, which is the selective deprotection of the incorporated protected lysine residue, or analog thereof, in the first polypeptide to reveal a free amine on the lysine residue, or analog thereof.
  • This step b) of the method may occur before all previously protected free amine residues from additional lysines or N-terminus' amines are deprotected ( Figure 1, 2 and 3).
  • the further lysines or N-terminal amino groups may have their side chains protected to allow for the specific modification of the target lysines, or analog thereof. This is accomplished using a reaction where the protecting group can reach, or at least approach, saturation (100%) of the further lysines present in the polypeptidic chain.
  • the second protecting group for the remaining lysines in the first polypeptide may be selected from the group consisting of FMoc, Boc or CBz.
  • the first polypeptide is further dissolved in an appropriate solvent.
  • This solvent may include, but is not limited to, the group of polar aprotic solvents, in particular from a variety such as dichloromethane, tetrahydrofuran or dimethyl sulfoxide (DMSO).
  • An appropriate base for this reaction may be selected from a variety as described above, including, but not limited to bases such as inorganic bases or Lewis bases.
  • nitrogen bases may be used such as pyridine, triethyl amine, quinuclidine and N-ethyldiisopropylamine (DIEA).
  • the selected protecting group is a fer/-butyloxycarbonyl group or any of the protecting groups mentioned above.
  • the polypeptide is treated with the chosen protecting agent, in this example this may be di(ieri-butyloxy) anhydride, and the reaction is conducted in DMSO as the selected polar aprotic solvent.
  • the chosen protecting agent in this example this may be di(ieri-butyloxy) anhydride
  • the reaction is conducted in DMSO as the selected polar aprotic solvent.
  • DIEA N- ethyldiisopropyl amine
  • Using an excess of DIEA may result in the formation of side products. This is probably due to deamination or dehydration under over alkaline condition.
  • Step b) can be performed in the following manner: if azidonorleucine or azidonorvaline was used in the incorporation step a), the polypeptide could now be treated with a reduction agent.
  • These reagents can include, but are not limited to, all azide reduction reagents known to the person skilled in the art, with the only requirement that they do not deprotect the second protecting group at the same time.
  • reagents can include, but are not limited to tns(2-carboxyethyl)phosphine, Pt0 2 , boron trifluoride diethearate and indium trichloride.
  • a different protecting group such as a CBz or a Boc group
  • the deprotection conditions can be selected from a different variety of reagents, known to the person skilled in the art.
  • deprotection agents can include catalytic hydrogenation by using a metal catalyst, such as palladium on charcoal (Pd/C) or palladium hydroxide on charcoal (Pd(OH) 2 /C).
  • Deprotection agents can include, but are not limited to, Bronsted acids or Lewis acids.
  • Bronsted acids can include, but are not limited to, sulfuric acid, hydrochloric acid or trifluoroacetic acid (TFA), in connection with a hydrogen-donor such as triethylsilane, tripropylsilane or triisopropylsilane.
  • Lewis acids can include, but are not limited to, boron trifluoride, aluminium chloride or zinc chloride.
  • the deprotection can also be effected using heat under conditions known to the person in the art.
  • the solvent may include, but is not limited to, the group of polar aprotic solvents, in particular from a variety such as dichloromethane, tetrahydrofuran or dimethyl sulfoxide (DMSO).
  • the reduction agent is tris(2- carboxyethyl)phosphine (TCEP) and DMSO was used as the polar aprotic solvent ( Figure 3). This step reveals a free amine on the target lysine residue or analog thereof, and the conjugatable moiety can now be installed on said free amine.
  • Step c) of the present method is the linking of a conjugatable moiety to the target lysine residue, or analog thereof, in the first polypeptide ( Figure 1 , 2 and 3).
  • the conjugatable moiety may be linked to the free amine on the target lysine residue, or analog thereof.
  • step c) the conjugatable moiety can be chosen from a group of compounds illustrated below in formula I:
  • R is independently hydrogen, optionally substituted heteroalkyl, hydroxyl, cyano, halogen, oxo, carboxy, alkoxycarbonyl-, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkenyl, optionally substituted carbocyclyl, or optionally substituted cycloalkenyl;
  • R is hydrogen or optionally substituted alkyl
  • n is an integer from 1 to 4.
