Peptide coupling using thiocycloheptyne derivatives

Abstract

The invention relates to novel compounds of the formula (I), optionally coupled to a peptidic or proteinaceous molecule, and their synthesis. The invention also relates to the use of the novel compounds in coupling reactions with linkers and other molecules. The invention further relates to the use of the novel compounds in bioorthogonal strain promoted cyclo addition (copper-free) that is click reactions.

Description

[0001] P129506PC00 Title: Peptide coupling using thiocycloheptyne derivatives Field of the invention The invention relates to peptidic and proteinaceous molecules. In particular, the invention relates to compounds and methods for coupling of peptidic and proteinaceous molecules to other molecules, such as drugs, drug delivery systems, targeting and imaging ligands, in particular using copper-free click chemistry. Background of the invention Chemical methods for site-specific functionalization of peptides and proteins are useful in a variety of synthesis, research and biomedical applications. Examples include functionalization of peptides and proteins with affinity tags to facilitate isolation, purification, and characterization, with imaging labels to enable detection, with several molecules such as polymers, ligands, small molecules, etc. to create fusion-proteins with the aim to modulate the biological activity, pharmacokinetic properties or targeting properties of either of the two. Several methods for the functionalization of peptides and proteins have been developed, that allow site-specific coupling while maintaining the structural integrity of the protein or peptide. Such methods include modification at cysteine residues achieved by reaction of the thiol group with electrophiles, introduction of non-natural amino acids with a specific reactivity in the protein, enzymatic coupling and modification of the N-terminus of proteins and peptides, many of the latter requiring specific N-terminal amino acids. MacDonald et al. (2015) describes site-specific modification of protein N- termini using 2-pyridinecarboxyaldehydes (2PCA).2PCA-biotin or 2PCA-PEG reagents are attached to multiple protein substrates, with the resulting biotinylated proteins being able to be captured by streptavidin resin, while the PEGylation of therapeutic proteins serves to improve in vivo characteristics. Li et al. (2018) use 2PCA with an azide-functionality (6-(azidomethyl)-2- pyridinecarboxyaldehyde; 6-AM-2PCA) for site-specific modification of antibodies. The azide-functionality of 6-AM-2PCA is click chemistry coupled to dibenzylcyclo- octyne (DBCO). These methods, however, have the disadvantage that the range of coupling partners for the protein is limited or require cumbersome additional steps. For instance, the method of Li et al. requires the presence of an alkyne moiety in the coupling partner, DBCO in Li et al. However, incorporation of such alkyne moiety site specifically may be complex and is not compatible or difficult with many coupling partners of interest. An example are peptide because alkyne-containing click chemistry partners DBCO en BCN are inherently labile due to the amide and cyclopropane moiety, respectively, under acidic peptide synthesis reaction condition (Chigrinova et al. 2013; Erickson et al.2021; Janson et al. 2020; La-Venia et al. 2021), and as a result of which they cannot be coupled to peptides during solid phase synthesis. Hence, with the method of Li et al. it is not possible to couple two peptides in a selective way. In addition, the yields of the final compound in the click reaction in Li et al. are relatively low (<50% to 79%). Another disadvantage of the method of Li et al. is that DBCO is a hydrophobic and relatively large reagent, which has a detrimental effect on the solubility of peptides / proteins, which may result in aggregation, which in turn will result in a reduced subsequent conjugation efficiency. MacDonald et al. require N-Hydroxysuccinimide (NHS) chemistry to introduce a chelator, fluorescent label or another small molecule such as biotin to the 2PCA molecule prior to N-terminal functionalization. Coupling of these molecules to the 2PCA is not possible after N-terminal functionalization of the peptide / protein with the 2PCA moiety. Further, MacDonald et al. showed a variance in the percentage modification which could be achieved per protein, ranging from 43-95%. Hence, there still is a need to increase the diversity of protein and peptide functionalization, in particular of novel, versatile, specific and tunable functionalization, that is broadly applicable to a wide range of peptides and proteins and coupling partners, does not affect their functionality and is cost- effective due to high functionalization and (almost) quantitative conjugation efficiency and consequently more economical use of materials. Summary of the invention It is an object of the present invention to expand the diversity and selectivity of functionalization of peptidic and proteinaceous molecules with other molecules, including peptides and proteins. It is a further object of the invention to provide compounds that allow such expansion. The compounds and methods of the invention allow virtually any peptidic or proteinaceous molecule to be specifically modified at the N-terminus followed by a strain promoted azide alkyne cycloaddition (SPAAC) reaction for coupling to virtually any molecule including another peptide protein in a site-specific manner, that is non-toxic in biological systems. The invention therefore provides a compound of Formula (I), wherein n and m are independently 0, 1, or 2 with the proviso that n + m is 2; R1-R8are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group wherein the (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group are optionally substituted with one or more substituents independently selected from the group consisting of halogen, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; or R1and R7, R1and R8, R2and R7, R2and R8, R3and R5, R3and R6, R4and R5, R4and R6form a cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group wherein the cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group is optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; and L is a linking group. In a further aspect, the invention provides a compound comprising a compound of formula (I) according to the invention coupled to a peptidic or proteinaceous molecule, in particular to the N-terminus of the peptidic or proteinaceous molecule. In particular, the compound comprising a compound of formula (I) according to the invention is site specifically coupled to the N-terminus of a peptidic or proteinaceous molecule. In a further aspect, the invention provides a compound comprising a compound of formula (I), (II) or (III) according to the invention, wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to a compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, 1,3-dipole or 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound. In a further aspect, the invention provides a use of a compound of formula (I), (II) or (III) according to the invention, in a bio-orthogonal, copper-free, click reaction. Such reaction results in the formation of a bioconjugate. In a further aspect, the invention provides a use of a compound of formula (I), (II) or (III) according to the invention for bioconjugation, in particular for coupling a molecule to a peptidic or proteinaceous molecule, preferably wherein the molecule is selected from the group consisting of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate. In a further aspect, the invention provides a method for coupling a molecule to a peptidic or proteinaceous molecule comprising reacting a compound of formula (I), (II) or (III) according to the invention with said molecule, wherein said molecule comprises a thiol, a 1,3-dipole or a 1,3-(hetero)diene, and wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to the compound comprising a thiol or a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide- alkyne coupling results in the formation of a triazole compound. Detailed description As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The compounds disclosed in this description and in the claims may comprise one or more asymmetric centers, and different diastereomers and / or enantiomers may exist of the compounds. The description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise. In addition, the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer. The compounds may occur in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise. The compounds disclosed in this description and in the claims may further exist as exo and endo stereoisomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual exo and the individual endo stereoisomer of a compound, as well as mixtures thereof. The compounds disclosed in this description and in the claims may exist as cis and trans isomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual cis and the individual trans isomer of a compound, as well as mixtures thereof. As an example, when the structure of a compound is depicted as a cis isomer, it is to be understood that the corresponding trans isomer or mixtures of the cis and trans isomer are not excluded from the invention of the present application. A used herein, “halogen” refers to fluoro, chloro, bromo, or iodo. Preferred halogen atoms are fluoro and chloro. As used herein, the term “a (Cx-y)alkyl” refers to a branched or unbranched alkyl group having x-y carbon atoms. For instance, (C1-6)alkyl means a branched or unbranched alkyl group having 1-6 carbon atoms, for example methyl, ethyl, propyl, isopropyl or butyl. Similarly, the term “(C1-2) alkyl” refers to an alkyl group having 1 or 2 carbon atoms. Preferred alkyl groups are methyl and ethyl. As used herein, the term (Cx-y)alkoxy refers to an alkoxy group having x-y carbon atoms, wherein the alkyl moiety is as defined above. For instance, the term (C2-6)alkoxy means an alkoxy group having 2-6 carbon atoms. Preferred alkoxy groups are methoxy and ethoxy. As used herein, the term “(Cx-y)alkenyl” refers to a branched or unbranched alkenyl group, i.e. having at least one double bond, having x-y carbon atoms. For instance, the term “(C2-6)alkylene” means a saturated alkylene group having 2-6 carbon atoms. