WO2015137883A1 - Method for protein conjugation - Google Patents

Abstract

Provided is a method for the conjugation of two or more polypeptides, by incorporating during translation a target lysine residue, or analog thereof, protected by a first protecting group in a first polypeptide, deprotecting said target lysine residue, or analog thereof, conjugating a conjugatable moiety to the target lysine residue or analog thereof, conjugating the target lysine residue, or analog thereof, and a second polypeptide.

Description

METHOD FOR PROTEIN CONJUGATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore patent application No. 1020140051 1W, filed 10 March 2014, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention generally relates to the field of biochemistry. In particular, 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. BACKGROUND OF THE INVENTION

[003] 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. In this area either therapeutic, diagnostic, and research agents, or their carriers are chemically conjugated to polypeptides that can selectively bind to targeted cells.

[004] Generally, the resulting conjugates are structurally and functionally heterogeneous because they are formed randomly via chemical reactions with few of several available chemical groups, usually ε-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.

[005] A classic example of polypeptide conjugation is ubiquitination which 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. Biologically, 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.

[006] Although conjugated polypeptides with homogenously conjugated polypeptides have been developed in a chemical manner, their applications have been limited due to reaction inefficiencies or impractical incorporation of the ligatable functionality for conjugation.

[007] In particular, existing technologies for the synthesis of conjugated peptides and proteins include Na-auxiliary-mediated chemical ligation, but these ligation functionalities can only be introduced via solid-phase-peptide-synthesis (SPPS) or ligation. They include genetically encoded orthogonal protection and activated ligation approach, but the ligation reaction requires the need for further protection groups on the polypeptide (a non-native ligation). Although these methods can generate the native isopeptide linkage of polypeptide conjugates, these methods are either labour-intensive or give low yields. To reduce labour, 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.

[008] There is therefore a need to provide conjugated polypeptides and alternative methods for their preparation that overcome, or at least ameliorate, one or more of the disadvantages described above. SUMMARY

[009] In a first aspect, there is provided a method of conjugating two or more polypeptides. The method comprises incorporating during translation a target lysine residue, or analog thereof, protected by a first protecting group in a first polypeptide. In another step, 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. Afterwards, the target lysine residue, or analog thereof, is conjugated to a second polypeptide.

[0010] In a second aspect, there is provided a conjugated polypeptide produced according to the method as described herein.

DEFINITIONS

[001 1] The following words and terms used herein shall have the meaning as indicated.

[0012] The term "analog thereof in the context of the lysine residue (i.e. 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 C₂. 4-alkyl bearing a terminal nitrogen functionality. This functionality can include azides, amines and carbamates. The lysine analog may be a valine residue.

[0013] The term "residue" 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. (CH₂)₄NH₃ ⁺ in case of lysine side chain) associated with the amino acid backbone (NH₂-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.

[0014] The term "functionality" or "functional group" used herein refers to a manipulable chemical moiety which contains heteroatoms that are active under the associated reaction conditions. It usually refers to an azide or amine.

[0015] The term "conjugatable moiety" or "ligation moiety" or "ligatable 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)CH₂N-. 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 -C¾-.

[0016] The term "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.

[0017] The term "isopeptide bond" as used herein, 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.

Thus, an isopeptide may form intramolecularly within a single protein or intermolecularly i.e. between two peptide/protein molecules. Non-limiting examples of isopeptide bonds are Gly- ε-Lys isopeptide bond or Ala-s-Lys isopeptide bond.

[0018] "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.

[0019] "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.

[0020] The phrase "protecting group" as used herein 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. In contrast to a protecting group, the term "deprotect" or deprotection" refers to the removal of at least one protecting group from the polypeptide of interest. [0021 ] "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.

[0022] The term "RNA", or "ribonucleic acid" refers to a polynucleotide or oligonucleotide which comprises at least one ribonucleotide residue.

[0023] The term "tRNA" or "tRNA molecule" refers to a specialized RNA molecule, which acts as a template to direct the synthesis of the polypeptide.

[0024] The term "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.

[0025] The term "tRNA synthetase/fRNA pair" refers to the pair of a tRNA molecule and a tRNA synthetase specific to that tRNA molecule.

[0026] The term "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). 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 1, Ufml, UniProt P61960.

[0027] "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. [0028] The term "alkyl" as used herein 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. For example, the term 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-dimethylpentyl, 1,2- dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1 ,1,2- trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. Alkyl groups may be optionally substituted.

