C-Terminal Peptide Modification

Abstract

The present invention provides a method for providing a cargo to a C-terminus (111) of a peptide (110), comprising a first step and a second step, the first step comprising reacting the C-terminus (111) of the peptide (110) with a first reactant (120) in the presence of a first catalyst (130) and a first radiation (140) to provide a first intermediate (150), the first catalyst (130) being configured to decarboxylate the C-terminus (111) of the peptide (110) in the presence of the first radiation (140), and the second step comprising exposing the first intermediate (150) to a second reactant (220).

Description

[Technical field]

[0001] The present invention relates to a method for providing a cargo at the C-terminus of a peptide.The present invention further relates to a peptide-cargo conjugate.The present invention further relates to a peptide array comprising the peptide-cargo conjugate.The present invention further relates to the use of the peptide-cargo conjugate. [Background technology]

[0002] Methods for providing cargo to proteins are known in the art. For example, US Patent Publication No. 2014256879 describes a method for assembling proteins from peptide fragments, where the peptide fragments have a C-terminal cyclic bis(2-sulfanylethyl)amino group. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Publication No. 2014256879 Summary of the Invention [Problem to be solved by the invention]

[0004] C-terminal modification of peptides, particularly proteins, can be important for sensors, arrays, (basic) research, sequencing applications, and the synthesis of protein-drug conjugates. However, it can be difficult to selectively modify the C-terminus of native peptides and proteins.

[0005] For example, prior art methods for C-terminal peptide modification can only accommodate a small range of C-terminal residues.

[0006] Prior art methods may further relate to the synthesis of (recombinant) mutant proteins bearing functional groups for further modification, however such methods may not be compatible with the modification of peptides derived from (natural) biological samples.

[0007] Furthermore, prior art methods may require solid phase synthesis methods or the use of non-natural coupling chemistries to attach the cargo to the peptide.

[0008] Prior art methods may further require specific solvents, which may not be compatible with peptides that are insoluble in such solvents, and may be particularly appropriate for relatively large peptides and proteins.

[0009] Thus, prior art methods may be quite limited in their compatibility with peptides, especially with respect to C-terminal residues and various types of cargo.

[0010] Thus, aspects of the present invention provide alternative methods of C-terminal peptide modification, which preferably further at least partially obviate one or more of the above-mentioned drawbacks. The present invention may have the object of overcoming or ameliorating at least one of the disadvantages of the prior art, or of providing a useful alternative method. [Means for solving the problem]

[0011] Thus, in a first aspect, the present invention may provide a method for providing a cargo at the C-terminus of a peptide, i.e., a method for providing a peptide-cargo conjugate. The method may comprise a first stage and a second stage. The first stage may particularly comprise reacting the C-terminus of the peptide with a first reactant in the presence of a first catalyst and a first radiation to provide a first intermediate. The first catalyst may particularly be configured to decarboxylate the C-terminus of the peptide in the presence of the first radiation. In an embodiment, the first reactant has a first chemical structure according to formula I,

[0012] [ka]

[0013] In particular, R and R' (or "R'") are each independently selected from the group consisting of H and an alkyl group, R" (or "R") is an electron withdrawing group comprising a functional group selected from the group consisting of an ester, a thioester, an amide, a ketone, a nitro, a sulfoxide, a sulfone, a phosphate ester, an acyl hydrazide, a cyano group, and a trihalogen methyl group, and n1 and n2 can each independently be selected from the range of 1 to 2. In embodiments, the second stage can include exposing the first intermediate to a second reactant. In embodiments, the second reactant has a second chemical structure according to Formula II:

[0014] [ka]

[0015] In particular, X may be O or NH and R1 may comprise a cargo, whereby the method, in particular the second step, may provide a protein-cargo conjugate.

[0016] The method of the present invention involves selectively decarboxylating the C-terminus of a peptide, which can introduce a radical, followed by attaching a reactive group to the C-terminus of the peptide, in particular to a bis(2-sulfanylacyl)amino functional group, such as a bis(2-sulfanylethyl)amino functional group. The reactive group can then be used in a second step to attach a cargo to the peptide. In particular, the method of the present invention can provide the advantage that a wide variety of cargoes, including other peptides, can be selectively covalently attached to the C-terminus of a native peptide. For example, this can be beneficial in the context of peptide sequencing, in particular protein sequencing, in the formation of antibody drug conjugates, and in providing peptide arrays, in particular protein arrays.

[0017] In particular, the method, and in particular the first reactant, may be compatible with most peptides, since C-terminal modification may be possible for peptides having any of the following standard residues as the C-terminal residue: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, the method is believed to be compatible with the naturally occurring non-standard amino acids pyrrolysine and selenocysteine, as well as the majority of unnatural amino acids.

[0018] Furthermore, the method may be carried out not only in water but also in organic solvents such as DMSO or DMF, which, taking into account the solubility of the peptide (or cargo), may allow the use of a wider range of substrates than prior art methods.

[0019] Thus, in certain embodiments, the present invention provides a method for providing a cargo at the C-terminus of a peptide, comprising a first stage and a second stage, the first stage comprising reacting the C-terminus of the peptide with a first reactant in the presence of a first catalyst and a first radiation to provide a first intermediate, the first catalyst being configured to decarboxylate the C-terminus of the peptide in the presence of the first radiation, and the first reactant having a first chemical structure according to formula I:

[0020] [ka]

[0021] wherein R and R' are each independently selected from the group consisting of H and an alkyl group; R'' is an electron withdrawing group comprising a functional group selected from the group consisting of an ester, a thioester, an amide, a ketone, a nitro, a sulfoxide, a sulfone, a phosphate ester, an acylhydrazide, a cyano group, and a trihalogenmethyl group; n1 and n2 are each independently selected from the range of 1 to 2; and the second step comprises exposing the first intermediate to a second reactant, the second reactant having a second chemical structure according to formula II;

[0022] [ka]

[0023] wherein X is O or NH and R1 comprises a cargo.

[0024] Thus, the present invention may provide a method for providing cargo at the C-terminus of a peptide.

[0025] The term "cargo" may refer to any compound suitable for binding to a peptide. For example, cargo may include a second peptide, an antibody, a drug, a transcription factor, a purification tag, a nanoparticle, a fluorescent dye (or "fluorescent probe"), a reporter molecule, and an anchor tag.

[0026] The term "peptide" may herein refer to a chain of amino acids of any length, in particular a chain of at least two amino acids, more particularly a chain of 10 or more amino acids. Thus, the term peptide may also refer to oligopeptides, polypeptides, and proteins. In an embodiment, the peptide may comprise a natural peptide, in particular a natural protein, i.e. an unmodified peptide (or protein) produced by a naturally occurring organism. The peptide may in particular comprise a number of amino acids selected from the range of 2-4000, such as 3-2500, in particular 3-1000. In a further embodiment, the peptide may comprise up to 500 amino acids, such as up to 100 amino acids, in particular up to 50 amino acids, such as up to (about) 40 amino acids. In a further embodiment, the peptide may comprise at least 2 amino acids, such as at least 3 amino acids, in particular at least 5 amino acids.

[0027] The method may in particular include a first stage and a second stage. As used herein, the term "stage" and similar terms may refer to a period (and "phase") of the method. The first stage and the second stage may in particular be separated in time, with the second stage being located in time after the first stage.