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl and pentenyl. Unsubstituted alkenyl groups may also contain a cyclic moiety. As used herein, the term “(Cx-y)alkynyl” refers to a branched or unbranched alkynyl group having x-y carbon atoms, wherein the triple bond may be present at different positions in the group, for example ethynyl, propanyl, 1-butynyl, 2- butynyl. For instance, the term “(C2-6)alkynyl” refers to a branched or unbranched alkynyl group having 2-6 carbon atoms. As used herein, “(Cx-y)cycloalkyl group” refers to a cyclic alkyl group having x-y carbon atoms, which can be a single ring or multiple rings, such as a fused bicyclic ring. For instance, the term “(C3-6)cycloalkyl” refers to a cyclic alkyl group having 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, which are preferred cycloalkyl groups. As used herein, “(Cx-y)heterocycloalkyl group” refers to a cyclic group comprising at least one heteroatom, preferably O, S or N, in the cyclic backbone, i.e. having x-y carbon atoms + heteroatoms. The group can be a single ring or multiple rings, such as a fused bicyclic ring. For instance, the term “(C3-12) heterocycloalkyl group” refers to a cyclic alkyl group having 3-6 carbon atoms, such as pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran, thiomorpholine. As used herein, “(Cx-y)aryl group” refers to cyclic aromatic groups with x-y carbon atoms and may include monocyclic, bicyclic and polycyclic structures. Optionally, the aryl groups may be substituted by one or more substituents further specified. Preferred examples of aryl groups include phenyl, naphthyl, anthracyl. A preferred aryl group is phenyl. As used herein, “(Cx-y)heteroaryl group” refers to cyclic aromatic groups with x-y carbon atoms + heteroatoms and may include monocyclic, bicyclic and polycyclic structures. Optionally, the aryl groups may be substituted by one or more substituents further specified. Preferred examples of heteroaryl groups include furan, pyridine, pyrazine, pyrrole, imidazole, pyrazole, oxazole and thiophene. As used herein, “(Cx-y)alkyl(hetero)aryl group” encompasses (Cx- y)alkylheteroaryl groups and (Cx-y)alkylaryl group having x-y atoms carbon atoms (in the case of (Cx-y)alkylaryl) or x-y carbon atoms + heteroatoms (in the case of (Cx-y)alkylheteroaryl groups in the alkyl and (hetero)aryl groups taken together. As used herein, “(Cx-y)(hetero)arylalkyl” encompasses (Cx-y)heteroarylalkyl groups and (Cx-y)arylalkyl group having x-y atoms carbon atoms (in the case of (Cx-y)arylalkyl) or x-y carbon atoms + heteroatoms (in the case of (Cx- y)heteroarylalkyl groups in the (hetero)aryl and alkyl groups taken together. With respect to substituents, the term “optionally substituted” indicates a group may be unsubstituted or substituted with the indicated number and type of the substituent(s). The term "independently substituted" means that if a group that is substituted with more than one substituent, these substituents may be the same or different from each other. The present inventors have surprisingly found that 2PCA can be introduced in a thiocycloheptyne derivative and used to site-specifically functionalize peptides and proteins at the N-terminus, with the aim to provide a wide range of bioconjugates. It was further found that introduction of 2PCA in a thiocycloheptyne derivative provides a site-specific modification tool that is highly versatile and applicable for efficient coupling of the most diverse range of peptidic compounds and coupling partners reported so far, ranging from small to large molecules, including peptides and proteins and supramolecular assemblies such as nanoparticles. The relatively small and hydrophilic TMTHSI-like-2PCA derivative can achieve a higher percentage of conversion than for example larger 2PCA-DBCO derivatives or 2PCA-BCN (bicyclo[6.1. 0]non-4-yne) derivatives or larger biotin derivatives and subsequent binding of large (strept)avidin moiety as used by MacDonald et al. (2015). Additionally, the synthesis of the alternative click reagent 2PCA-DBCO would lead to a relatively hydrophobic derivative, which has an adverse effect on solubility and hence reactivity of peptides and proteins. In addition, the resulting 2PCA-DBCO reagent might not be sufficiently stable for N- terminal introduction. The alternative click reagent 2PCA-BCN would also be relatively hydrophobic and less reactive as compared to the reagent of the present invention. Furthermore, with N-Hydroxysuccinimide (NHS) chemistry as required by e.g. MacDonald et al., a chelator, fluorescent label or other small molecule needs to be introduced to the 2PCA molecule prior to N-terminal functionalization of the peptide / protein. Introduction of these molecules is not possible after N-terminal 2PCA functionalization of proteins, whereas the presence of a thiocycloheptyne- 2PCA compound of the present invention allows for the clicking of any kind of small or large compound after N-terminal functionalization of peptides / proteins. Another advantage of the thiocycloheptyne compound is that it is resistant to strong acidic conditions, which opens up possibilities for its introduction under peptide synthesis conditions. The compounds and methods of the invention in particular allow for a wider range of applications than known so far because introducing an azide in the molecule or coupling partner is easier than e.g. introducing a strained alkyne. After specific introduction of strained alkyne, very high (>90%) yields are achieved in the subsequent reaction of the functionalized peptidic molecule with an azide-containing molecule, which reaction, additionally, proceeds much faster than known coupling reactions of functionalized proteins and their coupling partners. As demonstrated in the Examples the TMTHSI-like-2PCA derivative of the invention show a good biological and chemical stability. They remain stable in the presence of human serum (see Example 2 and figure 7). In addition, the stability over time under acidic conditions (pH 2) was maintained for at least 56 hours and at pH 7 for prolonged period of time (see Example 3 and figure 8). Since the derivatives of the invention are for use under physiological conditions, the acid sensitivity is only of importance during synthesis, work-up, and formulation steps. Since the conjugation of TMTHSI-like-2PCA to biomolecules works best at a pH between 7-8, TMTHSI-like-2PCA conjugates would only be exposed to an acidic environment during work-up and / or analysis. As the amount of the 2PCA conjugate in solution stays above 95% for about 56 hours, it will remain stable for the duration of work-up and analysis steps. Finally, peptide or protein functionality is not compromised when functionalized in accordance with the present invention. In a first aspect, the invention therefore provides a compound of Formula (I), wherein n and m are independently 0, 1, or 2 with the proviso that n + m is 2; R1-R8are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group wherein the (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group are optionally substituted with one or more substituents independently selected from the group consisting of halogen, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; or R1and R7, R1and R8, R2and R7, R2and R8, R3and R5, R3and R6, R4and R5, R4and R6form a cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group wherein the cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group is optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; and L is a linking group. Thiocycloheptyne derivatives of formula have been described in international patent application WO 2020 / 013696. Herein, this compound is used with a variety of linkers and reactive groups for coupling of two different coupling partners. In formula (I), (II) and (III), m and n are independently 0, 1, or 2 with the proviso that n + m is 2. I.e. m and n are both one, or m is 2 and n is 0 or m is 0 and n is 2. It is preferred that m is 1 and n is 1. Hence, a preferred compound of formula (I) is a compound of formula (IA): A preferred compound of formula (II) is a compound of formula (IIA): A preferred compound of formula (III) is a compound of formula (IIIA): In formula (I), (II) and (III), R1-R8are preferably independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-4)alkyl and (C1- 4)alkoxy, wherein the (C1-4)alkyl and (C1-4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy. More preferably, R1-R8are independently selected from the group consisting of hydrogen, halogen and C1-C4 alkyl, preferably methyl or ethyl. More preferably, R1-R8are independently selected from the group consisting of hydrogen, methyl and ethyl, more preferably hydrogen and methyl. In one particularly preferred embodiment, R1-R4are methyl and R5-R8are hydrogen. It is further preferred that m and n are both 1. Hence, a preferred compound of formula (I) is a compound of formula (1B): A preferred compound of formula (II) is a compound of formula (IIB): A preferred compound of formula (III) is a compound of formula (IIIB): In formula (I), (II) and (III), L is a linking group. As used herein, “linking group” and “linker” are used interchangeably and refer to a chemical group having functionality to connect different parts of a molecule or compound, typically two parts. In the present disclosure, the linking group connects the thiocycloheptyne moiety to the 2-pyridinecarboxaldehyde moiety (2PCA). A wide variety of linkers can be used and the selection of linkers can be based on other criteria, for instance in relation to the final applications of the compounds. Suitable linking groups are well known in the art and can be appropriately selected by a person skilled in the art. In one preferred embodiment, L is a degradable linker. As used herein the term “degradable linker” refers to a linker that over time and / or under specific circumstances such as physiological conditions is cleavable. The linker is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity. Suitable degradable linkers are selected from linkers comprising an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone. Another example of a suitable degradable linker is a linker comprising a valine–citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker. Such linkers are commonly used as enzymatically cleavable linkers. In one preferred embodiment, the linking group L comprise or is selected from linear or branched C1 - C24 alkylene groups, C2 - C24 alkenylene groups, C2 - C24 alkynylene groups, C3 - C24 cycloalkylene groups, C5 - C24 cycloalkenylene groups, C5 - C24 cycloalkynylene groups, C7 -C24 alkyl(hetero)arylene groups, C7 - C24 (hetero)arylalkylene groups, C5 - C24 (hetero)arylalkenylene groups, C9 - C24 (hetero)arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkenylene groups and (hetero)arylalkynylene groups optionally being substituted with one or more substituents independently selected from the group consisting of C1 – C12 alkyl groups, C2 - C12 alkenyl groups, C2 – C12 alkynyl groups, C3 – C12 cycloalkyl groups, C5 - C12 cycloalkenyl groups, C5 – C12 cycloalkynyl groups, C8 – C12 alkoxy groups, C2 – C12 alkenyloxy groups, C2 – C12 alkynyloxy groups, C3 – C12 cycloalkyloxy groups, halogen,, amino and oxo. In another preferred embodiment, L is a linear or branched carbon atom chain with a length of 1 - 50 atoms, whereby said chain length is determined by the number of atoms in the longest linear chain of atoms, and whereby said longest linear chain may comprise one or more heteroatoms and / or one or more saturated or unsaturated cyclic or heterocyclic moieties. Hence, L is preferably minimally a CH2 group. As an example, in the compound , the number of atoms in the longest linear chain of atoms of linking group L is 6. Preferably, the chain has a length of 1 – 40 atoms in the longest linear chain of atoms, more preferably 1 – 30 atoms, more preferably 1 – 25 atoms, more preferably 1 – 20 atoms, more preferably 1 – 15 atoms. Said longest linear chain may comprise one or more heteroatoms such as O, N, S and P. Preferably it may comprise one or more N and / or O atoms, more preferably one or more O and one or more N atoms. In a further preferred embodiment, the linear or branched carbon atom chain with a length of 1 - 50 atoms, preferably 1 – 40, 1 – 30, 1 – 25, 1 – 20 or 1 – 15 atoms, comprises one or more moieties independently selected from the group consisting of -S(O)2-, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl and combinations thereof in the longest linear chain, preferably 1 – 10 of said moieties. In one preferred embodiment the linear or branched carbon atom chain comprises 1, 2 or 3 of said moieties. In a further preferred embodiment, the linear or branched carbon atom chain comprises a (C3-12)heterocycloalkyl, preferably a (C3-8)heterocycloalkyl, more preferably a (C5-6)heterocycloalkyl, most preferably a C6-heterocycloalkyl. Preferred examples of (C3-12)heterocycloalkyl are pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran and thiomorpholine. Most preferred is piperazine. It is further preferred that said chain with a length of 1 - 50 atoms, preferably 1 – 40, 1 – 30, 1 – 25, 1 – 20 or 1 – 15 atoms, is not branched, i.e. the chain is linear, optionally with the exception of the atoms or heteroatoms in moiety or moieties -S(O)2-, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3- 12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl that are not comprised in the longest linear chain. In preferred embodiments L comprises a piperazine or polyethylene glycol (PEG) moiety. In a particularly preferred embodiment L, in particular the linear or branched carbon atom chain, comprises a piperazine, more preferably the moiety . In one particular preferred embodiment, the linking group L has the structure , whereby the -C(O)- is attached to the nitrogen atom of the thiocycloheptyne. In another preferred embodiment, linking group L comprises or is a polyethylene glycol (PEG) moiety, preferably −(O−CH2−CH2)n− wherein n is up to 13, more preferably n is an integer from 2 to 10. In one preferred embodiment, the linking group L comprises a piperazine and a PEG moiety, preferably the moiety moiety, preferably a PEG2 moiety (−(O−CH2−CH2)n− wherein n = 2). In another preferred embodiment, the linking group L has the , whereby the -C(O)- is attached to the nitrogen atom of the thiocycloheptyne. In a preferred embodiment, the compound of formula (I) is selected from: . The invention also provides methods for the preparation of compounds of formula (I), (IA) and (IB). Compounds of formula (I) can be prepared by methods known in the art. Methods for the preparation of a compound of the formula are described in detail in international patent application WO 2020 / 013696, which is incorporated herein by reference. Suitable methods for the preparation of compounds of formula (I), (IA) and (IB) are described in the examples herein. For instance, and in brief, 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7- tetrahydro-1H-1λ6-thiepine 1-oxide (TMTHSI) was derivatised with its succinimidyl carbamate using DSC in ACN to obtain TMTHSI-OSu. The tosylated form of piperazine-2PCA was reacted with TMTHSI-OSu in a ACN:DCM 1:1 mixture using DIPEA as base. The product was purified over silica, triturated from Et2O, filtered, washed with pentane and air-dried. Compounds of formula (I) wherein L is a ethylene glycol spacer can for instance be synthesized in a 7-step reaction as shown in scheme 4 in the examples herein, starting from an ethylene glycol derivative and pyridine bismethylene alcohol. As detailed herein above, compounds of formula (I), (IA) and (IB) can be suitably coupled to peptidic or proteinaceous molecules, preferably a peptide or protein. Also provided is therefore a compound comprising a compound of formula (I), (IA) and (IB) coupled to a peptidic or proteinaceous molecule, preferably a peptide or protein, in particular to the N-terminus of the peptidic or proteinaceous molecule. In particular, the compound comprising a compound of formula (I) according to the invention is site specifically coupled to the N-terminus of a peptidic or proteinaceous molecule. In particular provided herein are compounds of formula (II) or (III): In formula (II) and (III), m, n, R1-R8and L are as defined herein above. In a preferred embodiment, the compounds are compounds of formula (IIA) or (IIIA): In formula’s (II), (IIA), (IIB), (III), (IIIA) and (IIIB), X is a peptidic or proteinaceous molecule. As used herein “peptidic molecule” and “proteinaceous molecule” refer to a molecule comprising at least one amino acid residue, preferably comprising a plurality of amino acids residues. Typically, the term “peptidic molecule” is used for molecules containing relatively short amino acid chains, e.g. up to about 50 amino acids and the term “proteinaceous molecule” is used for molecules containing larger amino acids chains, e.g. from about 50 amino acids. In preferred embodiments, X is a peptide or protein. Before coupling to the compounds of formula (I), (IA) or (IB), the peptidic or proteinaceous molecule comprises at least two amino acid residues bound to each other via a peptide bond, i.e. the peptidic or proteinaceous molecule comprises at least a dipeptide. The N-terminal amino acid of this peptidic or proteinaceous molecule reacts with the 2PCA and forms a cyclic structure, in particular the 5- membered ring in compounds (II), (IIA), (IIB) or the 6-membered ring in compounds (III), (IIIA) and (IIIB). In the compounds of formula (II), (IIA), (IIB), (III), (IIIA) and (IIIB), X