TECHNICAL FIELD

The present disclosure relates to copper-catalysed cycloaddition reactions. In particular, the use of copper-catalysed cycloaddition reactions in bioconjugation reactions, e.g. bioconjugation reactions involving the use of an ynamine as a substrate.

BACKGROUND

Orthogonal control of chemical reactivity is an essential requirement for the selective modification of biomolecules. The ‘bio-orthogonality’ of these reactions - either produced in nature or synthetically - is contingent on reagent pairs reacting with each other whilst minimizing or avoiding any cross-reactivity with other natural and non-natural of functional groups. One example class of bio-orthogonal reaction available for chemoselective modification of biomolecules is the copper-catalysed azide-alkyne [3+2] cycloaddition (which is often abbreviated to CuAAC) or “click” reaction. This involves the use of a Cu (I) catalyst to promote a [3+2]cycloaddition with an azide to exclusively form a 1 ,4-disubstituted triazole.

A hallmark of CuAAC reactions is the reactive latency of terminal alkyne and azide reagents under physiological conditions, and it is contingent on activation of a terminal alkyne to form the corresponding Cu-acetylide by a Cu(l) catalyst. This sets the CuAAC apart from other classes of metal-free bio-orthogonal reactions, which are typically driven by exothermic second-order reaction kinetics as is the case for strain- promoted azide-alkyne cycloaddition (SPAAC) or inverse electron demand Diels-Alder (iEDDA) reactions. ^{[1] 1 1} As a consequence, the combined attributes of tuneable reaction kinetics, regiospecificity and synthetic tractability of incorporating alkyne and azide reagents into biomolecules has resulted in the CuAAC reaction being used throughout chemical biology, and particularly in the preparation of bioconjugates.

Whilst the CuAAC offers clear advantages over other bio -orthogonal reaction classes for in-cell biomolecule tagging and bioconjugation, one principle drawback is the need for micromolar concentrations of Cu(l) to produce the triazole product under physiologically-relevant conditions (Figure 1 a). ^[2] This becomes problematic as Cu(l) is toxic to cells at these concentrations, and so such catalytic systems typically require the use of a Cu(l)-stabilizing ligand e.g., 20-50 pM) to suppress toxicity. This issue is exacerbated by the prevalence of numerous Lewis basic functional groups in biomolecules, which act as competing Cu-chelating sites. This can often in the accumulation of Cu-mediated oxidative damage and residual Cu in the final product. Therefore, there is a fine balance between the formation of sufficient levels of a catalytically competent Cu(l) species for optimal triazole formation versus the potential to produce deleterious side-reactions arising from copper chelation to biomolecules and oxidative damage. Another reason for the need of high copper loadings in biological media to effect this reaction is that the step of Cu-acetylide formation is rate-determining for conventional terminal alkynes. As a result, the CuAAC reaction is profoundly affected by fluctuations in catalytically active Cu species when non -chelating alkynes and azide reagents are used.

Efforts to mitigate oxidative damage and improve CuAAC reaction kinetics have primarily focused on developing water-soluble Cu(l)-stabilizing ligands, Cu nanoparticles, and Cu-chelating azide groups.

Aromatic ynamines have recently been identified as alkyne surrogates for CuAAC reactions, which undergo chemoselective formation of 1 ,4-triazoles in the presence of other terminal alkynes. ¹³¹ In such reactions, ynamine chemoselectivity provides a shift in the rate determining step (RDS) away from acetylide formation towards the azide ligation step using a Cu(ll) pre-catalyst. ^[4] This results in reducing the Cu dependency of the ynamine activation step, and thereby affording a kinetic advantage relative to regular terminal alkynes in low [Cu] conditions (Figure 1 b).

There remains a need to develop novel cycloaddition reactions that address at least some of these drawbacks and/or that can widen the utility of these reactions in more complex molecules, in particular biomolecules such as peptides and oligonucleotides. In particular, there is a need to develop novel ligation reactions that can be performed in the presence of other bio-orthogonal groups to produce a discrete final product. Additionally, the ability to modulate reactivity by exploiting biomolecules in the molecular environment would provide an additional level of chemoselectivity when compared to the current state of the art set of bio-orthogonal reactions.