[0029] The term "alkenyl" as used herein, unless otherwise specified, 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.

[0030] The term "alkynyl" as used herein, unless otherwise specified, 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.

[0031] The term "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.

[0032] The term "amino" includes an amine group (i.e., -NH₂) or a substituted amine group.

[0033] The term "azide" or "azido" is intended to mean N₃ ^".

[0034] The term "carboxy" refers to the group -C(0)OH.

[0035] The term "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.

[0036] The term "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. [0037] "Keto" or "oxo" is intended to mean the radical=0.

[0038] The terms "halogen" and "halo" refer to a fluoro, chloro, bromo, or iodo moiety.

[0039] The term "aromatic group", or variants such as "aryl" or "arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like.

[0040] The term "heteroaryl" as used herein 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, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1 ,2,4-triazinyl, benzothiazolyl and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like. Heteroaryl groups may be optionally substituted.

[0041] The term "carbocyclyl" as used herein 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. . In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, 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." Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0042] The term "cycloalkenyl" as used herein 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. Non-limiting examples of 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.

[0043] The term "OMe" refers to an alkoxy group, wherein Me stands for CH₃.

[0044] The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups other than hydrogen provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a stable compound. 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, arylcarboxamidoalkyl, aroyl, aroyl- 4-alkyl, arylalkanoyl, acyl, aryl, arylalkyl, alkylaminoalkyl, a group R_{x y}N-, R_xOCO(CH₂)_m, R_xCON(R_y)(CH₂)_m, R_xR_yNCO(C¾)_m, R_xRyNS0₂(CH₂)_m or R_xS0₂NR_y(CH₂)_m (where each of R_x and R_y is independently selected from hydrogen or alkyl , or where appropriate R_xR_y forms part of carbocylic or heterocyclic ring and m is 0, 1 , 2, 3 or 4), a group R_xR_yN(CH₂)_p- or R_xRyN(CH₂)_pO- (wherein p is 1 , 2, 3 or 4); wherein when the substituent is R_xR_yN(CH₂)_p- or R_xR_yN(CH₂)pO, R_x with at least one CH₂of the (CH₂)_P portion of the group may also form a carbocyclyl or heterocyclyl group and R_y may be hydrogen, alkyl.

[0045] 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"), is the group that is directly connected to the core. Thus, 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.

[0046] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements. [0047] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a 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.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0048] Chemical peptide conjugation often suffers from the limitation that the ligation functionalities for conjugation of polypeptides can only be introduced via solid-phase- peptide-synthesis (SPPS) or ligation. Methodologies so far developed for utilizing genetically encoded orthogonal protection for introduction of the ligation functionality and activated ligation suffer from the limitation, that the ligation reaction requires the need for further protection groups on the polypeptide (a non-native ligation) and they are therefore labour- intensive or give low yields. It was surprisingly found that if a protected lysine or analog thereof was incorporated during translation, followed by deprotection of said lysine or analog thereof and conjugation with a ligation moiety, the ligation can be performed in a higher yield.

[0049] Thus, in one aspect, 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. In this method, 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). In this way, a polypeptide may be produced having a target lysine, or analog thereof, protected site-specifically by genetically encoded protection of that residue. In step b), 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). [0050] 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.

[0051] In the described method, the first step (step a) entails the provision of a nucleic acid which is encoding the first polypeptide. In the method described herein, 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.

[0052] 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. For example, 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. Alternatively, the tRNA synthetase may be from bacteria, for example from Desulfitobacterium hafniense PCP1 or Desulfotomaculum acetoxidans DSM 771. Alternatively, the tRNA synthetase may be from eukaryotes, such as yeast.

[0053] In another example, 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. Furthermore, the process may be performed when the tRNA synthetase is MetRS with LI 3 A mutation (MetRSL13A). The MetRSL13A system is especially useful for introducing an azide as a protection group on the lysine residue, or analog thereof. In particular, the MetRSL13A system is useful to incorporate an azidonorleucin or azidonorvaline into the first polypeptide.

[0054] Following this, one or more 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.

[0055] The target lysine, or analog thereof, (NPG¹) 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) (NPG²). 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.

[0056] 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 C₂-5-alkyl chain bearing a terminal nitrogen functionality. In particular, it 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.

[0057] In one example, the target (or protected) lysine residue, or analog thereof, incorporated by this method may be azidonorleucine (Anl) or azidonorvaline (Anv). In this case, 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.