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 1.
  • n may be 1 , 2, 3, or 4.
  • p may be 0, 1, 2, 3, 4, 5, or 6.
  • q may be 0 or 1.
  • the conjugatable moiety can additionally be chosen from a compound according to Formula 1 wherein R 1 is hydrogen, optionally substituted aryl or alkyloxycarbonyl.
  • the conjugatable moiety can also be selected from a variety of compounds wherein the optional substituent is halogen, alkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, hydroxymethyl, halomethyl, alkanoyloxy, alkenoyloxy, alkynoyloxy, alkanoyloxymethyl, alkenoyloxymethyl, alkynoyloxymethyl, alkoxymethyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylsulfonamido, alkenylsulfonamido, alkynylsulfonamido, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy, alkyloxycarbon
  • the conjugatable moiety may be of formula (la):
  • R 1 , R 2 , n and p are as defined herein.
  • the conjugatable moiety may be of formula (lb):
  • R 1 , R 2 , n and p are as defined herein.
  • the conjugatable moiety may be of formula (Ic):
  • R 1 , R 2 , n and p are as defined herein.
  • the conjugatable moiety may be of formula (Id):
  • the conjugatable moiety may be of formula (Ie):
  • R is hydrogen, phenyl substituted with one or more methoxy, nitro, or tert-butyloxycarbonyl.
  • the conjugatable moiety can be selected from the following compounds:
  • R is a substitutent selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, hydroxymethyl, halomethyl, alkanoyloxy, alkenoyloxy, alkynoyloxy, alkanoyloxymethyl, alkenoyloxymethyl, alkynoyloxymethyl, alkoxymethyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylsulfonamido, alkenylsulfonamido, alkynylsulfonamido, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy, alkyloxycarbonyl, carboalkyl, phenoxy, phenyl, thiophenoxy, benzyl, amino, hydroxyamino, alkoxyamino, alkylamino,
  • the conjugatable moiety which is used in step c) can be chosen from a variety of compounds, including but not limited to a G76C mutant, ⁇ - or ⁇ -thiol group, or a G76 with a Na-auxiliary.
  • conjugatable moiety As an example for the addition of the conjugatable moiety, the following compound can be added, which can attach to the target lysine, or analog thereof, to form the following chemical moiety:
  • the reaction product of the preceding step is dissolved in an appropriate solvent.
  • this solvent may include, but is not limited to, the group of polar aprotic solvents, in particular from a variety such as dichloromethane, tetrahydrofuran or dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • This solution is treated with the conjugatable moiety as described above until the reaction is found to be complete.
  • step ca the polypeptide obtained from the previous step c) is deprotected to effect the removal of the second protecting group.
  • This deprotection also allows the process to follow a 'native' conjugation, i.e. the linking of two proteins without the use of protecting groups.
  • the term 'protein' hereby and in the following is encompassed by the definition of 'polypeptide'.
  • This deprotection is effected by treating the polypeptide from step c) with a deprotection agent selected from a variety of agents known to the person skilled in the art.
  • deprotection agents can include, but are not limited to, Bronsted acids or Lewis acids.
  • Bronsted acids can include, but are not limited to, sulfuric acid, hydrochloric acid or trifluoroacetic acid (TFA), in connection with a hydrogen-donor such as triethylsilane, tripropylsilane or triisopropylsilane.
  • Lewis acids can include, but are not limited to, boron trifluoride, aluminium chloride or zinc chloride. The deprotection can also be effected using heat under conditions known to the person in the art.
  • an Fmoc-group has been used as protecting group in step aa)
  • the following conditions known to the person skilled in the art can be used: treating the polypeptide with a nitrogen-base, which can be either a tertiary, secondary or a primary amine.
  • a nitrogen-base which can be either a tertiary, secondary or a primary amine.
  • tertiary amines can include, but are not limited to DIEA and triethylamine.
  • secondary amines can include, but are not limited to piperidine and piperazine.
  • Examples for primary amines can include, but are not limited cyclohexylamine and ethanolamine.
  • the protection group used in step aa) is a CBz group, the deprotection is typically effected by hydrogenation using conditions known to the person skilled in the art.