[0028] The first stage may comprise reacting the C-terminus of the peptide with a first reactant in the presence of a first catalyst and a first radiation to provide, inter alia, a first intermediate.

[0029] The first catalyst may be specifically configured to decarboxylate the C-terminus of a peptide in the presence of a first radiation. In particular, the first catalyst may be a riboflavin catalyst, including riboflavin derivatives, such as riboflavin tetrabutyrate and lumiflavin, and an iridium catalyst, such as Ir[dF(CF 3 )ppy] 2 (dtbbpy)PF 6 The first catalyst may comprise one or more of, in particular riboflavin tetrabutyrate, or in particular lumiflavin. The first catalyst may in particular be a photocatalyst that decarboxylates the C-terminus of a peptide when exposed to the first radiation. Thus, the first catalyst may be configured to decarboxylate the C-terminus of a peptide in the presence of the first radiation. The first radiation may in particular comprise a first wavelength in the range of 250-600 nm, in particular in the range of 300-600 nm. In a further embodiment, the first radiation may comprise a first wavelength in the range of 375-525 nm, for example in the range of 400-525 nm. The first radiation may in particular comprise a first wavelength selected such that the C-terminus of a protein is decarboxylated by the first catalyst when exposed to the first radiation. That is, the first wavelength may be suitable for the first catalyst to decarboxylate the C-terminus of a protein when exposed to the first radiation.

[0030] In a further embodiment, when the first solvent (see below) comprises, and in particular consists of, an organic solvent, the first catalyst is (Ir[dF(CF 3 )ppy] 2 (dtbpy))PF 6(CAS number 870987-63-6), [Ir(dtbbpy)(ppy) 2 ]PF 6 (CAS number 676525-77-2), [Ir(dFppy) 2 (dtbbpy)]PF 6 (CAS number 1072067-44-7), [Ir(dFCF 3 ppy) 2 -(5,5'-dCF 3 bpy)]PF 6 (CAS number 1973375-72-2), [Ir(dF(Me)ppy) 2 (dtbbpy)]PF 6 (CAS number 1335047-34-1), [Ir{dFCF 3 ppy} 2 (bpy)]PF 6 (CAS number 1092775-62-6), [Ir(pF(Me)ppy) 2 -(4,4'-dtbbpy)]PF 6 (CAS number 808142-88-3), Ir(dFppy) 3 (CAS number 387859-70-3), Ir(dFFppy) 2 (dtbbpy)PF 6 (CAS number 2042201-18-1) and Ir[dFFppy] 2 -(4,4'-dCF 3 bpy)PF 6 (CAS No. 2030437-92-2). In such an embodiment, the first environment, in particular the first solvent, may particularly comprise 0.005 to 2 mM, for example 0.01 to 1 mM, of the first catalyst.

[0031] In a further embodiment, when the first solvent comprises water or when the first solvent comprises an organic solvent / water mixture, the first catalyst is selected from the group consisting of riboflavin tetrabutyrate (CAS No. 752-56-7), lumiflavin (CAS No. 1088-56-8), riboflavin (CAS No. 83-88-5), riboflavin 5'-monophosphate sodium salt (CAS No. 130-40-5), lumichrome (CAS No. 1086-80-2), riboflavin N-ethylcarbamate, 4-[2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridiamine] ... In one embodiment, the first environment, particularly the first solvent, may comprise a riboflavin catalyst comprising a riboflavin derivative selected from the group comprising 10-ethyl-3,7,8-trimethyl-benzo[g]pteridine-2,4(3H,10H)-dione, 7,8-dimethyl-10-{2-[bis(2-hydroxyethyl)amino]ethyl}isoalloxazine hydrochloride, 7,8-diethyl-10-{2-[bis(2-hydroxyethyl)amino]ethyl}isoalloxazine hydrochloride, and 7,8-diethyl-10-(1'-d-ribityl)isoalloxazine. In such an embodiment, the first environment, particularly the first solvent, may comprise 0.005 to 600 mM, for example 0.01 to 300 mM, of the first catalyst.

[0032] In further embodiments, the first catalyst may comprise a mesitylacridinium catalyst. In such embodiments, the first environment, particularly the first solvent, may particularly comprise 0.01 to 1 mM of the first catalyst.

[0033] In embodiments, the first reactant may have a first chemical structure according to Formula I:

[0034] [ka]

[0035] In an embodiment, R and R' are each independently selected from the group consisting of H and an alkyl group, and may in particular be selected from the group consisting of H and an alkyl group containing 1-10 C atoms, in particular 1-6 C atoms, e.g. 1-4 C atoms. In particular, the alkyl group may affect the solubility of the first reactant in various solvents, e.g. a large amount of alkyl group reduces the solubility of the first reactant in water. Furthermore, the first reactant may be more reactive (with respect to the formation of the first intermediate) when R and R' contain H or a small amount of alkyl group. Thus, in an embodiment, R and R' may in particular be selected from the group consisting of H and an alkyl group containing 1-6 C atoms, e.g. H and an alkyl group containing 1-4 C atoms, in particular H and an alkyl group containing 1-2 C atoms. In a further embodiment, at least one of R and R' may contain H, in particular R and R' both contain H.

[0036] In an embodiment, R'' may be an electron-withdrawing group, in particular an electron-withdrawing group comprising a functional group selected from the group consisting of ester, thioester, amide, ketone, nitro, sulfoxide, sulfone, phosphate ester, acylhydrazide, cyano group, and trihalogenmethyl group, in particular a functional group selected from the group consisting of ester, sulfoxide, sulfone, cyano group, and trihalogenmethyl group. In particular, the electron-withdrawing group may be selected such that the C=C bond shown in formula I may be (further) reactive with respect to the decarboxylated C-terminus of the peptide. In a further embodiment, the electron-withdrawing group may in particular comprise an ester group. In a further embodiment, the electron-withdrawing group may in particular comprise a ketone functional group. In a further embodiment, the electron-withdrawing group may in particular comprise a nitro functional group. In a further embodiment, the electron-withdrawing group may in particular comprise a sulfoxide functional group. In a further embodiment, the electron-withdrawing group may in particular comprise a phosphate ester functional group. In further embodiments, the electron withdrawing group may specifically include an acylhydrazide functional group. In further embodiments, the electron withdrawing group may specifically include a cyano functional group. In further embodiments, the electron withdrawing group may specifically include a trihalogenmethyl functional group, in particular a trihalogenmethyl functional group that includes a halogen selected from the group including F, Cl, and Br, and more particularly a trihalogenmethyl functional group that includes three identical halogens, for example, -CF 3 , -CCl 3 or CBr 3 , especially -CF 3 Examples of the trihalogen methyl functional groups include:

[0037] In further embodiments, R″ is an ester, a cyano group, or —CF 3 In embodiments, when R" comprises an ester, R" may specifically comprise a methyl ester. Thus, in certain embodiments, R" may comprise a methyl ester.

[0038] In embodiments, n1 and n2 may each be independently selected from the range of 1 to 2. In particular, in the second step (see below), the ring (bis(2-sulfanylacyl)amide functionality) may open due to protonation of the sulfur atom, and the number of carbon atoms between the nitrogen and sulfur in each "arm" may determine the distance of further reaction, with the presence of two carbon atoms between the N and S atoms appearing to be particularly effective. Thus, in further embodiments, at least one of n1 and n2 may be 1, and in particular, both n1 and n2 are 1.