SUMMARY

The present disclosure is based on identification of a bioconjugation reaction between a reactant comprising an ynamine moiety and a reactant comprising an azide moiety, wherein at least one of the reactants is a biomolecule. These reactions take place in the presence of a copper catalyst and a biomolecule and surprisingly, it has been found that these reactions can proceed with minimal to low levels degradation of the biomolecule, despite the presence of the copper catalyst. Thus, according to a first aspect, there is provided a method of forming a triazole moiety in a bioconjugation reaction to link a first and a second reactant, wherein the method comprises: contacting a first reactant comprising an ynamine moiety with a second reactant comprising an azide moiety in the presence of a copper catalyst and a reducing agent, wherein at least one of the first and second reactants is a biomolecule.

The first and second reactants may be contacted together under suitable conditions which allow them to react to form the triazole moiety. The reaction may be modulated, controlled, promoted and/or catalysed by a copper catalyst in the presence of reducing agent. In particular, the reaction may be modulated, controlled, promoted and/or catalysed by adding the copper catalyst and reducing agent to the first and second reactants. The reaction may take place in any suitable solvent. In some examples, the reducing agent may be selected from glutathione and sodium ascorbate. In some examples, the biomolecule may be selected from proteins, carbohydrates, lipids, nucleic acids, polypeptides, peptides, amino acids, polysaccharides, oligosaccharides, monosaccharides, polynucleotides, oligonucleotides, nucleotides, and antibodies. In some examples, the biomolecule may be selected from a peptide and an oligonucleotide.

Furthermore, the present disclosure relates to the identification that a reaction between a reactant comprising an ynamine moiety and a reactant comprising an azide moiety to provide a triazole-containing product can be modulated, controlled and/or promoted by the use of glutathione (GSH). In particular, it has been observed that the addition of glutathione to a copper-catalysed cycloaddition reaction can modulate, facilitate and/or promote the formation of the triazole product. This same phenomenon has not been observed for conventional alkynes.

Indeed, the conditions described herein have been shown to be highly chemoselective and to proceed in good yields even at low concentrations of copper catalyst. In particular, the reactions and conditions described herein may be compatible with more complex systems, tolerating the presence of many different functional groups. Consequently, this class of reaction may find particular utility in orthogonal reaction systems (e.g. as a bio-orthogonal tool in the sequential ligation of biomolecules such as peptides and oligonucleotides).

Glutathione (GSH) is the principle redox mediator in live cells, minimizing the production of deleterious reactive oxygen species (ROS) within a cellular environment. Typically present in millimolar concentrations (e.g. 0.1 -10 mM), GSH can modulate the oxidation state of Cu ions by chelating Cu(ll) as well as acting as a reducing agent to form GSH-Cu(l) complexes. Complexes can also be formed between Cu ions and glutathione disulfide (GSSG), which is influenced by the ratio of GSH/GSSG present. ^{[5] 6 6} One of the primary functions of GSH within a cell is to mediate the cell’s oxidation state and to deactivate electrophilic agents. This poses a considerable challenge for the application of bio-orthogonal reagents as tools for biomolecule tagging and bioconjugation as many major classes (including SPAAC and iEDDA) are susceptible to side reactions with GSH. However, the present inventors have unexpectedly identified that not only can ynamines show good stability to GSH across physiological concentration ranges, but also that GSH can act as an important mediator of ynamine- azide cycloaddition reactions. This same phenomenon has not been observed using conventional alkyne substrates.

According to a second aspect of the disclosure there is provided a method of promoting and/or catalysing a reaction between a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety to provide a product comprising a triazole moiety. The reaction may be modulated, controlled, promoted and/or catalysed by a copper catalyst in the presence of glutathione. In particular, the reaction may be modulated, controlled, promoted and/or catalysed by adding the copper catalyst and glutathione to the first and second reactants. The reaction may take place in any suitable solvent.

Alternatively, there is provided a method of forming a triazole-containing product, the method comprising reacting a first reactant comprising an ynamine moiety together with a second reactant comprising an azide moiety in the presence of a copper catalyst and glutathione.

In some examples of the various reactions described herein, the step of reacting together the first reactant and second reactant may comprise contacting the first and second reactants in the presence of a copper catalyst and reducing agent (e.g. glutathione). Thus, the ynamine and azide moieties may react together in the presence of the copper catalyst and the reducing agent (e.g. glutathione) to produce the triazole- containing product.

The method may be a click reaction. For example, the reaction may be a click ligation reaction.The method may comprise a cycloaddition reaction.