is the peptidic or proteinaceous molecule without the N- terminal amino acid that is reacted with 2PCA. Any peptidic or proteinaceous molecule as described herein preferably comprises one or more of a peptide and a protein, and optionally one or more of a non-amino acid moiety and a cleavable moiety. In one embodiment, the peptidic or proteinaceous molecule comprises or is a peptide or protein. Non-limiting examples are therapeutic peptides and proteins, including antibodies, antigens and ligands, targeting and imaging ligands, and any other biologically active peptide or protein. The term “biologically active peptide or protein” refers to a peptide or protein that exerts an activity when administered to a subject. The activity can be any activity, including, but not limited to, a therapeutic or prophylactic activity, a binding activity, a diagnostic activity or a targeting activity. In one preferred embodiment, the peptidic or proteinaceous molecule is a peptide or protein, meaning that it consists entirely of amino acid residues. In another embodiment, the peptidic or proteinaceous molecule comprises one or more non-amino acid moieties. In principle, any molecule can be coupled to at least a dipeptide to form a peptidic or proteinaceous molecule as defined herein. Non-limiting examples or non-amino acid moieties are oligonucleotides, peptidomimetics, targeting or imaging labels, saccharide, and any other biologically active molecule. These non-amino acids are attached to the at least one amino acid via a secondary amine. Hence, in a preferred embodiment, the peptidic or proteinaceous molecule is a peptide, protein or molecule coupled to at least a dipeptide, preferably the molecule is selected from the group consisting of oligonucleotides, peptidomimetics, targeting or imaging labels, saccharide, and any other biologically active molecule. As used herein, at least a dipeptide means a chain of at least two amino acids residues, wherein the residues are bound to each other via a peptide bond. In one embodiment, the peptidic or proteinaceous molecule comprises a cleavable moiety or linker, preferably between the at least dipeptide that is coupled to a compound of formula (I) of the invention and the peptide, protein or other molecule of interest. Such cleavable moiety or linker provides for release of the peptide, protein or other molecule of interest under the appropriate circumstances. The cleavable moiety or linker is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity. Suitable cleavable moieties or linkers are selected from the group consisting of an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone. Another example of a suitable cleavable moiety or linker is a linker comprising a valine–citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker. The peptide or proteinaceous molecule may comprise any number of amino acids and any non-amino acid moiety as the ability of the molecule to be coupled to a compound according to the invention is not limited by the size of peptidic or proteinaceous molecule and the nature or size of the non-amino acid moiety or moieties. For instance the molecule may comprises a peptide of between 2 and 50 amino acids, between 2 and 100 amino acids, between 2 and 500 amino acids or between 2 and 1000 amino acids. The peptidic or proteinaceous molecule may comprise a single, amino acid chain. Alternatively, the peptidic or proteinaceous molecule may comprise two or more amino acid chains that are connected via non- peptide bonds. The at least two amino acid residues bound to each other via a peptide bond in the peptidic or proteinaceous molecule before coupling to the compounds of formula (I), (IA) or (IB) must have a free N-terminus. This free N-terminus is available for coupling to the 2PCA moiety of a compound of the invention of formula (I), (IA) or (IB). The N-terminal amino acid of the peptidic or proteinaceous molecule before coupling to the compounds of formula (I), (IA) or (IB) can be any amino acid, either a naturally occurring amino acid or non-natural amino acid, and either a L-amino acid or a D-amino acid. It is, however, preferred that the amino acid is an alpha amino acid or a beta amino acid. Coupling of peptidic or proteinaceous molecule comprising an N-terminal alpha amino acid to a compound of formula (I), (IA) and (IB) results in a compound of formula (II). Coupling of peptidic or proteinaceous molecule comprising an N-terminal beta amino acid to a compound of formula (I), (IA) and (IB) results in a compound of formula (III). It is further preferred that in any peptidic or proteinaceous molecule that is coupled to a compound of formula (I), (IA) or (IB) of the present invention the second amino acid, which is thus the first amino acid in X, is not proline, more preferably not proline or another amino acid containing a tertiary amine. In formula’s (II), (IIA) and (IIB), R9is an amino acid side chain. In formula’s (III), (IIIA) and (IIIB) one of R9and R10is an amino acid side chain and the other of R9and R10is hydrogen or both of R9and R10are an amino acid side chain. The term “amino acid side chain” is well known in the art and refers to the characterizing substituent of the relevant amino acid. This term refers to the substituent bound to the alpha-carbon of the amino acid. In R9and / or R10the side chain can be any amino acid, either a natural or non-natural a-amino acid. Coupling of a peptidic or proteinaceous molecule to a compound of formula (I), (IA) or (IB) can be achieved by methods well known in the art. A suitable method for such coupling is described in the examples herein. In brief, (I), (1A) or (1B) is dissolved in phosphate buffer pH 7.4, with 10% ACN. The solution with concentration of 10 mM is added to the peptidic or proteinaceous molecule which is also dissolved in phosphate buffer pH 7.4, at 0.1mM concentration. This is reacted overnight at room temperature. The reaction mixture was purified using dialysis against 1 kDa MWCO membrane, PD Sephadex column G10 or G25, depending on size of the peptidic molecule. In all cases using 20 mM phosphate buffer pH 7.4 as eluent. Optionally, excess of the thiocycloheptyne-2PCA reagent can be removed by other known techniques, for instance by incubation with hydroxylamine-containing beads, which capture the aldehyde of the reagent by formation of an oxime. The alkyne group of the compounds of the invention is reactive and can be functionalised using for instance cycloaddition-type reactions, in particular to provide bioconjugates. The term “bioconjugate” refers to the coupling of two molecules, at least one of which is a biological molecule, in particular a peptide or protein. In one embodiment, the alkyne group can be reacted with a compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene. In certain embodiments, the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene is an azide- comprising compound, a nitrone-comprising compound or a nitrile oxide comprising compound. Most preferred is an azide-comprising compound. In preferred embodiments, the azide-comprising compound can be coupled using a copper-free click reaction. The coupling results in a triazole-type structure. Thus, the compounds of the invention are suitable for coupling of a peptide or proteinaceous molecule to another molecule. The compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene can be coupled to a thiocycloheptyne compound of the present invention before or after a peptidic or proteinaceous molecule is coupled thereto. Provided is therefore a compound comprising a compound according to any of the invention, wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to a compound comprising a thiol, a 1,3-dipole or a 1,3- (hetero)diene. Preferably the compound comprising a thiol, 1,3-dipole or 1,3- (hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound. The functionalisation can be with any compound, for instance with a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate, in particular comprising a thiol, 1,3-dipole or 1,3-(hetero)diene, preferably an azide, a nitrone or a nitrile oxide, more preferably an azide. Methods to introduce an azide into a compound that does not contain an azide are well known in the art and a skilled person is well able to select an appropriate reaction. A suitable method for introducing an azide at a primary amine is for instance described in Prescher and Bertozzi, (2005) and Meng et al. (2019). Hence, in a preferred embodiment the compound comprising a thiol, a 1,3- dipole or a 1,3-(hetero)diene comprises a one or more of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate. The molecule may be attached to the compounds of formula (I), (II) or (III) via a cleavable moiety or linker. In one embodiment, therefore, the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises a cleavable moiety or linker, preferably between the thiol, a 1,3-dipole or a 1,3-(hetero)diene that is coupled to a compound of formula (I), (II) or (III) of the invention and the peptide, protein or other molecule. Such cleavable moiety or linker provides for release of the molecule under the appropriate circumstances. The cleavable moiety or linker can be any cleavable moiety or linker described herein above. It is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions or by the enzymatic