[0039] Thus, in certain embodiments, the first reactant can have a first chemical structure according to Formula III.

[0040] [ka]

[0041] A first reactant according to formula III may be particularly advantageous for the methods of the present invention, given their reactivity and solubility.

[0042] In further embodiments, at least one of n1 and n2 may be 2, and in particular, both n1 and n2 are 2.

[0043] In an embodiment, in the first step, the peptide may be exposed to a first environment. In particular, the peptide (and the first catalyst) may be placed in a first mixture comprising a first solvent. In an embodiment, the first solvent may be selected from the group comprising water, dimethylsulfoxide, (DMSO) and dimethylformamide (DMF), acetonitrile, dimethylacetamide, N-methylpyrrolidone, and tetrahydrofuran, in particular from the group comprising water, DMSO, and DMF. In a further embodiment, the first solvent may (further) comprise an alcohol, in particular 1-butanol.

[0044] The first mixture may specifically comprise one or more of a first solvent, a peptide, and a first reactant. In embodiments, the first mixture may comprise a further additive (see below).

[0045] In an embodiment, the first solvent may comprise water. In such an embodiment, the first solvent may have a pH selected from the range of 0.5 to 8, for example from the range of 0.5 to 6, in particular from the range of 0.5 to 4.5. In a further embodiment, the first solvent may have a pH selected from the range of 1 to 4, for example from the range of 2 to 3. Thus, in an embodiment, the first environment may have a pH selected from the range of 0.5 to 8, for example from the range of 0.5 to 6, in particular from the range of 0.5 to 4.5. In a further embodiment, the first environment may have a pH selected from the range of 0.5 to 4.5, in particular from the range of 1 to 4, for example from the range of 2 to 3. In particular, it may be beneficial for the first stage to occur in a first environment having a pH of about 2 to 3, in particular (about) 2.5. In an embodiment, the first solvent may comprise at least 70 vol.%, such as at least 80 vol.%, particularly at least 90 vol.%, including 100 vol.%, water.

[0046] In a further embodiment, the first environment, in particular the first solvent, may have a pH of at least 0.5, in particular at least 1. In a further embodiment, the first environment, in particular the first solvent, may have a pH of at least 1.5, in particular at least 2. In a further embodiment, the first environment, in particular the first solvent, may have a pH of at most 8.5, such as at most 8, in particular at most 6, for example at most 4.5. In a further embodiment, the first environment, in particular the first solvent, may have a pH of at most 4, in particular at most 3.5, for example at most 3.

[0047] In a further embodiment, the first solvent may comprise an organic solvent, in particular an organic solvent selected from the group comprising DMSO, acetonitrile and DMF. In an embodiment, the first solvent may comprise at least 70 vol.%, for example at least 80 vol.%, in particular at least 90 vol.%, including 100 vol.% of an organic solvent. The term "organic solvent" as used herein also refers to a plurality of (different) organic solvents.

[0048] In an embodiment, the first environment, in particular the first mixture, more particularly the first solvent, may have a first temperature selected from the range of 5-45° C., in particular from the range of 10-40° C. In a further embodiment, the method may comprise, in particular in the first stage, controlling the first temperature to a maximum of 45° C., such as a maximum of 40° C. In a further embodiment, the method may comprise controlling the first temperature using a temperature control element, in particular a cooling element.

[0049] In embodiments in which the first solvent comprises water, the first mixture may further comprise a sequencing agent for reactive oxygen species. In particular, in embodiments, the first mixture may comprise 1-10 vol.%, e.g., 3-7 vol.% glycerin.

[0050] Further, in an embodiment, the first solvent may include water and an organic solvent, such as 1-butanol. In particular, the first solvent may include at least 50 wt.% water and 2-20 wt.%, for example 5-15 wt.% organic solvent. The organic solvent may facilitate dissolution of the first catalyst. In a further embodiment, the first solvent may include an aqueous phase and an organic phase.

[0051] In an embodiment, the first environment, in particular the first mixture, may comprise between 0.02 and 100 mM peptide, in particular between 0.05 and 50 mM peptide.

[0052] In a further embodiment, the first environment, in particular the first mixture, may comprise from 0.05 to 1000 mM, in particular from 0.1 to 500 mM, of the first reactant.

[0053] In an embodiment, the first environment, in particular the first mixture, may comprise a salt, in particular 0.05 to 100 mM salt, for example 1 to 50 mM salt, in particular 2 to 10 mM salt. In an embodiment, in particular in an embodiment in which the first solvent comprises an organic solvent, the salt may be selected from the group comprising phosphate, hydrogen phosphate, dihydrogen phosphate, formate, trifluoroacetate, acetate, fluoride and carbonate, in particular K 2 HPO 4 , Cesium formate, Sodium trifluoroacetate, CsF, CsOAc, Cs 2 CO 3 , CsF and K 2 CO 3 From the group comprising, more particularly, 2 HPO 4 , cesium formate and sodium trifluoroacetate.

[0054] Thus, the first step may yield a first intermediate. The first intermediate may in particular have a formula according to formula IB.

[0055] [ka]

[0056] In particular, R 3 may refer to the peptide, where R, R', R'', n1 and n2 are as defined for the first reactant.

[0057] The second stage may include exposing the first intermediate to a second reactant. In particular, the second stage may include reacting the first intermediate and the second reactant to provide a peptide-cargo conjugate. The second reactant may in particular have a second chemical structure according to formula II.

[0058] [ka]

[0059] In embodiments, X may be selected from the group including O and NH. In further embodiments, R1 may include a cargo.

[0060] In particular, the second chemical structure may resemble an N-terminal cysteine ​​residue and may be attached to the cargo via an amide (CONR) as in natural proteins with N-terminal cysteine ​​residues, or may be attached to the cargo via an ester (COO). In embodiments where the cargo is attached via an amide, R in CONR is H or an alkyl group, in particular H or CH 3 , and more specifically, may refer to H.

[0061] In an embodiment, in the second stage, the first intermediate may be exposed to a second environment. In particular, the first intermediate (and the second reactant) may be disposed in a second mixture, in particular in a second solvent. In an embodiment, the second solvent may in particular be selected from the group comprising water, dimethylsulfoxide (DMSO) and dimethylformamide (DMF).

[0062] The second mixture may specifically comprise one or more of the second solvent, the first intermediate, and the second reactant. In embodiments, the second mixture may comprise further additives (see below).

[0063] In an embodiment, the second solvent may comprise water. In an embodiment, the second solvent may comprise at least 70 vol.%, such as at least 80 vol.%, in particular at least 90 vol.%, including 100 vol.%, of water.