As used herein, a cycloaddition reaction may refer to a reaction in which two or more unsaturated moieties react together to form a cyclic product. The two or more unsaturated moieties may generally belong to different reactants but can also be present on the same reactant. There may be a net reduction in bond multiplicity following the cycloaddition reaction. In other words, the cyclic product of the cycloaddition reaction may comprise an increased number of sigma (o) bonds and a decreased number of pi (TT) bonds relative to the number of these bonds in the reactants.

As would be understood by the skilled person, a cycloaddition reaction may be pre-fixed with a notation to denote the number of atoms involved in the reaction. For example, a cycloaddition reaction may be denoted as an [x+y] cycloaddition, wherein x is the number of atoms comprised on the first unsaturated moiety and y is the number of atoms comprises on the second unsaturated moiety.

In the present case, the reaction may be a [3+2] cycloaddition reaction. That is to say, three atoms from a first reactant (e.g. the azide moiety) and two atoms from a second reactant (e.g. the ynamine moiety) may be involved in the cycloaddition reaction.

The [3+2] cycloaddition may provide a five-membered ring product, such as a triazole. In particular, as stated above, the reaction may provide a product comprising a triazole moiety. The triazole may sometimes be referred to as a 1 ,2,3-triazole, wherein the three nitrogen atoms are at adjacent positions within the five-membered ring.

The cycloaddition reaction may produce one or more regioisomers. For example, the cycloaddition reaction may produce a product comprising a 1 ,4-substituted triazole or a 1 ,5-substituted triazole moiety (illustrated in Figure 1 ).

The reaction may be regioselective or regiospecific and/or may proceed regioselectively or reg iospecif ical ly . In other words, the reaction may favour the formation of one or more particular regioisomers over one or more other regioisomers. The reaction may favour the formation of a 1 ,4-substituted triazole. For example, the reaction may favour the formation of the 1 ,4-substituted triazole over the 1 ,5-substituted triazole. For instance, the molar amount of the 1 ,4-substituted triazole product obtained following the reaction may be greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, greater than about 99% or greater than about 99.9% of the total molar amount of triazole product obtained.

Thus, according to a further aspect there is provided a method of forming a 1 ,4- substituted triazole-containing product comprising the step of: contacting or reacting together a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety in the presence of a copper catalyst and glutathione.

As stated above, the reaction may proceed chemoselectively. In particular, the present inventors have identified a set of conditions under which a cycloaddition reaction between an ynamine and an azide will proceed, but under which alkyne reagents have shown no, or substantially no, reaction. By way of example, the first reactant comprising an ynamine moiety may preferentially react with the second reactant comprising the azide moiety even when the reaction is conducted in the presence of an alkyne moiety. In other words, under the reaction conditions described in the present disclosure, the azide moiety reacts preferentially with the ynamine moiety over an alkyne moiety. Consequently, the chemoselectivity of this method may be of particular use in sequential bioconjugation reactions.

As described above, the various reactions disclosed herein may be catalysed and/or promoted by a copper catalyst. The catalytically active species in the cycloaddition reactions described herein is typically Cu(l). Therefore, as used herein “copper catalyst” may embrace any copper species that is comprises, consists essentially of, or consists of a Cu(l) salt (such as a copper halide (e.g. copper iodide or copper bromide)). Additionally or alternatively, as used herein, “copper catalyst” may be generated in situ, e.g. by reduction of a Cu(ll) species or by oxidation of Cu(0) to produce the catalytically active species.

In some examples of the disclosure, the copper catalyst is a copper (II) salt. Representative examples include, but are not limited to, copper (II) acetate (Cu(OAc) ₂ ), copper (II) halides, copper (II) oxide, copper (II) carbonate, copper (II) acetylacetonate, copper (II) sulfate, copper (II) carboxylate (e.g. copper benzoate, copper malonate, copper (II) pyrrole carboxylates), copper (II) proline, copper (II) triflate and the like. In particular, the copper catalyst may be copper (II) acetate.

In some examples of the disclosure, the copper catalyst may comprise metallic copper. Representative examples include, but are not limited to, copper nanoparticles and copper tubing (e.g. copper derived from copper tubing used in the process and/or copper tubing used to house the reaction). In some examples, the copper catalyst may not be metallic copper.

In some examples of the reactions disclosed herein, copper ligands may be added, e.g. ligands which may coordinate to Cu ( I) and/or stabilise Cu ( I) . Suitable ligands will be known to those skilled in the art. Representative examples include, but are not limited to, THPTA (tris-hydroxypropyltriazolylmethylamine) and tris(benzyltriazolylmethyl)amine (TBTA).