[0058] After genetic incorporation of the protected lysine residue, or analog thereof, into the polypeptide (step a) and before deprotection of said first protecting group (step b), 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). This enables the target lysine, or analog thereof, to be selectively deprotected, leaving the other protecting groups on the additional lysines intact. In this way, the target lysine, or analog thereof, is site-specifically deprotected and can therefore be modified whilst the other lysines remain unaffected. Accordingly, 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). If 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. In particular, 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. [0059] In one example, 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.

[0060] 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).

[0061] 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. A variety of protecting groups have been described immediately above. As mentioned above, the second protecting group for the remaining lysines in the first polypeptide may be selected from the group consisting of FMoc, Boc or CBz. In a typical reaction according to this method, 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. In particular, nitrogen bases may be used such as pyridine, triethyl amine, quinuclidine and N-ethyldiisopropylamine (DIEA). In one example, the selected protecting group is a fer/-butyloxycarbonyl group or any of the protecting groups mentioned above. Accordingly, 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. As an exemplary base, N- ethyldiisopropyl amine (DIEA) is added in a minimal amount in order to adjust the pH to mild alkaline. 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. [0062] After installing the second protecting group, the initially protected target lysine residue, or analog thereof, in the first polypeptide can now be deprotected using conditions known to the person skilled in the art (Figure 1 and 2, step b). 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. These reagents can include, but are not limited to tns(2-carboxyethyl)phosphine, Pt0₂, boron trifluoride diethearate and indium trichloride. If a different protecting group has been used for step a), 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. In particular, if a CBz group is used, 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). If a Boc group is used in the first protection step, the deprotection can be effected by using a variety of conditions known to the person skilled in the art. Deprotection agents can include, but are not limited to, Bronsted acids or Lewis acids. Examples for 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. Examples for 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. In a typical deprotection reaction according to this method, 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). In one example, 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.

[0063] 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.

[0064] In step c), the conjugatable moiety can be chosen from a group of compounds illustrated below in formula I:

(Formula I)

wherein 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; and

q is an integer from 0 to 1.

[0065] n may be 1 , 2, 3, or 4.

[0066] p may be 0, 1, 2, 3, 4, 5, or 6.

[0067] q may be 0 or 1.

[0068] The conjugatable moiety can additionally be chosen from a compound according to Formula 1 wherein R¹ 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, alkyloxycarbonyl, carboalkyl, phenoxy, phenyl, thiophenoxy, benzyl, amino, hydroxyamino, alkoxyamino, alkylamino, dialkylamino, aminoalkyl, N- alkylaminoalkyl, N,N-dialkylaminoalkyl, phenylamino and benzylamino. The conjugatable moiety can be further selected from a group of compounds.

[0069] In one embodiment, the conjugatable moiety may be of formula (la):

wherein R¹, R², n and p are as defined herein.

[0070] In one embodiment, the conjugatable moiety may be of formula (lb):

wherein R¹, R², n and p are as defined herein.

[0071] In one embodiment, the conjugatable moiety may be of formula (Ic):

wherein R¹, R², n and p are as defined herein.

[0072] In one embodiment, the conjugatable moiety may be of formula (Id):

[0073] In one embodiment, the conjugatable moiety may be of formula (Ie):

[0074] In one embodiment, R is hydrogen, phenyl substituted with one or more methoxy, nitro, or tert-butyloxycarbonyl. In particular, the conjugatable moiety can be selected from the following compounds:

wherein 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, dialkylamino, aminoalkyl, N-alkylaminoalkyl, Ν,Ν-dialkylaminoalkyl, phenylamino and benzylamino.

[0075] Additionally, 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, γ- or δ-thiol group, or a G76 with a Na-auxiliary.

[0076] 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:

[0077] [0078] In a typical reaction to introduce the conjugatable moiety, the reaction product of the preceding step is dissolved in an appropriate solvent. In a typical conjugating moiety- conjugating reaction according to this method, 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). In one example, DMSO was used as the polar aprotic solvent. This solution is treated with the conjugatable moiety as described above until the reaction is found to be complete.

[0079] Before proceeding to the ligation of the two polypeptides (step d), two additional steps may be carried out. In the first additional step (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. If a Boc group has been used in the step aa), deprotection agents can include, but are not limited to, Bronsted acids or Lewis acids. Examples for 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. Examples for 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. If 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. Examples for tertiary amines can include, but are not limited to DIEA and triethylamine. Examples for 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. If 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. In one example, 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. In this example, 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.