[0064] In such an embodiment, the second solvent may have a pH selected from the range of 1 to 11, for example, 2 to 10, in particular, 2 to 9. In a further embodiment, the second solvent may have a pH selected from the range of 3 to 7, in particular, 3.5 to 7, for example, 4 to 6.5. In a further embodiment, the second solvent may have a pH selected from the range of 4.5 to 6.5, in particular, 5 to 6. Thus, in an embodiment, the second environment may have a pH selected from the range of 1 to 11, for example, 2 to 10, in particular, 2 to 9. In a further embodiment, the second environment may have a pH selected from the range of 3 to 7, in particular, 3.5 to 7, for example, 4 to 7. In a further embodiment, the second environment may have a pH selected from the range of 4.5 to 6.5, in particular, 5 to 6. In particular, it may be beneficial for the second step to occur in an environment having a slightly acidic pH, for example in the range of 3.5 to 6, in particular in the range of 4 to 5.5. In particular, the slightly acidic conditions may promote an N-S conversion, which may result in a first intermediate that forms a thioester group in situ. The thioester group may specifically react with a second reactant to provide a peptide-cargo conjugate.

[0065] In further embodiments, the second environment, in particular the second solvent, may have a pH of at least 1, in particular at least 2, for example at least 3. In further embodiments, the second environment, in particular the second solvent, may have a pH of at least 3.5, in particular at least 4. In further embodiments, the second environment, in particular the second solvent, may have a pH of at most 11, in particular at most 10, for example at most 7.5. In further embodiments, the second environment, in particular the second solvent, may have a pH of at most 7, in particular at most 6.5, for example at most 6, for example at most 5.5.

[0066] Thus, in an embodiment, in a second step, the first intermediate may undergo an N-S conversion to provide a thioester group, which reacts with a second reactant to form a peptide-cargo conjugate.

[0067] In particular, in the second step, the first intermediate is reacted with a reducing agent, in particular tris(2-carboxyethyl)phosphine hydrochloride (TCEP), tris(hydroxymethyl)phosphine (CAS number 2767-80-8), tris(hydroxypropyl)phosphine (CAS number 4706-17-6), tris(4-methoxyphenyl)phosphine (CAS number 855-38-9), (4-hydroxyphenyl)diphenylphosphine (CAS number 5068-21-3), (2-hydroxyphenyl)diphenylphosphine (CAS number 60254-10-6), triphenylphosphine- The mixture may be exposed to a reducing agent, in particular TCEP, selected from the group including 3,3',3''-trisulfonic acid trisodium salt hydrate (CAS 335421-90-4), triphenylphosphine-3,3',3''-trisulfonic acid (CAS 91171-35-6), tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate (CAS 1166403-40-2), trimethyl 3,3',3''-phosphine triyltripropanoate (CAS 29269-17-8), tris(3-chlorophenyl)phosphine (CAS number 29949-85-7). The reducing agent may reduce the disulfide bond in the first intermediate to provide an SH group, which may facilitate NS conversion. In a further embodiment, the second mixture may contain 10 to 100 mM of a reducing agent, in particular TCEP.

[0068] In an embodiment, the second mixture may include a thiol catalyst, in particular an alkanethiol catalyst, or in particular an aromatic thiol catalyst. The thiol catalyst may facilitate the rapid second stage. In a further embodiment, the thiol catalyst may include one or more of 4-mercaptophenylacetic acid (MPAA; CAS No. 39161-84-7), 2-mercaptomethanesulfonic acid sodium salt (MESNA; CAS No. 19767-45-4) and 2,2,2-trifluoroethanethiol (TFET; CAS No. 1544-53-2), in particular MPAA, or in particular one or more of MESNA and TFET. In particular, the presence of an alkanethiol catalyst, for example MESNA and TFET, may facilitate stable conversion at acidic pH, for example a pH of about 4.

[0069] The thioester group formed can be specifically reacted with a second reactant to form a peptide-cargo conjugate.

[0070] In an embodiment, the second environment, in particular the second mixture, may have a second temperature selected from the range of 25-70°C, such as from the range of 35-55°C.

[0071] In a further embodiment, the second solvent may comprise an organic solvent, in particular an organic solvent selected from the group comprising dimethylsulfoxide (DMSO), acetonitrile and dimethylformamide (DMF). In a further embodiment, the second solvent may comprise at least 70 vol.%, such as at least 80 vol.%, in particular at least 90 vol.%, including 100 vol.%, of an organic solvent.

[0072] In an embodiment, the second environment, in particular the second mixture, may comprise between 0.05 and 200 mM of the first intermediate, in particular between 0.1 and 100 mM of the first intermediate.

[0073] In a further embodiment, the second environment, in particular the second mixture, may comprise from 0.05 to 400 mM, in particular from 0.1 to 200 mM, of the second reactant.

[0074] The presence of oxygen may be detrimental to providing the first intermediate and / or to providing the protein-cargo conjugate in the second step. In particular, the presence of oxygen may be detrimental to the photocatalytic decarboxylation of peptides in the first step. In particular, in the first step, the presence of oxygen may cause a decrease in the yield of the first intermediate. Furthermore, oxygen may oxidize S in the first reactant to S=O, which may occur during irradiation with the first radiation. If both S are oxidized to S=O, NS conversion to thioester may no longer be possible in the second step. Furthermore, in the second step, the presence of oxygen may also cause oxidation of SH groups, which may cause ring closure by forming disulfide structures. Thus, in both steps, the presence of oxygen may interfere with the method of the present invention.

[0075] Thus, in an embodiment, the first environment, in particular the first mixture, may comprise a degassed buffer. In particular, the first environment, in particular the first mixture, may comprise 10 ppm or less of dissolved oxygen, for example 5 ppm or less of dissolved oxygen, in particular 2 ppm or less of dissolved oxygen. In a further embodiment, the first environment, in particular the first mixture, may comprise 1 ppm or less of dissolved oxygen, for example 0.8 ppm or less of dissolved oxygen, in particular 0.6 ppm or less of dissolved oxygen, for example 0.5 ppm or less of dissolved oxygen. In a further embodiment, the first mixture may comprise up to 0.4 ppm of dissolved oxygen, for example 0.2 to 0.4 ppm of dissolved oxygen.

[0076] In an embodiment, the method may comprise the step of controlling, in particular reducing, the (dissolved) oxygen concentration in the first environment, in particular in the first mixture, e.g., in the first solvent. In particular ... 2 , or particularly may include the step of sparging (or "purging") the first mixture with Ar.

[0077] In a further embodiment, the method further comprises the step of: 2Or it may involve carrying out the first stage in a degassed buffer solution under Ar, in particular Ar. Argon may be particularly suitable as it is heavier than air.

[0078] In a further embodiment, the second environment, in particular the second mixture, may comprise a degassed buffer. In particular, the second environment, in particular the second mixture, may comprise 10 ppm or less of dissolved oxygen, for example 5 ppm or less of dissolved oxygen, in particular 2 ppm or less of dissolved oxygen. In a further embodiment, the second environment, in particular the second mixture, may comprise 1 ppm or less of dissolved oxygen, for example 0.8 ppm or less of dissolved oxygen, in particular 0.6 ppm or less of dissolved oxygen. In a further embodiment, the second mixture may comprise up to 0.4 ppm of dissolved oxygen, for example 0.2-0.4 ppm of dissolved oxygen.

[0079] In an embodiment, the method may include the step of controlling, in particular reducing, the (dissolved) oxygen concentration in the second environment, in particular in the second mixture. In particular, the method may include the step of controlling, in particular reducing, the (dissolved) oxygen concentration in the second environment, in particular in the second mixture, by using an inert gas, in particular N 2 , or particularly, may include sparging the second mixture with Ar.

[0080] In a further embodiment, the method further comprises the step of: 2 Or it may involve carrying out the second stage in a degassed buffer solution under Ar, more particularly Ar. Argon may be particularly suitable as it is heavier than air.