  • Suitable catalysts can include, but are not limited to Pd/C or Pd(OH) 2 /C.
  • the second protection group installed in step aa) is a Boc group and the deprotection therefore requires typical Boc deprotection conditions as known to the person skilled in the art.
  • trifluoroacetic acid (TFA) in connection with triisopropylsilane, was selected as the deprotection agent. This step may reveal all additional lysines in the polypeptide, as well as the N-terminus amines.
  • the conjugatable moiety can be deprotected thereby activating the first polypeptide by revealing a thiol moiety.
  • the deprotection may involve a ring-opening reaction wherein an alkylene group is removed.
  • the alkylene may be a methylene group.
  • the depro ected conjugatable moiety may be of Formula II:
  • R 1 is independently hydrogen, optionally substituted heteroalkyl, hydroxyl, cyano, halogen, oxo, carboxy, alkoxycarbonyl-, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkenyl, optionally substituted carbocyclyl, or optionally substituted cycloalkenyl;
  • R 2 is hydrogen or optionally substituted alkyl
  • n is an integer from 1 to 4.
  • r is an integer from 1 to 6;
  • q is an integer from 0 to 1.
  • r may be 1 , 2, 3, 4, 5, or 6.
  • the deprotected conjugatable moiety may be of formula (Ila):
  • R 1 , R 2 and r are as defined herein.
  • the deprotected conjugatable moiety may be of formula (lib):
  • R 1 , R 2 and r are as defined herein.
  • the deprotected conjugatable moiety may be of formula (He):
  • the deprotected conjugatable moiety may be of formula (lid):
  • R is as defined herein.
  • Reagents to effect this deprotection are selected from a variety of hard nucleophiles, including but not limited to methoxyamine and semicarbazides. Another reagent, which may be used, is zinc in an acetic acid solution. This deprotection reaction is useful for setting up the conjugatable moiety to react with the second polypeptide. In one example, methoxyamine was used as the deprotection reagent.
  • the deprotected conjugatable moiety may be the following compound:
  • the first polypeptide can be conjugated to a second polypeptide thioester.
  • the thioester which is needed in the second polypeptide for ligation, can be generated through the thiolysis of a second polypeptide with thioester-forming reagents.
  • Reagents of choice can include, but are not limited to reagents such as 3-mercaptopropionic acid, mercaptophenyl acetic acid, or sodium mercaptoethanesulfonate (MESNa).
  • MESNa has been used to generate the thioester on the second polypeptide.
  • thiol additives may be used for the coupling reaction, such as MESNa, MPAA, 2,2,2-trifluoroethanediol or 2- mercapto-acetamide.
  • the first polypeptide and the second polypeptide thioester is dissolved in a ligation buffer containing the coupling additive, which can be selected from the thiol additives described immediately above.
  • an additive of choice may be the aromatic thiol mercaptophenyl acetic acid (MPAA).
  • the concentration of MPAA that may be used is from about 1 mM to about 50 mM, or from about 1 mM to about 45 mM, or from about 1 mM to about 40 mM, or from about 1 mM to about 35 mM, or from about 1 mM to about 30 mM, or from about 1 mM to about 25 mM, or from about 1 mM to about 20 mM, or from about 1 mM to about 15 mM, or from about 1 mM to about 10 mM, or from about 1 mM to about 5 mM, or from about 5 mM to about 50 mM, or from about 10 mM to about 50 mM, or from about 15 mM to about 50 mM, or from about 20 mM to about 50 mM, or from about 25 mM to about 50 mM, or from about 30 mM to about 50 mM, or from about 35 mM to about 50 mM, or from about 40 mM
  • the reaction is run towards completion, which may be monitored by an analytical method, such as HPLC.
  • HPLC an analytical method
  • a minimum concentration of the additive, such as MPAA, may be used to avoid potential overlaps of the polypeptide peaks and the additive peaks on the HPLC profiles. Accordingly, yields as high as 85% can be achieved for this linking reaction in a short reaction time.
  • the Na-mediated native chemical ligation is highly efficient in the present method.
  • the auxiliary of the conjugatable moiety may be cleaved off (step da). This cleavage reaction can be effected by treating the conjugated polypeptide from step d) with a cleaving agent.