[0080] In the second additional step (step cb), 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:

Formula II

wherein R¹ 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;

R² is hydrogen or optionally substituted alkyl;

n is an integer from 1 to 4;

r is an integer from 1 to 6; and

q is an integer from 0 to 1.

[0081] r may be 1 , 2, 3, 4, 5, or 6.

0082] In one embodiment, the deprotected conjugatable moiety may be of formula (Ila):

wherein R¹, R² and r are as defined herein.

[0083] In one embodiment, the deprotected conjugatable moiety may be of formula (lib):

wherein R¹, R² and r are as defined herein.

[0084] In one embodiment, the deprotected conjugatable moiety may be of formula (He):

wherein R is as defined herein.

[0085] In one embodiment, the deprotected conjugatable moiety may be of formula (lid):

wherein 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.

[0086] The deprotected conjugatable moiety may be the following compound:

[0087] For carrying out the conjugation reaction, such as an Na-auxiliary-mediated chemical polypeptide conjugation (step d), (Figures 1 , 2 and 3), the first polypeptide can be conjugated to a second polypeptide thioester.

[0088] 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). In one example, MESNa has been used to generate the thioester on the second polypeptide. After installation of the thioester on the second polypeptide, a variety of thiol additives may be used for the coupling reaction, such as MESNa, MPAA, 2,2,2-trifluoroethanediol or 2- mercapto-acetamide. In a typical conjugation reaction, 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. In one example, 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 to about 50 mM, or from about 45 mM to about 50 mM, or about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0089] The reaction is run towards completion, which may be monitored by an analytical method, such as HPLC. 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. As shown, the Na-mediated native chemical ligation is highly efficient in the present method. [0090] In a final step, 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. Examples for 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. Examples for 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. In one example, trifluoroacetic acid (TFA), in connection with triisopropylsilane, was selected as the cleavage agent. The particular isopeptide bond created by the method as detailed throughout this disclosure may have the following structural formula (Formula III):

Formula III

2 is hydrogen or optionally substituted alkyl; and

r is an integer from 1 to 6; wherein

* indicates the point of attachment to the second polypeptide; and

** indicates the ε-nitrogen of the target lysine residue, or analog thereof, which is the point of attachment to the first polypeptide.

[0091] Formula III may also be depicted as:

Polypeptide 2

[0092] In Formula III, the dashed-line box indicates the isopeptide linkage of the polypeptide conjugates:

[0093] Figure 2 shows a more detailed depiction of the functional elements of the method, as described above. In particular, 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). 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). 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.

[0094] By this method, in one example, the conjugated polypeptide was obtained in an overall yield of 35%.

[0095] For the first polypeptide, which is used in the disclosed method, it is possible to use a polypeptide which can be easily denatured and renatured without losing its properties as a protein. In this regard, 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. In one example 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.

[0096] The second polypeptide as described herein may be ubiquitin or a ubiquitin-like protein or histone. Examples for 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.

[0097] This method can be carried out with the target lysine residue, or analog thereof, being a K48 lysine residue in ubiquitin. [0098] In more detail with respect to this example, 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).

[0099] Meanwhile, 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.

[00100] To demonstrate the methodology, in one example, 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.

[00101] The method illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. The term "about" in connection with polypeptides shall relate to ±10% or ±5% or ±2% or ±1% of the value which is associated with it and refers in the present disclosure to 4 amino acid units. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00102] The method has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the method. This includes the generic description of the method with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [00103] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the method are described in terms of Markush groups, those skilled in the art will recognize that the method is also thereby described in terms of any individual member or subgroup of members of the Markush group.

BRIEF DESCRI PTION OF THE DRAWINGS

[00104] The accompanying drawings illustrate disclosed embodiments and serves to explain the principles of the disclosed embodiments. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the disclosure. The method will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[00105] Figure 1 shows a highly schematized depiction of the functional elements of the method.

[00106] Figure 2 shows a more detailed depiction of the functional elements of the method.

[00107] Figure 3 shows an example of how the method can be performed.

[00108] Figure 4 CI 8 analytical HPLC (A) and deconvoluted ESI-MS (B) of the key intermediates during the installation of conjugatable moiety to the side chain of ubiquitin K48.

[00109] 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.