[0081] The first and second steps may in particular be carried out in (essentially) the same solvent, even if the solvents differ, for example in pH or salt concentration, for example having different pH (in the case of water). For example, both the first and second steps may be carried out in water, in particular adding a base to the first mixture to provide the second mixture. However, in embodiments, the first and second steps may be carried out in different solvents. In such embodiments, the method may further comprise an intermediate step. The intermediate step may in particular comprise a step of separating the first intermediate from the remainder of the first reactant and / or peptide and / or first catalyst, in particular the first catalyst, or in particular the (remaining of) the first reactant, or in particular the (remaining of) the peptide. The intermediate step may in particular comprise a step of separating the first intermediate from the first solvent. In an embodiment, the intermediate step may comprise extraction, in particular extraction with an organic solvent, for example extraction with one or more of ethyl acetate, dichloromethane or an ether, for example an ether selected from the group comprising diethyl ether, methyl tert-butyl ether and diisopropyl ether.

[0082] The intermediate stage may in particular be located in time between the first stage and the second stage.

[0083] As shown above, the method of the present invention can be compatible with a wide variety of C-terminal residues, including (natural) proteinogenic amino acids, such as standard amino acids, as well as natural amino acids that are not naturally incorporated into proteins, such as ornithine, as well as unnatural amino acids. Thus, in embodiments, the peptide can comprise a C-terminal residue selected from the group comprising alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, ornithine and valine, in particular the group comprising alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0084] For some C-terminal amino acids, e.g., cysteine ​​and selenocysteine, it may be beneficial to protect the C-terminal amino acid before the first step. Thus, in an embodiment, the method may further comprise a preparatory step temporally positioned before the first step, the preparatory step comprising protecting the side chains of the residues of the peptide, in particular the cysteine ​​residues, or in particular the selenocysteine ​​residues. Methods for capping such residues are known in the art. For example, the preparatory step may comprise protecting the side chains of the residues using one or more of iodoacetamide, acrylamide, maleimide, alpha-haloacetamide, and capping by disulfide formation with a small molecule activated disulfide. In particular, the preparatory step may comprise protecting each cysteine ​​and selenocysteine ​​residue of the peptide.

[0085] In a further embodiment, the preparation step may include blocking residues, particularly cysteine ​​or selenocysteine ​​residues, using iodoacetamide or maleimide.

[0086] In a further embodiment, the preparation step may include protecting residues, in particular cysteine ​​or selenocysteine ​​residues, by forming disulfides with thiols.

[0087] In an embodiment, the cargo may comprise a second peptide. Thus, in an embodiment, the peptide-cargo conjugate may comprise a peptide-peptide conjugate, in particular a fusion peptide. In a further embodiment, the second peptide may have an N-terminus according to formula II, in particular an N-terminal cysteine ​​residue. Thus, the method of the invention may facilitate providing a fusion peptide (or protein), in particular where the peptide and optionally the second peptide are naturally occurring. Thus, the method of the invention may facilitate providing a non-recombinant fusion peptide (or protein).

[0088] In a further embodiment, the cargo may comprise an antibody. In particular, as known to those skilled in the art, an antibody may comprise multiple polypeptides and therefore bind to this peptide as well as other peptides. In particular, the antibody may be configured to target a (specific) target location in an animal body, in particular in the human body, and the peptide may have a (suitable) medical or therapeutic function at the target location. This may facilitate the provision of the peptide to a specific site in an animal body, in particular in the human body, by the peptide-cargo conjugate. Thus, the method of the present invention may facilitate the provision of a peptide-antibody conjugate, in particular an antibody-drug conjugate.

[0089] In embodiments, the cargo may comprise a tag suitable for selectively positioning the (resulting) peptide-cargo conjugate on a target location.

[0090] Various specific examples of cargo are described herein, however, it will be apparent to one of skill in the art that the invention is not limited to such examples and can further encompass a wide variety of other cargo compounds.

[0091] In a further aspect, in particular, the present invention may provide a peptide-cargo conjugate obtainable using the method of the present invention. The peptide-cargo conjugate may in particular have a chemical structure according to formula IV:

[0092] [ka]

[0093] In the formula, R, R', R 3 , R″, n1 and R 1 may be the same as defined above for the peptide, the first reactant, and the second reactant. In certain embodiments, R 3may be a peptide, R and R' may be independently selected from the group consisting of H and an alkyl group, R'' may be an electron withdrawing group comprising a functional group selected from the group consisting of an ester, a thioester, an amide, a ketone, a nitro, a sulfoxide, a sulfone, a phosphate ester, an acylhydrazide, a cyano group, and a trihalogen methyl group, n1 may be selected in the range of 1 to 2, R 1 may include a cargo.

[0094] In embodiments, the cargo may comprise an antibody. Thus, the peptide-cargo conjugate may comprise a peptide-antibody conjugate.

[0095] In further embodiments, the peptide may comprise (one or more polypeptides of) an antibody. In such embodiments, the cargo may comprise, for example, a drug, e.g., a peptide drug. In particular, the C-terminus of an antibody may not typically be involved in guest binding, and thus this functionalization may not interfere with antibody binding. As antibodies generally have four C-termini, loading of well-defined cargo may be facilitated.

[0096] In embodiments, the peptide-cargo conjugate may be for use as an antibody-drug conjugate. In particular, the peptide-cargo conjugate may be for use as a pharmaceutical, in particular the peptide has a medically relevant activity. An antibody-drug conjugate (ADC) is a construct of an antibody that is linked to a (cytotoxic) drug, in particular via a covalent linker. The purpose of the antibody may be (selective) targeting, and the drug may act, for example, to kill tumor (or other) cells. The linker may act to maintain the binding of the drug to the antibody, for example to avoid release of the drug in the absence of the target cell or tissue. It may therefore be important that drugs are attached via stable linkages and in controllable numbers to the antibody (which may make attachment to multiple amine or carboxylic acid side chains of the antibody less favorable as this may result in heterogeneous mixtures and the properties of the antibody may be altered). Thus, the methods of the invention may be particularly suitable for providing antibody-drug conjugates, also taking into account that the C-terminus of the antibody may not typically be involved in guest binding and therefore this functionalization may not interfere with antibody binding.

[0097] In embodiments, the cargo may comprise a second peptide. Thus, the peptide-cargo conjugate may comprise a fusion peptide, in particular a non-recombinant fusion peptide.

[0098] In further embodiments, the cargo may comprise a tag suitable for selectively positioning the peptide-cargo conjugate on a target location, e.g., on a surface. In further embodiments, the tag may comprise an antibody. Such embodiments may facilitate sequencing of the peptides and may facilitate providing peptide arrays.