  • Cleavage agents that may be used can include, but are not limited to Bronsted acids or Lewis Acids.
  • Bronsted acids can include, but are not limited to hydrochloric acid, sulfuric acid or trifluoroacetic acid (TFA), in connection with a hydrogen-donor such as triethylsilane, tripropylsilane or triisopropylsilane.
  • Lewis acids can include, but are not limited to boron trifluoride, aluminium chloride or zinc chloride.
  • the cleavage reaction can also be effected using heat using conditions as known to the person skilled in the art.
  • trifluoroacetic acid (TFA) in connection with triisopropylsilane, was selected as the cleavage agent.
  • TFA trifluoroacetic acid
  • r is an integer from 1 to 6;
  • ** indicates the ⁇ -nitrogen of the target lysine residue, or analog thereof, which is the point of attachment to the first polypeptide.
  • Formula III may also be depicted as:
  • Figure 2 shows a more detailed depiction of the functional elements of the method, as described above.
  • it shows the incorporation of the protected lysine residue, or analog thereof, into a first polypeptide (1. PP), followed by treating further lysine residues as well as any N-terminal amino groups within the first polypeptide with a second protecting group (PG).
  • PG second protecting group
  • This is followed by deprotection of the first, translationally incorporated lysine residue, or analog thereof, and subsequent reaction of the free amine with a conjugatable moiety (CM).
  • CM conjugatable moiety
  • Deprotection of the second protecting group is followed by the ring-opening reaction on the conjugatable moiety.
  • the first polypeptide is now ligated with the second polypeptide (2. PP) and in a final step the residual functionality from the ligation moiety is cleaved to reveal the conjugated polypeptide.
  • the conjugated polypeptide was obtained in an overall yield of 35%.
  • the polypeptide used herein may be a small to medium sized protein.
  • Exemplary polypeptides to be modified include ubiquitin, histones and/or small transcription factors.
  • the polypeptide to be modified is not more than a few hundred amino acids long; for example 400 amino acids or fewer, or about 300 amino acids or fewer.
  • the first polypeptide as described in the present method may be ubiquitin.
  • the second polypeptide as described herein may be ubiquitin or a ubiquitin-like protein or histone.
  • ubiquitin-like proteins can include, but are not limited to small ubiquitin-related modifier 1-3, Interferon-induced 17 kDa protein and other examples as defined above.
  • This method can be carried out with the target lysine residue, or analog thereof, being a K48 lysine residue in ubiquitin.
  • the chemically synthesized diubiquitin was refolded through dialysis against refolding buffer to generate native K48-linked diubiquitin. The native state of the refolded diubiquitin was confirmed by circular dichroism (CD) spectrometry. To test whether the chemically synthesized diubiquitin was biological active, the diubiquitin was analyzed by western blot using ubiquitin monoclonal antibody P4D1 ( Figure 6).
  • the present method also shows an application of genetically incorporated Anl besides the very-well recognized click reaction, which is applied in bioconjugation or in constraining protein secondary structures, or cell surface labeling.
  • K48-linked diubiquitin with native isopeptide linkage was synthesized with an overall yield of 35%.
  • the histone protein H2A was also ubiquitinated at 119 using this method.
  • the present method can overcome the shortcomings of chemical ubiquination and makes it less labor intensive.
  • Figure 1 shows a highly schematized depiction of the functional elements of the method.
  • Figure 2 shows a more detailed depiction of the functional elements of the method.
  • Figure 3 shows an example of how the method can be performed.
  • Figure 5 (A) C8 analytical HPLC monitored ligation reaction between peptide 6 and ub(l -75)-MES. (B) The raw and deconvoluted ESI-MS of 7.
  • FIG. 1 A) SDS-PAGE (coomassie blue staining) and western blot of the chemically synthesized K48-linked diubiquitin 8. B) The raw and deconvoluted ESI-MS of 8. C) Deubiquitinase assays of 8 performed with IsoT and A20CD monitored by C8 analytical HPLC. Peak a: 8; peak b: ubiquitin 1; peak c: wild type ubiquitin.