[001 10] Figure 6 (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.

EXPERIMENTAL SECTION

[001 1 1] The numbers for the compounds used in the following experimental section correspond to the numbers used in Figure 3 in the present disclosure. It is therefore advised to refer to Figure 3 for better understanding. The synthesis of a conjugated polypeptide as depicted in Figure 3 starts with a first receptor polypeptide 1, wherein one amino acid is replaced with Anl (azidonorleucine). All amino functionalities in the first receptor polypeptide are subsequently protected as a teri-butyloxy group, using di(tert-butyloxy) anhydride, to give globally protected peptide 2. The azide in Anl is further reduced to free amine, resulting in peptide 3, which is reacted with compound 9, thereby conjugating the conjugatable moiety to the free amine and producing adduct 4. Previously Boc-protected amines are deprotected to give polypeptide 5 and a further deprotection step of the conjugatable moiety using methoxylamine gives product 6. This product is now ligated with ubiquitin (l-75)-MES in a minimum presence of MPAA to result in the conjugated polypeptide 7. In a final step, the auxiliary is cleaved using TFA and the conjugated K48- linked diubiquitin 8 is obtained.

Reagents and conditions for Figure 3 : i) Boc anhydride, DIEA, DMSO; ii) 1 M TCEP in H₂0, 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 MeONH₂, 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/ ¾0 (95/2.5/2.5), 86%.

The analytical data such as HPLC profiles and ESI-MS traces for the following examples are depicted in Figure 4 and Figure 5.

Figure 4 shows the HPLC and ESI-MS data for key compounds. In particular, CI 8 analytical HPLC (A) and deconvoluted ESI-MS (B) of the key intermediates during the installation of conjugatable moiety to the side chain of ubiquitin K48.

(A): (a) purified ubiquitin 1; (b) crude ubiquitin 5; c): purified ubiquitin 5; d) purified ubiquitin 6. Gradient: 0-80% buffer B (90% acetonitrile, 10% H₂0 containing 0.045% TFA) in buffer A (H₂0 containing 0.045% TFA) for 40 min.

B) a-f: Deconvoluted ESI-MS of ubiquitin 1-6. Average mass of 1, calculated 8617.70, observed 8618.3; average mass of 2: 8 Boc added, calculated 9418.62, found 9417.0, 7 Boc added, calculated 9318.50, found 9315.9; average mass of 3: 8 Boc added, calculated 9392.63, found 9390.7, 7 Boc added, calculated 9292.51, found 9290.8; average mass of 4: calculated 9557.84; found 9555.7 (7 Boc added product was observed); average mass of 5: calculated 8857.03; found 8856.9; average mass of 6: calculated 8845.02; found 8845.2.

[00112] Figure 5 (A) C8 analytical HPLC monitored ligation reaction between peptide

6 and ub(l-75)-MES with MPAA as the thiol additive at 0 h, 7 h and 12 h. 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.

[00113] (B) The raw and deconvoluted ESI-MS of 7. Average mass calculated

17334.7; found 17337.

[001 14] Figure 6 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. Average mass calculated 17138.43; found 17141.0.

C) Deubiquitinase assays of 8 performed with IsoT and A20C_D monitored by C8 analytical HPLC. Peak a: 8; peak b: ubiquitin 1; peak c: wild type ubiquitin.

Example 1 - Step a)

The synthesis of K48-linked diubiquitin starts with a receptor ubiquitin with K48 replaced by Anl (ubiquitin 1). In this example, 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. To express ubiquitin 1, the codon coding for K48 of the ubiquitin is mutated to that of Met. To ensure the initiator Met can be removed after protein expression within the cells, two extra residues Alanine and Serine are introduced after initiator Met. The expression of 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).

To install the ligation conjugatable moiety at the K48 side chain of ubiquitin, all the free amines of ubiquitin 1 are first protected with Boc anhydride. In a typical reaction, 8.8 mg of 1 is dissolved in 300 μΐ, of dimethyl sulfoxide (DMSO). 3.6 μΕ of Boc anhydride is added. 1.6 μΐ, of N-ethyldiisopropylamine (DIEA) is added to adjust the pH to mild alkaline. The reaction is completed within 1 h at room temperature (Figure 4 B trace b). Example 2 - Step b)

After protection, the azide group of ubiquitin 2 is reduced to amine by tris(2- carboxyethyl)phosphine hydrochloride (TCEP). This is done by dissolving the protected ubiquitin 2 in 300 μΐ, DMSO containing 30 μΐ, \ M aqueous TCEP. The reduction is finished within 3 h, which is confirmed by ESI-MS (Figure 4 B trace c).