In some examples, the reaction may take place under batch conditions. In some examples, the reaction may not take place under flow conditions. The reactions as disclosed herein may be modulated, controlled, promoted and/or catalysed by a relatively low concentration of copper catalyst (e.g. a relatively low concentration of copper catalyst in the reaction mixture). In particular, in the presence of a reducing agent (e.g. glutathione), the reaction between the ynamine moiety and the azide moiety may proceed relatively quickly and/or in good yield at relatively low concentrations of copper catalyst. This may be beneficial in certain bioconjugation applications as high levels of copper can lead to oxidative damage of cells. In addition, the present inventors have observed that under the equivalent low concentrations of copper catalyst, no cycloaddition reaction was observed if the ynamine substrate were replaced with a conventional alkyne substrate. Thus, without being bound by theory, the present inventors hypothesise that the relatively higher reactivity of an ynamine moiety under the copper catalysed conditions can facilitate the use of the disclosed methods in bioconjugation reactions, as the lower levels of copper lead to less degradation of the highly functionalized and/or relatively sensitive biomolecules (e.g. oligonucleotides and peptides).

In some examples, the concentration of the copper catalyst may be less than or equal to about 1 mM, less than or equal to about 750 pM, less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM.

In some examples, the concentration of the copper catalyst may be greater than or equal to about 0.1 pM, greater than or equal to about 1 pM, or greater than or equal to about 10 pM.

By way of further example, the concentration of the copper catalyst may be between about 0.1 pM and about 1 mM, between about 1 pM and about 750 pM, or between about 10 pM and about 500 pM. In some examples, the concentration of the copper catalyst may be between about 50 pM and about 250 pM, e.g. about 100 pM.

In some examples, the concentration of the reducing agent (e.g. glutathione or sodium ascorbate) may be less than or equal to about 10 mM, less than or equal to about 5 mM, less than or equal to about 1 mM, less than or equal to about 750 pM, less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM. The concentration of the reducing agent (e.g. glutathione or sodium ascorbate) may be greater than or equal to about 0.1 pM, greater than or equal to about 1 pM, or greater than or equal to about 10 pM.

By way of further example, the concentration of the reducing agent (e.g. glutathione or sodium ascorbate) may be between about 0.1 pM and about 1 mM, between about 1 pM and about 750 pM, or between about 10 pM and about 500 pM. In some examples, the concentration of the reducing agent (e.g. glutathione or sodium ascorbate) may be between about 50 pM and about 250 pM, e.g. about 100 pM.

The molar ratio of the copper catalyst and the reducing agent (e.g. glutathione or sodium ascorbate) may modulate and/or control the cycloaddition reactions described herein. In some examples, the molar ratio of the copper catalyst and the reducing agent (e.g. glutathione or sodium ascorbate) may be between 5:1 and 1 :5, between 3:1 and 1 :3, between 2:1 and 1 :2. In some examples, the molar ratio of the copper catalyst and the reducing agent (e.g. glutathione or sodium ascorbate) may be approximately 1 :1 .

Surprisingly, the present inventors have also identified that the reaction between the ynamine moiety and the azide moiety can proceed at relatively low concentrations of copper catalyst even in the absence of glutathione or other reductant (e.g. sodium ascorbate).

Thus, according to a further aspect of the disclosure, there is provided a method of promoting and/or catalysing a reaction between a first reactant comprising an ynamine moiety and a second reactant comprising an azide moiety to provide a product comprising a triazole moiety, wherein the copper catalyst is present at a concentration of less than or equal to about less than or equal to about 500 pM, less than or equal to about 400 pM, less than or equal to about 350 pM, less than or equal to about 250 pM, less than or equal to about 100 pM, or less than or equal to about 50 pM. In some examples, the copper catalyst may be present at a concentration between about 10 pM and about 500 pM.

Without being bound by theory, the inventors hypothesise that (in contrast with cycloaddition reactions between alkynes and azides), the cycloaddition reaction between the ynamine and azide does not require high concentrations of the Cu(l) species - which is typically produced by excess reductant in the alkyne-azide cycloaddition. As noted above, high concentrations of Cu(l) can be highly damaging to biomolecules as it can lead to oxidative damage.