activity. Suitable cleavable moieties or linkers are selected from the group consisting of an ester, orthoester, amide, carbonate, carbamate, anhydride, ketal, acetal and hydrazone. Another example of a suitable cleavable moiety or linker is a linker comprising a valine– citrulline (VCit) dipeptide linker or a glutamic acid–valine–citrulline tripeptide linker. The invention further provides a use of a compound of any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB), in a bioorthogonal, copper-free, click reaction. Copper-free click chemistry is a bioorthogonal reaction, as by eliminating cytotoxic copper catalysts, the reaction is performed without toxicity to biological systems such as cells and tissues. Also provide is a use of a compound of any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) for coupling a molecule to a peptidic or proteinaceous molecule, preferably wherein the molecule is selected from the group consisting of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate. Also provided is a method for coupling a molecule to a peptidic or proteinaceous molecule comprising reacting a compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) with said molecule, wherein said molecule comprises a thiol a 1,3-dipole or a 1,3-(hetero)diene, and wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to the compound comprising a thiol or a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound. In some embodiments, the molecule that is coupled to a compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is a drug delivery vehicle, in particular a nanoparticle. I.e. the compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is coupled to a nanoparticle at the alkyl group of the thiocycloheptyne of this compound. Drug delivery systems are increasingly used in pharmaceutical science. The use of these drug delivery systems such as nanoparticles or liposomes is developing rapidly in the pharmaceutical sciences. Pharmaceutical sciences are using these systems to reduce toxicity and side effects of drugs, improve delivery and adding imaging ligands aids in the monitoring of the effectiveness. Nanoparticles, typically having diameters of < 100nm have been made from a wide variety of materials and anticipated applications in medicine include drug delivery, both in vitro and in vivo diagnostics, nutraceuticals and production of improved biocompatible material. Nanoparticles can be customised for particular purposes ad a wide variety of materials is available. Most pharmaceutically interesting nanoparticles are based on (bio)polymeric materials that can be in a variety of forms. Source materials may be of biological origin like phospholipids, lipids, lactic acid, dextran, chitosan, or have more “chemical” characteristics like various (co)polymers. These nanoparticles are typically functionalised by connecting, coupling, binding (covalently) active ingredients (drugs, ligand, imaging ligands etc.). The functionalisation of many of these nanoparticles can be achieved by a wired variety of chemistries such as copper free click chemistry using the thiocycloheptyne derivatives of the present invention. In certain embodiments, the nanoparticle is a self-assembling polymeric micelle, preferably from thermosensitive block copolymers. Particularly, copolymers based on PEG-b-poly(N- hydroxyalkyl methacrylamide-oligolactates) with partially methacrylated oligolactate units are preferred, but also other (meth)acrylamide esters can be used to construct the thermosensitive block, e.g. esters, and optionally (oligo)lactate esters, of HPMAm (hydroxypropyl methacrylamide) and HEMAm (hydroxyethylmethacrylamide), and N- (meth)acryloyl amino acid esters. Also preferred thermo-sensitive block copolymers are derived from monomers containing functional groups which may be modified by derivatised and underivatised methacrylate groups, such as HPMAm-lactate polymers; that is, this modification encompassing the incorporation of linker moieties. Other types of functional thermosensitive (co)polymers, which can be used, are hydrophobically modified poly(N-hydroxyalkyl) (meth) acrylamides, copolymer compositions of N-isopropylacrylamide (NIPAAm) with monomers containing reactive functional groups (e.g., acidic acrylamides and other moieties such as N- acryloxysuccinimide) or similar copolymers of poly(alkyl) 2-oxazalines, etc.. Further preferred thermo-sensitive groups can be based on NIPAAm and / or alkyl-2- oxaxolines, which monomers may be reacted with monomers containing a reactive functional group such as (meth)acrylamides or (meth)acrylates containing hydroxyl, carboxyl, amine or succinimide groups. Suitable thermo-sensitive polymers are described in US-B-7,425,581 and in EP-A- 1776400, which is incorporated by reference herein. Further, in WO 2010 / 033022 and WO2013 / 002636, which are incorporated by reference herein. In WO2012 / 039602, which is incorporated by reference herein, drug-polymer matrix particles are described using such polymers. Moreover, in WO 2012 / 039602 biodegradable linker molecules are described that may be used in these known polymer matrix particles. Typically, nanoparticles based on thermosensitive block-copolymers as outlined hereinabove can be linked to the compounds of the invention using azide- alkyne copper- free coupling. Examples thereof are described in WO2017 / 086794, which is incorporated by reference herein. Thus, in certain preferred embodiments of the invention, a nanoparticle is prepared wherein a compound of the current invention is coupled to an azide- containing nanoparticle. In certain embodiments, the nanoparticle is a self- assembling polymeric micelle, preferably from thermosensitive block copolymers. In some embodiments, the molecule that is coupled to a compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is an antibody. I.e. the compound any of formula’s (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA) or (IIIB) of the invention is coupled to an azide-containing antibody at the alkyl group of the thiocycloheptyne of this compound. Antibody-drug conjugates (ADCs) are increasingly developed in the pharmaceutical sciences. Antibody−drug conjugates combine the specificity and targeting potential of monoclonal antibodies (mAbs) with e.g. the potency of cytotoxic molecules. Such ADCs are for instance developed for the treatment of cancer, where they aim to target and kill tumor cells while sparing non-tumor cells. An example of an antibody-drug conjugate in accordance with the invention is therefore a bioconjugate of a cytotoxic peptide that is functionalized with a thiocycloheptyne-2PCA compound of the invention. Any cytotoxic peptide can be functionalized with a thiocycloheptyne-2PCA compound of the invention and subsequently coupled to an antibody to form an ADC. Features may be described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments. The invention will be explained in more detail in the following, non-limiting examples. Brief description of the drawings Figure 1: HPLC-trace of TMTHSI-2PCA reaction with octreotide. Figure 2: HPLC trace of the TMTHSI-2PCA reaction with LTX. Figure 3: TP10-2PCA-glycol-TMTHSI (right peak) and TP10-2PCA-glycol- TMTHSI clicked with azidoethanol (left peak indicated with arrow) resulting in a peak shift. Figure 4: Reaction of Evasin-3 with 16 (top) and chromatogram of reaction mixture after 26 hours (bottom) Figure 5: HPLC trace of Evasin-3-TMTHSI 20. Figure 6: Reaction of Evasin-3 TMTHSI 20 with CDP-azide 21 (top) and trace of purified product with mass inserted from deconvoluted MS spectrum of peak (bottom). Figure 7: Decrease of the radiochemical purity of B2 over time. Dashed, grey line: control experiment in a His / Suc buffer; Solid, black line: experiment in a 1:1 solution of human serum : His / Suc buffer at 4 °C. N=2. Figure 8: Graph representing the decrease in % C1 over time. Dashed, grey line shows the stability of C1 at pH 7; the solid, black line shows the stability of C1 at pH 2. N=2. Examples Example 1 We investigated whether it is possible to combine the 2- pyridinecarboxaldehyde (2PCA) reagent with our recently published TMTHSI-click handle (Weterings et al. 2020). The first click construct (3), that we evaluated was obtained in 50% yield by combining 2PCA (2) and TMTHSI via a piperazine linker in a reaction with our earlier described TMTHSI-hydroxy succinimide derivative 1 (Scheme 1). Scheme 1. Synthesis of TMTHSI-2PCA 3 Octreotide 4, a clinically used peptide for the treatment of acromegaly, was used for studying this new TMTHSI-2PCA reagent 3. Since octreotide is a cyclized peptide, containing several functionalities including a lysine side chain NH2 and a disulfide bridge, it is a valid model for evaluation of this potential N-specific reagent 3. After reaction with 20 eq. of TMTHSI-2PCA 3, a >90% conversion was achieved (Scheme 2) based on the decrease of octreotide peak and it was selectively functionalized on the N-terminus as was proven by LCMS (Figure 1). Not only was the expected mass of the modified octreotide observed, but also proof of the occurrence of cyclization, resulting from the N-terminal specific Edman- degradation-like cyclization reaction (Rutjes, 2015) was apparent from the presence of two diastereomers, having the same mass and which could be separated by LCMS.