Example 3 - Step c)

To introduce the conjugation moiety, the reduction product ubiquitin 3 is dissolved in 330 μΕ DMSO. 2.6 mg of compound 9 and 6 μΕ 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/H₂0 (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).

After the installation of the conjugatable moiety, 5 is deprotected by methoxylamine. For the deprotection step, 2.9 mg of 5 is dissolved in 750 iL buffer (6 M Gdn-HCl, 0.2 M phosphate, 0.4 M MeONH₂, pH 4). After 5 h at 37°C, 2.5 mg of deprotected product 6 is obtained after HPLC purification (yield 85%) (Figure 4 A trace d and B trace f).

Example 4 - Step d

To form diubiquitin 7, 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).

For the auxiliary-mediated ligation, the aromatic thiol mercaptophenyl acetic acid (MPAA) is tested as thiol additive. For a typical ligation reaction, 2.8 mg of ub(l-75)-MES and 1.7 mg of ubiquitin 6 are dissolved in 200 μΐ, of ligation buffer (6 M Gdn-HCl, 0.2 M phosphate, 25 mM MPAA, 25 mM TCEP, pH 8). To avoid the possible overlap of MPAA peak and ubiquitin peaks on HPLC profiles, a minimum concentration of MPAA is chosen. The ligation undergoes efficiently in the presence of such minimum concentration of MPAA. After 7 h at room temperature, majority of ubiquitin 6 is consumed and formed the ligation product. After another 5 h, almost all of compound 6 is consumed (Figure 5). 2.9 mg of ligation product 7 is obtained with an isolated yield of 85%. The final step for the synthesis of 48-linked diubiquitin 8 is to remove the auxiliary. To do this, 2.9 mg of 7 is treated with a cocktail containing TFA/TIS/H₂O (95/2.5/2.5) for 20 min. After purification, 2.5 mg of 8 is obtained with an isolated yield of 86%. The ESI-MS and C8 analytical HPLC analysis of diubiquitin 8 are shown in Figure 5 B and C (trace a). The overall yield for the diubiquitin synthesis is 35%.

[001 15] To generate native K48-linked diubiquitin, 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. To test whether the chemically synthesized diubiquitin is biological active, 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. Deubiquitinase assays with two ubiquitin deconjugating enzymes, isopeptidase T (IsoT) and A20 catalytic domain (A20_CD) are also performed. It is observed that with a 350:1 substrate enzyme ratio, IsoT could effectively hydrolyze almost all the K48 diubiquitin within 2 h at 37°C. Compared to IsoT, A20_CD is found to be less efficient in hydrolyzing K48 diubiquitin. With a 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-

APPLICATION

[001 16] In conclusion, there is provided an efficient chemical ligation-based method for protein conjugation. Different from all the previously reported chemical ligation conjugation methods, the present method utilizes a ligation reaction facilitated by a conjugatable moiety which is post-synthetically installed on a recombinant protein containing a genetically incorporated lysine residue, or analog thereof.

[001 17] 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. [001 18] Thus 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.

[001 19] In another example, the method can be repeated to allow one to link several proteins together. In the case that both the polypeptidic chain and the protein are ubiquitin, it is possible to use the invention to produce ubiquitin chains, for example homogenously and/or heterogeneously linked.

[00120] Thus another aspect of the disclosure are the resulting 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.

[00121] Another such examples are the homogenously linked ubiquitin chains obtainable by the method. As shown above, 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.

[00122] 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. Thereby, following this protocol, the synthesis of 48-linked diubiquitin through the combination of genetic Azidonorleucin incorporation and Na-auxiliary-mediated native chemical ubiquitination can be achieved.

[00123] 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.

[00124] As such, 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.

Claims

Claims

1. A method of conjugating two or more polypeptides, said method comprising:

(a) incorporating during translation a target lysine residue, or analog thereof, protected by a first protecting group in a first polypeptide;

(b) deprotecting said target lysine residue, or analog thereof;

(c) conjugating a conjugatable moiety to the target lysine residue, or analog thereof; and

(d) conjugating the target lysine residue, or analog thereof, and a second polypeptide. 2. The method according to the preceding claim, wherein step (a) comprises:

(i) providing a nucleic acid encoding the first polypeptide which nucleic acid comprises an orthogonal codon encoding the target lysine or analog thereof;

(ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase and tRNA pair capable of recognising said orthogonal codon and incorporating said target lysine residue, or analog thereof, protected by a first protecting group into the first polypeptide.