The various reactions according to the present disclosure may be carried out or conducted in a solvent. The solvent may be any solvent that is compatible with the reactants and products and/or any solvent that allows, facilitates or enables the formation of the triazole-containing product. For instance, the solvent may be any solvent that promotes the formation of the triazole-containing product. In particular, the reaction may be carried out in any solvent that is capable of promoting the cycloaddition reaction. The solvent may be or comprise a polar solvent, a non-polar solvent and/or an aqueous solvent. The solvent may be or comprise a water miscible solvent. Thus, the reaction may proceed in an aqueous solution.

The solvent may comprise a single solvent or mixture of different solvents.

Suitable solvents may include, but are not limited to, an alcohol, water, a fluorinated solvent, acetonitrile (MeCN), dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), or any combination thereof. Representative examples of an alcohol solvent used in the reaction may include, but are not limited to, methanol (MeOH), ethanol (EtOH), propanol (such as iso-propanol (iPrOH)), butanol (such as iso-butanol or tert-butanol), or ethylene glycol. Representative examples of a fluorinated solvent, used in the reaction may include a Ci-C ₆ alcohol in which one or more hydrogen atoms have been replaced with a fluorine atom, such as 2,2,2-Trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), hexafluoro-2-methyl-2-propanol (HFMP), 2- (nonafluorobut-1 -yl)ethan-1 -ol, 2-(perfluorobut-1 -yl)ethan-1 -ol (PFH), 1 ,1 ,1 - trifluoropropan-2-ol (TFP), perfluoro-tert-butanol (PTB), 1 ,6-dihydroxy-2,2,3,3,4,4,5,5- octafluorohexane (DHP), 2,2,3,3,3-pentafluoro-1 -propanol (PFP) and 2,4,6- trifluorophenol.

In some examples, the solvent may be an aqueous buffered solution such as a phosphate buffered saline solution (e.g. Dulbecco’s phosphate -buffered saline (DPBS)).

In some examples, certain co-solvents have been observed to promote and/or facilitate the formation of the triazole-containing product (e.g. when added to water or an aqueous buffered solution). For example, certain co-solvents may increase the rate of reaction and/or conversion to the triazole-containing product. In particular, protic fluorinated solvents (such as TFE or HFIP) were observed to increase the rate of reaction and/or conversion to the triazole-containing product. Without being bound by theory, it is hypothesised that enhanced polarity and hydrogen-bonding character of these solvents may act to facilitate and/or promote the cycloaddition reaction between the ynamine and azide moieties. In other examples, a Ci-C ₆ alcohol (such as methanol) has been found to be an effective co-solvent. In view of the above, the solvent may comprise an aqueous solution (e.g. an aqueous buffered solution) and a co-solvent which is a protic fluorinated solvent (such as trifluoroethanol or hexafluoropropanol).

The aqueous solution may comprise the co-solvent in an amount of between about 1 % and about 50% by volume, between about 2% and about 30% by volume, or between about 5% and about 20% by volume. In some examples, the aqueous solution may comprise about 10% by volume of the co-solvent. By way of further example, the aqueous buffered solution may comprise approximately 10% by volume of a protic fluorinated solvent (such as trifluoroethanol or hexafluoropropanol).

In particular examples, where the reaction is a bioconjugation reaction and the biomolecule is an oligonucleotide, the solvent may be water and the co-solvent may be a Ci-C ₆ alcohol (such as methanol). The co-solvent may be present in an amount between about 1 % and 20% by volume. In these and other examples, and without being bound by theory, the use of an alcohol as a co-solvent may assist in limiting degradation of the oligonucleotide.

In some cases, the reactions of the present disclosure may be carried out at temperatures above 0 °C, above 5 °C, above 10 °C, above 15 °C or above 30°C. In some cases, the reaction may be carried out at a temperature between about 0 °C and about 100 °C. For example, the reaction may be carried out at a temperature between about 5 °C and 50 °C, between about 10 °C and 30 °C, or between about 15 °C and 25 °C. In some cases, the reaction may be carried out at about 20 °C.

In some cases, the reactions of the present disclosure may be carried out under atmospheric pressure. For example, at a pressure in the region of 101.325 kPa or 1 atmosphere.