[0099] Thus, in a further aspect, the present invention provides a peptide array comprising the peptide-cargo conjugate according to the present invention, in particular the peptide-cargo conjugate may comprise a tag. In particular the peptide array may comprise a library of peptides bound to a solid surface, e.g. a glass slide. Such arrays may be used on a large scale for the identification of peptide drug candidates, enzyme inhibitors, and enzyme substrates for profiling antibodies and mapping epitopes and receptor-ligand interactions. The most common method for generating such arrays is by covalent attachment, which may require reliable and selective binding chemistry, such as those described in Szymczak et al., "Peptide Arrays: Development and Application", Analytical Chemistry, 2018, which is hereby incorporated by reference. Usually, suitable chemical groups are introduced in peptide synthesis, but may be difficult to introduce into native peptides (peptides isolated from biological sources) by prior art methods. However, the method of the present invention may facilitate the selective C-terminal introduction of suitable chemical groups in peptides, facilitating the provision of peptide arrays. Thus, in embodiments, the cargo may comprise a tag suitable for selective placement of the peptide-cargo conjugate on a target location, in particular on a surface. For example, the tag may be suitable for click chemistry. In embodiments, the tag may be selected from the group including polynucleotides (configured to hybridize with a complementary polynucleotide at the target location), biotin tags (suitable for hybridizing with avidin or streptavidin at the target location) and azide-containing peptides (suitable for chemical immobilization by click chemistry on cyclooctyne-modified surfaces). It will be clear to those skilled in the art that many specific choices can be made for the cargo to allow placement of the peptide-cargo conjugate at the target location of the peptide array.

[0100] In a further aspect, the present invention may provide for the use of peptide-cargo conjugates for peptide sequencing. In particular, next-generation peptide / protein sequencing technologies, such as those described in Alfaro et al., "The emerging landscape of single-molecule protein sequencing technologies", Nature Methods, 2021, hereby incorporated by reference, may involve either immobilization of the peptide on a surface or binding of the peptide to a molecular entity whose translocation through a nanopore is facilitated. In both scenarios, it is important that binding occurs at a single specific location on the peptide to provide reliable positioning on the surface or when passing through the nanopore. As will be apparent to the skilled artisan, binding to either the N-terminus or C-terminus of the peptide may be particularly advantageous. The method of the present invention facilitates providing a bond between the peptide and a surface or a molecular entity at the C-terminus of the peptide, which may facilitate peptide sequencing of the peptide. In particular, in such an embodiment, the cargo may comprise a polynucleotide, in particular a single-stranded polynucleotide. The polynucleotide may be configured to position the peptide on the surface, for example, via hybridization with a (complementary) polynucleotide bound to the surface. The polynucleotide may be further configured to facilitate passage through the nanopore by the peptide-cargo conjugate.

[0101] The embodiments described herein are not limited to a single aspect of the invention. For example, an embodiment describing a method may further relate to, for example, a (resulting) peptide-cargo conjugate. Similarly, an embodiment of a peptide-cargo conjugate may further relate to an embodiment of a method. In particular, an embodiment of a method describing a structural formula with a particular group (e.g., a specifically selected potential R″ group) may indicate that a peptide-cargo conjugate may, in an embodiment, have such a group (e.g., a specifically selected potential R″ group). [Brief description of the drawings]

[0102] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: [Figure 1-1] An embodiment of the method is illustrated diagrammatically in Figure 1A. The diagram is not necessarily to scale. [Figure 1-2] An embodiment of the method is illustrated diagrammatically in Figure IB. The diagram is not necessarily to scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] In FIG. 1A, an embodiment of a method for providing a cargo at the C-terminus 111 of a peptide 110, i.e., a peptide-cargo conjugate, is shown diagrammatically. In the illustrated embodiment, the method comprises a first stage and a second stage, the first stage comprising reacting the C-terminus 111 of the peptide 110 with a first reactant 120 in the presence of a first catalyst 130 and a first radiation 140 to provide a first intermediate 150. Furthermore, the first catalyst 130 may be configured to decarboxylate the C-terminus 111 of the peptide 110 in the presence of the first radiation 140. In the illustrated embodiment, the first reactant 120 has a first chemical structure according to formula I:

[0104] [ka]

[0105] In particular, in the formula, R and R' are each independently selected from the group consisting of H and an alkyl group, and in particular, R'' is an electron-withdrawing group comprising a functional group selected from the group consisting of an ester, a thioester, an amide, a ketone, a nitro, a sulfoxide, a sulfone, a phosphate ester, an acylhydrazide, a cyano group, and a trihalogenmethyl group, and in particular, n1 and n2 are each independently selected in the range of 1 to 2.

[0106] In the illustrated embodiment, the second stage includes exposing the first intermediate 150 to a second reactant 220, and in particular, in the second stage, the first intermediate 150 is exposed to a second environment 200 having a pH selected from the range of 3.5 to 7. In an embodiment, the second reactant 220 may have a second chemical structure according to Formula II:

[0107] [ka]

[0108] In particular, where X is O or NH and R1 comprises a cargo. Thus, the method of the invention, in particular the second stage, may comprise reacting the first intermediate 150 with a second reactant 220 to provide a peptide-cargo conjugate.

[0109] In an embodiment, the first catalyst 130 is a riboflavin catalyst, such as riboflavin tetrabutylate, and an iridium catalyst, such as Ir[dFCF 3 ppy] 2 dtbbpyPF 6 In particular, the first catalyst may include riboflavin tetrabutyrate.

[0110] The first environment 100 may in particular comprise a first mixture comprising a first solvent 101. In an embodiment, the first solvent 101 may comprise one or more of DMF, DMSO and water, in particular water. Thus, in an embodiment, the first stage may be carried out in the first solvent 101.

[0111] Similarly, the second environment 200 may particularly include a second mixture including a second solvent 201. In an embodiment, the second solvent 201 may include one or more of DMF, DMSO, and water, particularly water. Thus, in an embodiment, the second stage may be carried out in the second solvent 201. In a further embodiment, the second stage may be carried out in an inert gas, such as N 2 This can be done in the following degassed buffer:

[0112] In the embodiment shown, for visualization purposes, only the carboxylic acid group of the C-terminal residue at the C-terminus 111 of peptide 110 is shown. In an embodiment, peptide 110 may include a C-terminal residue selected from the group including alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

[0113] In particular, the first step was carried out using peptide 110 having the following C-terminal residues: proline, asparagine, arginine, valine, lysine, leucine, phenylalanine, serine, alanine and glycine, respectively. First intermediates were successfully obtained for each of proline, asparagine, arginine, valine, lysine, leucine, phenylalanine, serine and alanine.

[0114] The second step was carried out using the first intermediate derived from a peptide having asparagine or arginine as the C-terminal residue, which in both cases successfully yielded peptide-cargo conjugates.

[0115] The second step was further carried out using the first intermediates derived from peptides with proline, valine, lysine, leucine, phenylalanine, serine and alanine as the C-terminal residues, which successfully yielded peptide-cargo conjugates (see below).

[0116] In an embodiment, the cargo may include a second peptide, in particular the second peptide having an N-terminal cysteine ​​residue, i.e. the second reactant 220 may include an N-terminus 222, the second reactant 220 having a cysteine ​​residue at the N-terminus.

[0117] In a further embodiment, the cargo may comprise an antibody.

[0118] 1A further illustrates a schematic representation of a peptide-cargo conjugate obtained using the method of the present invention. In the embodiment shown, peptide-cargo conjugate 250 has a chemical structure according to formula IV:

[0119] [ka]

[0120] In particular, R and R′ are each independently selected from the group consisting of H and alkyl groups, and in particular R 3 is a peptide, in particular R″ is an electron withdrawing group comprising a functional group selected from the group consisting of ester, thioester, amide, ketone, nitro, sulfoxide, sulfone, phosphate ester, acylhydrazide, cyano group, and trihalogenmethyl group, in particular n1 is independently selected in the range of 1 to 2, for example n1 is 1, in particular X is O or NH, in particular R 1 includes cargo.