  • Reagents and conditions for Figure 3 i) Boc anhydride, DIEA, DMSO; ii) 1 M TCEP in H 2 0, DMSO; iii) 9, DIEA, DMSO; iv) TFA/TIS/ H20 (95/2.5/2.5), 56% (four steps); v) 6 M Gdn-HCL 0.2 M phosphate, 0.4 M MeONH 2 , pi I 4.0, 85%; vi) Ub(l-75)-MES, 6 M Gdn'HCl, 0.2 M phosphate, 25 mM TCEP, 25 mM MPAA, pH 8.0, 85%; vii) TFA/TIS/ 3 ⁇ 40 (95/2.5/2.5), 86%.
  • Figure 4 shows the HPLC and ESI-MS data for key compounds.
  • Peak a the mixture of 6 and ub(l-75)-MES; peak b: ub(l-75)-OH; peak c: ligation product 7; peak d: ub(l-75)-MES.
  • K48-linked diubiquitin starts with a receptor ubiquitin with K48 replaced by Anl (ubiquitin 1).
  • Anl has been incorporated into proteins through an engineered methioninyl-tRNA synthetase (MetRS) in Met-auxotrophic E, Coli cells. It is found that MetRS with LI 3 A mutation (MetRSL13A) could also catalyze the incorporation of Anl. There are no other Met residues except the initiator Met present in ubiquitin sequence.
  • the codon coding for K48 of the ubiquitin is mutated to that of Met.
  • ubiquitin 1 is done with MetRSLDA in the presence of 1 mM Anl. After purification, homogeneous ubiquitin 1 is obtained with a yield of 10 mg/L.
  • Electrospray ionization mass spectrometry (ESI-MS) analysis of the protein confirms that the initiator Met has been completely removed ( Figure 4).
  • TCEP tris(2- carboxyethyl)phosphine hydrochloride
  • the reduction product ubiquitin 3 is dissolved in 330 ⁇ DMSO. 2.6 mg of compound 9 and 6 ⁇ of DIEA are added. After 45 min, the crude product ubiquitin 4 is obtained by ether precipitation ( Figure 4 B trace d). Finally, the crude protein 4 is treated with 200 of TFA/TIS/H 2 0 (95/2.5/2.5) for 20 min for global Boc deprotection. After ether precipitation, the crude deprotection product 5 is analyzed by CI 8 analytical HPLC ( Figure 4 A, trace b). The analytical HPLC showed that majority of the desired product 5 is formed. After HPLC purification, 4.9 mg of 5 is obtained. The overall yield for the first four steps is 56%. The analytical HPLC and deconvoluted ESI-MS of 5 is shown in Figure 4 (A trace c and B trace e).
  • ubiquitin 6 is ligated with an ubiquitin thioester containing the first 75 residues (ub(l -75)-MES) through auxiliary-mediated ligation.
  • the thioester is generated through the thiolysis of a second polypeptide with sodium mercaptoethanesulfonate (MESNa).
  • the aromatic thiol mercaptophenyl acetic acid is tested as thiol additive.
  • MPAA aromatic thiol mercaptophenyl acetic acid
  • a minimum concentration of MPAA is chosen. The ligation undergoes efficiently in the presence of such minimum concentration of MPAA.
  • the chemically synthesized diubiquitin 8 is refolded through dialysis against refolding buffer.
  • the native state of the refolded diubiquitin is confirmed by circular dichroism (CD) spectrometry.
  • CD circular dichroism
  • the diubiquitin is analyzed by western blot using ubiquitin monoclonal antibody P4D1 ( Figure 6). A single band corresponding to the diubiquitin is detected by western blot.
  • IsoT isopeptidase T
  • A20 CD is found to be less efficient in hydrolyzing K48 diubiquitin.
  • substrate enzyme ratio of 24: 1 only about 60% of the diubiquitin is hydrolyzed by A20 CD after 2 h at 37°C. After incubation for another 2 h, 85%) of the diubiquitin is hydrolyzed by A20 CD -
  • the method is useful in conjugation of polypeptide(s) and/or the study of same. It is useful if the polypeptide and/or the other polypeptide is ubiquitin.
  • Ubiquitin is a small protein that is easily denatured and renatured, allowing for its ease of production by recombination and selective protein chemistry according to the method of the present invention.