The first and second reactants may be present in any molar amount relative to one another that allows, facilitates or enables the formation of the triazole-containing product. For example, the first and second reactants may be present in a molar ratio of between 5:1 to 1 :5. For instance, the first and second reactants may be present in a molar ratio of 2:1 , 1 .5:1 , 1 .2:1 , 1 .1 :1 , 1 :1 , 1 :1.1 , 1 :1 .2, 1 :1 .5, 1 :2. In some instances, the first and second reactants may be present in an equimolar amount. Thus, the first and second reactants may be present in a 1 :1 molar ratio.

In some examples of the reactions described herein, the reaction proceeds in the presence of a magnesium salt, such as a magnesium halide salt. Representative examples include, but are not limited to, magnesium fluoride, magnesium chloride, magnesium bromide, and magnesium iodide. Without being bound by theory, in particular examples, where the reaction is a bioconjugation reaction and the biomolecule is an oligonucleotide, the addition of a magnesium salt may be particularly useful as it can displace any copper species bound to the phosphate backbone of the oligonucleotide, ensuring that the catalytic species is available to promote the reaction between the ynamine and the azide.

The magnesium salt may be present in the reaction at a concentration between about 0.1 mM and about 100 mM, between about 1 mM and about 50 mM, or between about 5 mM and about 30 mM, such as about 20 mM.

In some examples and unless otherwise stated, the reactions described herein may proceed in the absence of sodium ascorbate. As used herein, the absence of sodium ascorbate may mean that there is substantially no sodium ascorbate present in the reaction mixture. For instance, the amount of sodium ascorbate may be negligible or trace.

In some cases, the first and second reactants may both be comprised on the same molecule. For example, a molecule may comprise both an ynamine and an azide moiety that are spatially arranged such that they have the ability to react together.

As stated above, the first reactant comprises an ynamine moiety. The ynamine may be an aliphatic or aromatic ynamine. In some examples, the ynamine may be an aromatic ynamine. With the context of the present disclosure, in an aromatic ynamine, the nitrogen of the ynamine group may be substituted with an aromatic group or may form part of an aromatic ring (e.g. the nitrogen atom of the ynamine group may be a ring atom comprised within an aromatic ring).

The first reactant comprising an ynamine moiety may be represented according to formula (I):

R ¹ R ² N — = — R ³ (I) wherein R ¹ and R ² may each be independently selected from H, optionally substituted aryl, optionally substituted heteroaryl and Ci-C ₆ alkyl, on the proviso that at least one of R ¹ and R ² is an optionally substituted aryl or optionally substituted heteroaryl group; or wherein R ¹ and R ² may together form an optionally substituted heteroaryl ring; and wherein R ³ may be H or Ci-Ce alkyl.

In those examples where the first reactant is the biomolecule, the structure shown in formula (I) is covalently bound to the biomolecule by way of a substitution on one of the aryl or heteroaryl rings. In particular, a covalent bond to an atom on the biomolecule may replace a hydrogen bond at any chemically suitable position on the aryl or heteroaryl rings on the structure shown in formula (I) providing valencies are satisfied.

By way of particular example, R ¹ and R ² may together form an optionally substituted heteroaryl ring; and R ³ may be H, or Ci-C ₆ alkyl.

In those instances, where R ¹ and R ² together form a heteroaryl ring, the nitrogen atom of the ynamine is incorporated into the heteroaryl ring. In some cases, R ¹ and R ² may together form a five- to fourteen-membered heteroaryl ring containing at least N ring heteroatom (i.e. incorporating the nitrogen of the ynamine group as shown in formula (I)) and optionally containing an additional one to three ring heteroatoms each independently selected from N, O and S. The heteroaryl may be optionally substituted with from one to five substituents each independently selected from Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, halo, Ci- C ₆ alkoxy, aryl and heteroaryl, wherein the aryl and heteroaryl substituents are each optionally substituted with one to three substituents each independently selected from Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, halo, and Ci-C ₆ alkoxy.

In some further examples, R ¹ and R ² may together form an imidazole ring or a benzimidazole ring incorporating the nitrogen of the ynamine group. The imidazole or benzimidazole ring may comprise one or more (e.g. one to five or one to three) substituents. For example, the imidazole or benzimidazole ring may comprise one or more Ci-C ₆ alkyl substituents, such as a methyl group, one or more halo substituents, one or more Ci-C ₆ alkoxy substituents, or a combination thereof.

In those cases, where R ³ is H, the first reactant may be referred to as comprising a terminal ynamine moiety.