[0121] In a further embodiment, the peptide-cargo conjugate 250 has a chemical structure according to formula IV-2:

[0122] [ka]

[0123] In particular, R and R′ are each independently selected from the group consisting of H and alkyl groups, and in particular R 3 is a peptide, in particular R″ is an electron withdrawing group comprising a functional group selected from the group consisting of ester, thioester, amide, ketone, nitro, sulfoxide, sulfone, phosphate ester, acylhydrazide, cyano group, and trihalogenmethyl group, in particular X is O or NH, in particular R 1 includes cargo.

[0124] In embodiments, the peptide-cargo conjugate may be for use as a medicament, in particular where the peptide has a medically relevant (biological) function, and in particular where the cargo comprises an antibody. In further embodiments, the peptide-cargo conjugate may be for use as an antibody drug conjugate.

[0125] FIG. 1B illustrates a schematic of a particular embodiment of the method in which a first reactant 120 has a first chemical structure according to structure III.

[0126] [ka]

[0127] In particular, in the illustrated embodiment, n1=1 and n2=1. Further, in the illustrated embodiment, one or more of R and R' are H. In particular, both R and R' are H. Further, in the illustrated embodiment, R'' is an ester, a cyano group, and -CF 3 In particular, R'' comprises an ester, and in particular, R'' comprises a methyl ester.

[0128] 1B further illustrates a schematic representation of a peptide-cargo conjugate obtained using the method of the present invention. In the embodiment shown, peptide-cargo conjugate 250 has a chemical structure according to formula IV:

[0129] [ka]

[0130] where R and R′ are H, and R 3 is a peptide, R″ is an ester, in particular a methyl ester, n1 is 1, R 1 includes cargo.

[0131] In embodiments, the peptide-cargo conjugate may be for use as a medicament, in particular where the peptide has a medically relevant (biological) function, and in particular where the cargo comprises an antibody. In further embodiments, the peptide-cargo conjugate may be for use as an antibody drug conjugate. [Example]

[0132] The methods of the present invention have been successfully used to provide cargo at the C-terminus (111) of various peptides (110) using a linker according to structure III.

[0133] [ka]

[0134] Unless otherwise specified, the following procedures were used.

[0135] Preparation of peptide stock solutions - Peptides were dissolved in anhydrous DMF or DMSO and an amount of peptide stock solution (1 eq, corresponding to 0.25 μmol) was transferred to a 4 mL dram vial and dried overnight at ambient temperature under vacuum (approximately 1 mbar).

[0136] Degassing of solvents and solutions - All solvents used (1-butanol, H 2 O and DMF) and bulk stock solutions (riboflavin catalyst in buffer, glycerol, and 1-butanol) were degassed separately and then mixed with the peptide residue in a vial.

[0137] First phase - Step 1a - Photochemical reaction - A magnetic stir bar, 100 μL glycerol (20% v / v), 25 μL (10.0 eq, 2.5 μmol) of 0.1 mol / L cesium formate buffer pH 3.5 and 215 μL water were added to the peptide solution in a drum vial. The vial was flushed with argon and an ultrasonic bath was used to stimulate dissolution of the peptide. Then, 20 μL (10.0 eq, 2.5 μmol) of the first reactant (or "linker-molecule") stock solution (in DMF) was added to the vial and the reaction mixture was degassed by sparging with nitrogen for 15 minutes. Finally, under a flow of argon, 40 μL (3.0 eq, 0.75 μmol) of degassed riboflavin catalyst solution (in 1-BuOH) was added, the vial was flushed with argon, wrapped in parafilm, and the mixture was irradiated for 16 h using a blue LED (40 W, Kessil, at a distance of approximately 4 cm).

[0138] Thiol addition - To reduce, and in particular to prevent, aza-Michael addition during the drying process, a thiol catalyst was added to the mixture. 2-mercaptoethanol was added to 50 eq (12.5 μmol, 0.978 mg, 0.88 μL) of sample, and the mixture was mixed and stirred for about 30 minutes, then dried under vacuum for approximately 90 minutes.

[0139] First stage - Step 1b - Sample work-up - The residue from step 1a is purified by H using an ultrasonic bath. 2 The mixture was dissolved in 480 μL of HO and divided into two portions. Each portion was then extracted three times with 800 μL of diethyl ether. Both aqueous layers, dried from diethyl ether under a stream of argon, were then combined, mixed, and two portions of 200 μL (up to 0.1 μmol of conjugate) were transferred to two separate tubes. (The remaining aqueous layer was used for MALDI and LC-MS analysis.) The separate fractions were closed with perforated caps and lyophilized.

[0140] Second stage - Step 2 NCL (Native Chemical Ligation with Cargo) - Preparation of ligation buffer pH 4.0: Add 573.2 mg (6 mmol) of guanidine hydrochloride to a separate Eppendorf tube and 500 µL of sodium phosphate buffer (100 mM Na 2 HPO 4 , pH 9). The entire solution was then transferred to a tube containing 33.64 mg (200 μmol) of 4-mercaptophenylacetic acid (MPAA) and dissolved using an ultrasonic bath. 840 μL of this solution was then added to the tube along with 48.16 mg (168.0 μmol) of tris(2-carboxyethyl)phosphine hydrochloride and mixed until dissolved. 60 μL of 6 M NaOH was added to this solution and the solution was mixed. The mixture was adjusted to pH 4.0 using 6 M NaOH. The ligation buffer was then degassed by sparging with nitrogen for 30 minutes.

[0141] Sample preparation - 90.0 µL of degassed ligation buffer was transferred to a tube containing cargo with an N-terminal Cys residue (0.2 µmol cargo as dry residue) and mixed until dissolved. Note: In the following, the reaction mixture was flushed with argon whenever the reaction tube was open. Then, the entire mixture (approximately 6 M guanidine hydrochloride, 200 mM 4-mercaptophenylacetic acid (MPAA), 200 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 100 mM sodium phosphate buffer, up to 1.1 mM peptide, 2.2 mM cargo, pH 4.0) was transferred to a tube containing materials from step 1 and mixed. Finally, the entire mixture was transferred into a 0.5 mL reaction tube, flushed with argon, filled into a sealed glass vial with the lid closed under argon, and stirred at 50 °C for approximately 7-9 days.

[0142] For HPLC analysis and purification, the reaction mixture was 2 The mixture was acidified with 45.0 μL of 10% TFA in O, mixed and transferred into a 1.5 mL reaction tube. The mixture was then diluted with Et 2 MPAA was removed by extraction with 3 x 1 mL of O. The remaining aqueous layer was injected into the HPLC.

[0143] Experiment 1 Peptide-cargo conjugates were successfully formed by the methods of the invention using a linker according to structure III for each combination of peptide in Table 1 and cargo in Table 2.

[0144] [Table 1]

[0145] [Table 2]

[0146] Experiment 2 Peptide-cargo conjugates were successfully formed by the methods of the invention using a linker according to structure III for each combination of peptide in Table 3 and cargo in Table 4.