  • the present method is a tool that allows ubiquitination in a specific manner of any protein. When the effects of such ubquitination are known, such as for example by linking a protein to a polyubiquitin linked by K48 of the ubiquitin polypeptide, this can be helpful in studying their proteosomal degradation.
  • the method can be repeated to allow one to link several proteins together.
  • both the polypeptidic chain and the protein are ubiquitin
  • polypeptidic chains that are obtainable from the methods described herein, such as a polypeptidic chain linked specifically to a protein by an isopeptide bond.
  • examples are ubiquitinated proteins, whereby the reaction in step (d) is to link the ubiquitin to another protein by peptide bond formation.
  • homogenously linked ubiquitin chains obtainable by the method.
  • a homogenously linked ubiquitin chain has been obtained according to the method where the covalent link is an isopeptide bond between a lysine residue, or analog thereof, at position 48 and the C-terminus of another ubiquitin polypeptide. It is to be understood that the chain can be continued by further homogenous linkages, further obtainable by the method described herein.
  • Another aspect of the method is the homogenously linked ubiquitin obtained according to the method disclosed where the covalent link is an isopeptide bond between a lysine residue, or analog thereof, at position 48 and the C-terminus of another ubiquitin polypeptide.
  • the linkages can be continued for more than 2 links.
  • Said ubiquitin chain can be used as a medicament. It can be used in activating or promoting a response to DNA damage.
  • the chains have been shown to be linked to the BRCAl /Bardl E3 ligase complex and thus the ubiquitin chains can be used in preventing or treating cancer, preferably where the cancer is early-onset breast or ovarian cancer.
  • the ubiquitin chains can be treated as an oncological medicament and can be used in pharmaceutical compositions and administered by means well known in the art in the field of oncological pharmacy.

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Abstract

Cette invention concerne un procédé de conjugaison de deux polypeptides ou plus, par incorporation pendant la traduction d'un résidu lysine cible, ou d'un analogue de celui-ci, protégé par un premier groupe de protection, dans un premier polypeptide, déprotection dudit résidu lysine cible, ou de son analogue, conjugaison d'un fragment conjugable au résidu lysine cible ou à son analogue, conjugaison du résidu lysine cible, ou de son analogue, et d'un second polypeptide.
PCT/SG2015/050033 2014-03-10 2015-03-10 Procédé de conjugaison de protéines WO2015137883A1 (fr)

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Publication number Priority date Publication date Assignee Title
US11897917B2 (en) 2017-09-27 2024-02-13 The University Of York Bioconjugation of polypeptides

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010068278A2 (fr) * 2008-12-10 2010-06-17 The Scripps Research Institute Production de conjugués peptide-support utilisant des acides aminés non naturels réactifs chimiquement
WO2010139948A2 (fr) * 2009-06-04 2010-12-09 Medical Research Council Procédés
WO2011117583A2 (fr) * 2010-03-24 2011-09-29 Medical Research Council Procédé
WO2012085279A2 (fr) * 2010-12-23 2012-06-28 Universiteit Gent Procédé de réticulation de peptides
WO2012175924A2 (fr) * 2011-06-24 2012-12-27 Medical Research Council Incorporation de lysines substituées dans des polypeptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010068278A2 (fr) * 2008-12-10 2010-06-17 The Scripps Research Institute Production de conjugués peptide-support utilisant des acides aminés non naturels réactifs chimiquement
WO2010139948A2 (fr) * 2009-06-04 2010-12-09 Medical Research Council Procédés
WO2011117583A2 (fr) * 2010-03-24 2011-09-29 Medical Research Council Procédé
WO2012085279A2 (fr) * 2010-12-23 2012-06-28 Universiteit Gent Procédé de réticulation de peptides
WO2012175924A2 (fr) * 2011-06-24 2012-12-27 Medical Research Council Incorporation de lysines substituées dans des polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NGUYEN D.P. ET AL.: "Genetically Encoded 1,2-aminothiols Facilitate Rapid and Site- Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 133, no. 30, 2011, pages 11418 - 11421 *
TANRIKULU I.C. ET AL.: "Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 36, 2009, pages 15285 - 15290, XP055223428 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11897917B2 (en) 2017-09-27 2024-02-13 The University Of York Bioconjugation of polypeptides

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