[0002] Scheme 2. Reaction of TMTHSI-2PCA 3 with octreotide 4. Next, we exposed the oncolytic LTX-315 peptide (Sveinbjørnsson et al. 2017; Zhou et al. 2016) 6 containing several (five) lysine residues to TMTHSI-2PCA 3 (Scheme 3). Here too, a LTX peptide adduct 7 was formed having only one TMTHSI-linker moiety (Figure 2). As expected, no (stable) Schiff base adducts were formed with the side chains of the lysine residues. As above, the N-terminal modification was also apparent from the formation of a diastereomeric mixture (Figure 2).

[0003] Scheme 3. Reaction of TMTHSI-2PCA 3 with LTX 6 containing several lysine residues. Thus, the TMTHSI-2PCA 3 reagent is a suitable reagent for N-specific introduction of a TMTHSI-containing linker into a multifunctional peptide. For larger peptides and proteins we envisioned a TMTHSI derivative with a larger distance between the aldehyde reacting with the N-terminus and the TMTHSI-moiety, that is 16 (Scheme 4). The larger distance formed by an ethylene glycol spacer may be also favourable for water solubility of the adduct, in addition to the creation of a suitable distance between the strain promoted azide alkyne cycloaddition reaction and the site of attachment of the reagent in a sizable peptide or protein. The TMTHSI-glycol-2PCA derivative 16 was obtained in a straightforward 7-step synthesis starting from ethylene glycol derivative 8 and pyridine bismethylene alcohol 11 (Scheme 4). Scheme 4. Synthesis of TMTHSI-glycol-2PCA 16. This TMTHSI-glycol-2PCA derivative 16 was used in the N-terminal modification of the peptide Angiopep217 (Scheme 5). Angiopep2 is applied as a conjugate for intracellular delivery of oligonucleotides and blood-brain barrier transport (Lei et al. 2022). Excess of TMTHSI-glycol-2PCA can be removed by incubation, after completion of the reaction, with hydroxylamine containing beads (Scheme 5). These beads capture the aldehyde of TMTHSI-glycol-2PCA by formation of an oxime, which is illustrated in the work-up of the reaction of peptide TP10 (sequence) with hydroxyl amine beads. Scheme 5. Capture of excess of TMTHSI-glycol-2PCA 16 using hydroxyl amine beads. At this point we wanted to establish that the TMTHSI-moiety was still intact and reactive. We did this by addition of azido ethanol to the worked-up reaction mixture of the TP10 peptide (AGYLLGKINLKALAALAKKIL, membrane- transporting peptide (Soomets et al.2000; Zhang et al.2019; Islam et al.2014) N- terminal modification, Scheme 6. This resulted in a complete shift of the diastereomeric peptides to a more polar region of the HPLC-trace (Figure 3). Scheme 6. Click reaction of the TMTHSI-glycol-2PCA peptide construct with azido ethanol Finally, we wanted to show that TMTHSI-glycol-2PCA was capable of specifically reacting with the N-terminus of a larger protein. Evasin-3 was selected because N-terminal modification with this linker will enable attachment for modified vaccination purposes.(Figure 4, top). 12 equivalences of 16 where reacted with Evasin-3 for 26 hours. While no full conversion of Evasin-319 was seen in these conditions (Figure 4, bottom), separation on HPLC was possible and therefore we could isolate Evasin-3-TMTHSI 20 by collecting the HPLC fraction. This resulted in a pure product (Figure 5) with a final yield of 40%. Considering that 12 equivalences of 16 were used and purification by preparative HPLC was performed, a yield of 40% of pure product is very satisfactory, and is anticipated to be significantly higher upon further process development For vaccine application, Evasin-3-TMTHSI 20 was clicked with azide- functionalised CDP 21 (Figure 6). Two equivalents of Evasin-3-TMTHSI 20 were added dropwise to 1 equivalent of CDP-azide 21. After reacting for 3.5 hours, the reaction mixture was purified using HPLC and the product peak was collected. This resulted in a pure Evasin-CDP conjugate 22, as was confirmed by MS. Overall yield of the conjugate was 60%. Example 2. Biological stability of TMTHSI-2PCA conjugates with BSA in human serum The aim of this Example was to determine the stability of TMTHSI-2PCA BSA conjugates under physiological conditions. - Bovine serum albumin (BSA) was used as model protein, conjugation will occur with the 2PCA moiety - In order to track the stability, DFO-N3 was attached to the TMTHSI moiety via the alkyne functionality making89Zr labelling possible - The stability was determined by incubating the product in serum at 4 °C and by monitoring the decrease in radiochemical purity of BSA-2PCA-DFO-89Zr (B2) B1 synthesis: An Eppendorf tube was loaded with: - 439.5 µl 0.1 M PBS (pH = 7.4) - 10.5 µl TMTHSI-PEG2-2PCA (50 mM) - 50 µl BSA (0.1 g / ml) The reaction mixture was incubated in a thermoshaker at 37 °C for 2.5 days. The reaction mixture was purified using a PD10 cartridge column according to the following method: - Pipette 500 µl of the BSA modification solution onto the column - Pipette 1750 µl 0.1 M PBS onto the column - Pipette 1750 µl PBS onto the column; this fraction is caught The fraction caught from the PD-10 column was further purified using the following 30 kDa spin filter method: - Fill three 30 kDa spin filters with 500 µl, and one 30 kDa spin filter with 250 µl, and centrifuge the solution at 10000 rcf for 3 minutes - Wash the spin filter with 200 µl 1.0 M PBS, and centrifuge at 10000 rcf for 3 minutes - Repeat the washing step a total of 3 times - Reverse the spin filter into a new Eppendorf vial, and centrifuge at 10000 rcf for 3 minutes - Top the caught solution to 500 µl using PBS. 89Zr pre-labelling An Eppendorf was loaded with 20 MBq89Zr (volume depends on production date). The89Zr was topped to 20 µl with oxalic acid (1.0 M, volume depends on volume 89Zr). To this solution was added Na2CO3 (2.0 M, 9 µl) and was further topped with 1.0 M HEPES buffer (131 µl). A solution of DFO-N3 (0.5 mM, 0.1 eq, volume depends on final concentration modified BSA) was added to the89Zr solution, and the resulting solution was stirred in a thermoshaker at 20 °C for 15 minutes. B2 synthesis: To the89Zr pre-labelling solution was added 350 µl of the modified BSA solution. The resulting solution was incubated at 20 °C in a thermoshaker for 1 hour. The89Zr labelled modified BSA was rebuffered to a 240 mM Histidine / sucrose (His / Suc) buffer using a PD-10 column according to the following method: - Pipette the 500 µl labelling solution onto the column - Pipette 1750 µl 240 mM His / Suc buffer onto the column - Pipette 1500 µl 240 mM His / Suc buffer onto the column; this fraction is caught Stability of B2 over time: The caught fraction of B2 was filtered into a sterile bottle, along with 1.5 mL of human serum. B2 in a His / Suc buffer was used as control. A 100 µl aliquot was taken immediately after filtration for the t=0 hours measurement. The material was incubated at 4 °C. Subsequent 100 µl aliquots were taken at