[0147] [Table 3]

[0148] [Table 4]

[0149] Both DNA-cargo sequences were ordered from Biomers.net. The DNA-cargo was equipped with a 5'-terminal cysteine ​​group with a linker according to formula (V).

[0150] [ka]

[0151] Prior to use, the cysteine ​​groups of the DNA-cargo sequences were protected with tert-butyl sulfide groups, which form a disulfide with the S of the cysteine ​​group. In a second step, the addition of TCEP results in reduction of the disulfide bond. Thus, in experiment 2, both DNA-cargos were equipped with a 5'-terminal cysteine.

[0152] The term "plurality" refers to two or more than two. Moreover, the terms "plurality" and "multiple" may be used interchangeably.

[0153] The terms "substantially" or "essentially" and similar terms herein will be understood by those skilled in the art. The terms "substantially" or "essentially" may also include the embodiment by "entirely", "completely", "all", etc. Thus, the adjectives substantially or essentially may also be omitted in the embodiment. Where applicable, the terms "substantially" or "essentially" may also relate to 90% or more, including 100%, e.g., 95% or more, particularly 99% or more, and even more particularly 99.5% or more. Moreover, the terms "about" and "approximately" may also relate to 90% or more, including 100%, e.g., 95% or more, particularly 99% or more, and even more particularly 99.5% or more. It should also be understood that for numerical values, the terms "substantially", "essentially", "about" and "approximately" may relate to the value(s) to which they refer in the range of 90% to 110%, for example 95% to 105%, in particular 99% to 101%.

[0154] Additionally, the term "comprise" includes embodiments where the term "comprise" means "consist of."

[0155] The term "and / or" specifically relates to one or more of the items referred to before and after "and / or." For example, the phrase "item 1 and / or item 2" and similar phrases may relate to one or more of item 1 and item 2. In some embodiments, the term "comprising" may refer to "consisting of," while in other embodiments, it may also refer to "containing at least the defined species and any one or more other species."

[0156] Moreover, the terms first, second, third, etc. are used in the specification and claims to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable, under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in sequences other than those described or illustrated herein.

[0157] Apparatus, instruments, or systems of operation may be specifically described herein. As will be apparent to one of skill in the art, the present invention is not limited to methods of operation or apparatus, instruments, or systems of operation.

[0158] The term "further embodiments" and similar terms may refer to embodiments that include features of the embodiments previously discussed, but may also refer to alternative embodiments.

[0159] It should be noted that the above embodiments illustrate, rather than limit, the present invention and that those skilled in the art can design many alternative embodiments without departing from the scope of the appended claims.

[0160] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0161] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout this description and claims, the words "comprise, comprising, include, including, contain, containing, etc., are to be construed in their inclusive sense as opposed to an exclusive or exhaustive sense, i.e., in the sense of "including, but not limited to."

[0162] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0163] The invention can be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of such measures cannot be used to advantage.

[0164] The invention also provides a control system that can control a device, apparatus, or system or can carry out a method or process as described herein. Still further, the invention also provides a computer program product that, when executed on a computer operatively linked to or contained within a device, apparatus, or system, controls one or more controllable elements of such a device, apparatus, or system.

[0165] The present invention further applies to an apparatus, device or system comprising one or more of the characterized features described herein and / or shown in the accompanying drawings. The present invention further relates to a method or process comprising one or more of the characterized features described herein and / or shown in the accompanying drawings. Moreover, when a method or an embodiment of this method is described as being performed in an apparatus, device or system, it is understood that the apparatus, device or system is suitable for or configured to perform the method or embodiment of this method, respectively.

[0166] The various aspects discussed in this patent may be combined to provide additional advantages. Moreover, those skilled in the art will appreciate that the embodiments may be combined, and that two or more embodiments may be combined. Moreover, some of the features may form the basis of one or more divisional applications.

Claims

1. A method for providing a cargo to a C-terminus (111) of a peptide (110), comprising a first step and a second step, the first step comprising reacting the C-terminus (111) of the peptide (110) with a first reactant (120) in the presence of a first catalyst (130) and a first radiation (140) to provide a first intermediate (150), the first catalyst (130) being configured to decarboxylate the C-terminus (111) of the peptide (110) in the presence of the first radiation (140), the first reactant (120) having a first chemical structure according to formula I, 【Chemistry 1】 wherein R and R' are each independently selected from the group consisting of H and alkyl groups; R″ is an electron-withdrawing group comprising a functional group selected from the group consisting of ester, thioester, amide, ketone, nitro, sulfoxide, sulfone, phosphate ester, acylhydrazide, cyano group, and trihalogenmethyl group; n1 and n2 are each independently selected from the range of 1 to 2; the second stage comprises exposing the first intermediate (150) to a second reactant (220), the second reactant (220) having a second chemical structure according to formula II: 【Chemistry 2】 In the formula, X is O or NH, and R １ comprises a cargo.

2. 2. The method of claim 1, wherein n1=1 and n2=1.

3. The method according to claim 1 or 2, wherein R and R' are selected from the group consisting of H and alkyl groups containing 1 to 6 C atoms.

4. The method of claim 3, wherein one or more of R and R' is H.

5. R″ is an ester, a cyano group, or —CF ３ The method of any one of claims 1 to 4, comprising a functional group selected from the group consisting of:

6. The method of any of claims 2 to 5, wherein the first reactant (120) has a first chemical structure according to structure III. 【Chemistry 3】

7. The method of any of claims 1 to 6, wherein the first catalyst (130) comprises riboflavin tetrabutyrate.

8. The method according to any of the preceding claims, wherein the first radiation (140) is selected from the range of 375 to 525 nm.

9. 9. The method according to any of the preceding claims, wherein the first step is carried out in a first mixture comprising a first solvent (101), said first solvent (101) comprising one or more of DMF, DMSO and water.

10. 10. The method of claim 9, wherein the first mixture comprises no more than 1 ppm dissolved oxygen.

11. The method of any of claims 1 to 10, further comprising an intermediate stage, said intermediate stage comprising separating the first intermediate (150) from the first reactant (120).

12. The method according to any of claims 1 to 11, wherein the second step is carried out in a degassed buffer solution under an inert gas.

13. 13. The method of any of claims 1 to 12, wherein the peptide (110) comprises a C-terminal residue selected from the group comprising alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine and valine.

14. The method of any one of claims 1 to 13, wherein the cargo comprises a second peptide, said second peptide having an N-terminal cysteine ​​residue.

15. The method of any one of claims 1 to 14, wherein the cargo comprises an antibody.

16. A peptide-cargo conjugate (250) obtainable using the method according to any one of claims 1 to 15, having a chemical structure according to formula IV, 【Chemistry 4】 wherein R and R′ are each independently selected from the group consisting of H and alkyl groups; R ３ is a peptide, R″ is an electron withdrawing group comprising a functional group selected from the group consisting of ester, thioester, amide, ketone, nitro, sulfoxide, sulfone, phosphate ester, acylhydrazide, cyano group, and trihalogenmethyl group, n1 is 1, X is O or NH, R １ wherein the peptide-cargo conjugate (250) comprises a cargo.

17. A peptide-cargo conjugate (250) according to claim 16 for use as an antibody drug conjugate.

18. A peptide array comprising the peptide-cargo conjugate (250) of claim 16.

19. 17. Use of a peptide-cargo conjugate (250) according to claim 16 for peptide sequencing.

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