3. The method according to any one of the preceding claims, wherein the target lysine residue, or analog thereof, comprises 2 to 4 carbon atoms.

4. The method according to any one of claims 1 to 3, wherein said analog thereof of the target lysine residue is a target valine residue.

5. The method according to any one of claims 2 to 4, wherein the tRN A synthetase

comprises MetRS.

6. The method according to any one of claims 2 to 5, wherein the tRNA synthetase

comprises MetRSL13A. 7. The method according to claim 2, wherein the tRNA synthetase is a pyrrolysine-tRNA synthetase.

8. The method according to any one of claims 1 to 6, wherein the target lysine

residue, or analog thereof, protected by a first protecting group is selected from the group consisting of azidonorleucine and azidonorvaline.

9. The method according to any of the preceding claims, further comprising step (aa) treating the first polypeptide of step (a) to selectively protect further lysine residue(s) with a second protecting group, said second protecting group being different or orthogonal to the first protecting group, wherein step (aa) occurs before step (b).

10. The method according to claim 9, wherein step (aa) comprises treating the first polypeptide of step (a) with a deprotection reagent.

11. The method according to any one of the preceding claims, wherein step (b)

comprises selective deprotection of the target lysine residue, or analog thereof, and the further lysine residue(s) of step (aa) are not deprotected.

12. The method according to any one of the preceding claims, wherein the

conjugatable moiety comprises an auxiliary.

13. The method according to any one of the preceding claims, wherein the

conjugatable moiety is of formula (I):

wherein R is independently hydrogen, optionally substituted heteroalkyl, hydroxyl, cyano, halogen, oxo, carboxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyenyl, 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; and

q is an integer from 0 to 1. The method according to claim 13, wherein the conjugatable moiety is of formula (Ie)

wherein R¹ , R², n, p and q are as defined in claim 13.

15. The method according to claims 13 to 14, wherein R¹ is hydrogen, optionally substituted aiyl or alkyloxycarbonyl.

16. The method according to any one of claims 13 or 15, wherein the optional substituent is selected from the group consisting of halogen, alkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, hydroxymethyl, halomethyl, alkanoyloxy, alkenoyloxy, alkynoyloxy, alkanoyloxymethyl, alkenoyloxymethyl, alkynoyloxymethyl, alkoxymethyl, aikoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylsulfonamido, alkenylsulfonamido, alkynylsulfonamido, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy, alkyloxycarbonyl, carboalkyl, phenoxy, phenyl, thiophenoxy, benzyl, amino, hydroxyamino, alkoxyamino, alkylamino, dialkylamino, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, phenylamino and benzylamino.

17. The method according to any one of claims 13 to 16, wherein R¹ is hydrogen, phenyl substituted with one or more methoxy, or tert-butyloxycarbonyl.

18. The method according to any one of claims 13 to 17, wherein the conjugatable moiety is selected from the group consisting of:

19. The method according to any one of the precedmg claims, wherein the first polypeptide is ubiquitin or histone.

20. The method according to any one of the preceding claims, wherein the second polypeptide comprises a C-terminal thioester.

21. The method according to any one of the preceding claims, wherein the second polypeptide is ubiquitin or ubiquitin-like protein.

22. The method according to any one of the preceding claims, wherein the method further comprises a step (ca) deprotecting said further lysine residue(s).

23. The method according to claim 22, wherein step (ca) occurs before step (d).

24. The method according to any one of claims 22 to 23, wherein step (ca) comprises treating the product obtained by step (c) with a deprotection agent.

25. The method according to any one of the preceding claims, wherem the method further comprises a step (cb) deprotecting the conjugatable moiety.

26. The method according to claim 25, wherein the deprotected conjugatable moiety is of formula (II):

wherein R¹, R², p and q are as defined in claim 13, and

r is an integer from 1 to 6. 27. The method according to any one of the preceding claims, further comprising step (da) cleaving the auxiliary, resulting in formula (III):

Polypeptide 2

Polypeptide 1

wherein R² is as defined in claim 13 and r is as defined in claim 26.

28. A conjugated polypeptide produced according to any one of the preceding claims.