t=1, 24, 48, 72, 144, and 168 hours. The sample for spin filter analysis was prepared by taking 3 µl of the aliquot and diluting it by adding 6 µl 240 mM His / Suc buffer. Spin filter analysis was performed in duplo, according to the following methodology per sample: - 3 µl of the diluted spin filter aliquot is pipetted onto a 30 kDa spin filter, together with 100 µl 240 mM His / Suc buffer with 5% DMSO - The spin filter was centrifuged at 14000 rcf for 7 minutes - The washing and centrifuge steps are performed a total of 3 times - After the last centrifuging step, the spin filter and Eppendorf vial are separated into different tubes, and the activity is measured with a Hidex automatic gamma-counter, type 425-601. The counting window is between 400-1100 keV, with the counting time set to one minute - The full gamma-counter procedure lasts ~15 minutes and is performed at room temperature The radiochemical purity of B2 is determined by looking at the radiochemical purity of the tube containing the spin filter. Monitoring the stability of B2 according to the spin filter method described above yielded the radiochemical purity results shown in Figure 7. Although the control experiment (dashed, grey line) was not monitored as long as the experiment in human serum (solid, black line), the decrease in radiochemical purity follow similar trends in both experiments. Additionally, considering the standard deviation, the difference in radiochemical purity over time between the control and serum experiments does not appear to be significant. The results indicate that the stability of B2 is not affected upon being exposed to human serum. The observed decrease in radiochemical purity can be caused by the instability of89Zr in DFO, or by other degeneration caused by the radiation from89Zr. Example 3. Chemical stability of TMTHSI-2PCA with model peptides under rigorous acidic conditions The aim of this Example was to evaluate the stability of TMTHSI-2PCA conjugates at acidic pH values. - Tuftsin was used as a model peptide - Since 2PCA absorbs well at 267 nm, there was no need to use a chromophore to label the tuftsin-CliCr construct (C1) via the alkyne - The stability was determined by monitoring the decrease in C1 over time (vs. Control experiment) as well as generation of TMTHSI-PEG-2PCA Aqueous solutions with pH values of 2 and 7 were prepared using oxalic acid (0.86 mM and 1.4 µM respectively). C1 (2 mg, 1.89 µl) was dissolved in 200 µl of an oxalic acid solution. From this stock solution, 2 aliquots of 60 µl were made, resulting in 0.57 µmol C1 per aliquot. LC-MS of each aliquot was measured after 1, 5, 24, 32, 56, and 144, and 168 hours. The degradation of C1 was determined by integrating the peaks corresponding to C1 and TMTHSI-PEG-2PCA at 267 nm, and taking the average value of each measurement. Monitoring the stability of C1 over time under acidic conditions yielded the results depicted in Figure 8. After a week, 85.88% (0.49 µmol) and 80.73% (0.46 µmol) of C1 remains intact at pH 7 and 2, respectively. Figure 8 also shows that the amount of C1 decreases slightly faster at a pH value of 2 (solid, black line) compared to pH 7 (dashed, grey line). However, since TMTHSI-2PCA will be used under physiological conditions, the acid sensitivity is only of importance during synthesis, work-up, and formulation steps. Since the conjugation of TMTHSI-2PCA to biomolecules works best at a pH between 7-8, TMTHSI-2PCA conjugates would only be exposed to an acidic environment during work-up and / or analysis (Timmers et al. 2023). As Figure 8 shows, the amount of C1 in solution stays above 95% for about 56 hours, meaning that the 2PCA conjugate will remain stable for the duration of work-up and analysis steps. References Chigrinova, M.; McKay, C. S.; Beaulieu, L.-P. B.; Udachin, K. A.; Beauchemin, A. M.; Pezacki, J. P. Rearrangements and addition reactions of biarylazacyclooctynones and the implications to copper-free click chemistry. Organic & Biomolecular Chemistry 2013, 11 (21), 3436-3441 Erickson, P. W.; Fulcher, J. M.; Spaltenstein, P.; Kay, M. S. Traceless Click- assisted native chemical ligation enabled by protecting dibenzocyclooctyne from acid-mediated rearrangement with copper (I). Bioconjugate chemistry 2021, 32 (10), 2233-2244 Islam, M. Z.; Ariyama, H.; Alam, J. M.; Yamazaki, M. Entry of Cell- Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating across Lipid Membrane and Its Induced Pores. Biochemistry 2014, 53 (2), 386–396. Janson, N.; Krüger, T.; Karsten, L.; Boschanski, M.; Dierks, T.; Müller, K. M.; Sewald, N. Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs. ChemBioChem 2020, 21 (24), 3580-3593 La–Venia, A.; Dzijak, R.; Rampmaier, R.; Vrabel, M. An Optimized Protocol for the Synthesis of Peptides Containing trans‐Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes. Chemistry–A European Journal 2021, 27 (54), 13632-13641 Lei, Y.; Chen, S.; Zeng, X.; Meng, Y.; Chang, C.; Zheng, G. Angiopep-2 and Cyclic RGD Dual-Targeting Ligand Modified Micelles across the Blood–Brain Barrier for Improved Anti-Tumor Activity. J. Appl. Polym. Sci. 2022, 139 (24), 52358. Meng G, Guo T, Ma T, Zhang J, Shen Y, Sharpless KB, Dong J. Nature. 2019; 574(7776):86-89. doi: 10.1038 / s41586-019-1589-1. Prescher, J. A. and Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13– 21. Rutjes, F. P. J. T. How to Pick a Single Amine? Nat. Chem. Biol. 2015115 2015, 11 (5), 306–307. Soomets, U.; Lindgren, M.; Gallet, X.; Hällbrink, M.; Elmquist, A.; Balaspiri, L.; Zorko, M.; Pooga, M.; Brasseur, R.; Langel, Ü. Deletion Analogues of Transportan. Biochim. Biophys. Acta 2000, 1467 (1), 165–176. Sveinbjørnsson, B.; Camilio, K. A.; Haug, B. E.; Rekdal, Ø. LTX-315: A First- in-Class Oncolytic Peptide That Reprograms the Tumor Microenvironment. Future Med. Chem. 2017, 9 (12), 1339–1344. M. Timmers, W. Peeters, N.J. Hauwer, C.J.F. Rijcken, T. Vermonden, I. Dijkgraaf, R.M.J. Liskamp, Specific N-Terminal Attachment of TMTHSI Linkers to Native Peptides and Proteins for Strain-Promoted Azide Alkyne Cycloaddition, Chem. Commun., 2023, 59, 11397-11400, DOI: https: / / doi.org / 10.1039 / D3CC03397J Weterings, J.; Rijcken, C. J. F.; Veldhuis, H.; Meulemans, T.; Hadavi, D.; Timmers, M.; Honing, M.; Ippel, H.; Liskamp, R. M. J. TMTHSI, a Superior 7- Membered Ring Alkyne Containing Reagent for Strain-Promoted Azide–Alkyne Cycloaddition Reactions. Chem. Sci.2020, 11 (33), 9011–9016. Zhang, C.; Ren, W.; Liu, Q.; Tan, Z.; Li, J.; Tong, C. Transportan-Derived Cell-Penetrating Peptide Delivers SiRNA to Inhibit Replication of Influenza Virus in Vivo. Drug Des. Devel. Ther.2019, 13, 1059–1068. Zhou, H.; Forveille, S.; Sauvat, A.; Yamazaki, T.; Senovilla, L.; Ma, Y.; Liu, P.; Yang, H.; Bezu, L.; Müller, K.; et al. The Oncolytic Peptide LTX-315 Triggers Immunogenic Cell Death. Cell Death Dis. 2016732016, 7 (3), e2134–e2134.

Claims

Claims 1. Compound of Formula (I),wherein n and m are independently 0, 1, or 2 with the proviso that n + m is 2; R1-R8are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group wherein the (C1-6)alkyl, (C1-6)alkoxy, (C2-6)alkenyl, (C2-6)alkynyl, (C3- 12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6- 12)heteroaryl group, (C7-12)alkyl(hetero)aryl group and (C7-12)(hetero)arylalkyl group are optionally substituted with one or more substituents independently selected from the group consisting of halogen, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; or R1and R7, R1and R8, R2and R7, R2and R8, R3and R5, R3and R6, R4and R5, R4and R6form a cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group wherein the cycloalkyl, cyclo(hetero)aryl, cycloalkyl(hetero)aryl or cyclo(hetero)arylalkyl group is optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo, amino, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy; and L is a linking group.

2. Compound according to claim 1 wherein m is 1 and n is 1.

3. Compound according to any one of the preceding claim wherein R1-R8are independently selected from the group consisting of hydrogen, halogen, hydroxyl, oxo, (C1-4)alkyl and (C1-4)alkoxy, wherein the (C1-4)alkyl and (C1-4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxyl, (C1-4)alkyl, (C1-4)alkoxy, preferably wherein R1-R4are methyl and R5-R8are hydrogen.

4. Compound according to any one of the preceding claims wherein L is a linear or branched carbon atom chain with a length of 1 - 50 atoms, whereby said chain length is determined by the number of atoms in the longest linear chain of atoms, and whereby said longest linear chain may comprise one or more heteroatoms and / or one or more saturated or unsaturated cyclic or heterocyclic moieties.

5. Compound according to claim 4 wherein the linear or branched carbon atom chain comprises one or more moieties independently selected from the group consisting of -S(O)2-, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl and combinations thereof in the longest linear chain.

6. Compound according to any of the preceding claims, wherein the moiety.

7. Compound according to any one of the preceding claims of formula (IA):wherein L is as defined in any one of claims 1-6.

8. A compound comprising a compound according to any one of claims 1-7 coupled to the N-terminus of a peptidic or proteinaceous molecule.

9. Compound according to claim 8 of formula (II) or (III):wherein m, n, R1-R8and L are as defined in any one of claims 1-7; in formula (II) R9is an amino acid side chain; in formula (III), one of R9and R10is an amino acid side chain and the other of R9and R10is hydrogen or both of R9and R10are an amino acid side chain; andX is a peptidic or proteinaceous molecule.

10. Compound according to claim 9 of formula (IIB) or formula (IIIB):wherein L is as defined in any one of claims 1-7 and X and R9are as defined in claim 9.

11. A compound comprising a compound according to any of the claims 1-10, wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to a compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably the compound comprising a thiol, 1,3-dipole or 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound.

12. Compound according to claim 10, wherein the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises one or more of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate.

13. Use of a compound as defined in any one of claims 1-10, in a bio- orthogonal, copper-free, click reaction.

14. Use of a compound according to any one of claim 1-10 for bioconjugation, in particular for coupling a molecule to a peptidic or proteinaceous molecule, preferably wherein the molecule is selected from the group consisting of a drug, small molecule, an antibody, a protein, a peptide, a nucleic acid molecule, including an oligonucleotide, an antisense oligonucleotide and mRNA, a ligand, an imaging label incl. radioactive label, a targeting ligand, a delivery agent, a drug delivery vehicle, such as a nanoparticle, a carrier compound and a solid support, such as a surface plasmon resonance (SPR) plate.

15. A method for coupling a molecule to a peptidic or proteinaceous molecule comprising reacting a compound according to any one of claims 1-10 with said molecule, wherein said molecule comprises a thiol, a 1,3-dipole or a 1,3- (hetero)diene, and wherein the alkyne group of the thiocycloheptyne of Formula (I), (II) or (III) is coupled to the compound comprising a thiol or a 1,3-dipole or a 1,3- (hetero)diene, wherein preferably the compound comprising a thiol, a 1,3-dipole or a 1,3-(hetero)diene comprises an azide, a nitrone or a nitrile oxide, more preferably an azide whereby the azide-alkyne coupling results in the formation of a triazole compound.

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