In some examples, the first reactant comprising an ynamine moiety may be represented according to formula (la): wherein R ⁶ and R ⁷ are each independently selected from the group consisting of H, optionally substituted aryl, and optionally substituted heteroaryl; or wherein R ⁶ and R ⁷ together form an optionally substituted aryl or an optionally substituted heteroaryl ring; and wherein R ⁸ is selected from H and Ci-Ce alkyl. Again, in those examples where the first reactant is the biomolecule, the structure shown in formula (la) is covalently bound to the biomolecule by way of a substitution on one of the aryl or heteroaryl rings. In particular, a covalent bond to an atom on the biomolecule may replace a hydrogen bond at any chemically suitable position on the aryl or heteroaryl rings on the structure shown in formula (la) providing valencies are satisfied.

In particular, in some examples of formula (la), R ⁸ is H.

In some examples, where R ⁶ and R ⁷ are each independently selected from the group consisting of H, optionally substituted aryl and optionally substituted heteroaryl, there may be a proviso that at least one of R ⁶ and R ⁷ is optionally substituted aryl or optionally substituted heteroaryl.

In some examples, R ⁶ and R ⁷ are each independently selected from the group consisting of H, a six- to ten-membered ring aryl, and five- to ten-membered ring heteroaryl containing from one to three heteroatoms each independently selected from N, O and S, wherein the aryl and heteroaryl are optionally substituted with from one to five substituents each independently selected from Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, halo, and Ci-C ₆ alkoxy.

For the avoidance of any doubt, in those examples of formula (la) where R ⁶ and R ⁷ together form an optionally substituted aryl or heteroaryl ring, the two N ring heteroatoms of the imidazolyl core of formula (la) are comprised within the fused ring system. As such, in some examples, where the R ⁶ and R ⁷ groups may together form an aryl ring (i.e. a ring formed of carbon atoms), the overall ring system would be considered heteroaromatic as the rings are fused together. In other cases, where the R ⁶ and R ⁷ groups may together form a heteroaryl ring, this ring formed by the R ⁶ and R ⁷ groups of formula (la) may contain may contain up to three additional heteroatoms (e.g. one or two additional heteroatoms) each independently selected from N, O and S.

In some cases, R ⁶ and R ⁷ may together form a six- to ten-membered aryl ring which is optionally substituted with from one to five substituents each independently selected from Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, halo, and Ci-C ₆ alkoxy. (In such cases, and as is made clear above, such an aryl ring would be fused with the imidazolyl core of formula (la) and so the resulting ring system would be a nine- to fourteen-membered heteroaromatic group).

In some cases, R ⁶ and R ⁷ may together form a five- to ten-membered heteroaryl ring containing from one to three heteroatoms each independently selected from N, O and S, which is optionally substituted with from one to five substituents each independently selected from Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, halo, and Ci-C ₆ alkoxy. (In such cases, and as is made clear above, such a heteroaryl ring would be fused with the imidazolyl core of formula (la) and so the resulting ring system would be an eight- to fourteen-membered heteroaromatic group).

In some examples the first reactant comprising the ynamine moiety comprises a structure according to formula (Ic): wherein p is 0, 1 , 2 or 3; and

R ¹³ is selected from Ci-C ₆ alkyl, halo, Ci-C ₆ haloalkyl, and Ci-C ₆ alkoxy.

In some examples of formula (Ic), when the first reactant is the biomolecule, the structure shown above is covalently bound to the biomolecule by way of a substitution on the heteroaryl ring.

As mentioned above and described in more detail below, the ynamine moiety may sometimes be appended, conjugated or linked (e.g. covalently linked) to a biomolecule or a tagging moiety. In some examples, compounds of formulae (I) and (la) may be covalently bonded to the biomolecule or tagging moiety by way of a substitution to the aryl or heteroaryl ring(s) defined in formulae (I) and (la). When an aryl or heteroaryl group is substituted with a biomolecule or tagging moiety, any hydrogen atom(s) on the aryl or heteroaryl ring may be replaced with the covalent bond to the biomolecule or tagging moiety, providing valencies are satisfied.

Representative examples of the first reactant comprising an ynamine moiety are set out below:

Additional representative examples of the first reactant comprising an ynamine moiety are set out below: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ; X ¹ represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues;

L is a covalent linker; p is 0, 1 , 2 or 3; and R ¹³ is selected from Ci-C ₆ alkyl, halo, Ci-C ₆ haloalkyl, and Ci-C ₆ alkoxy. By way of further example, the first reactant may be selected from one of the following examples. wherein X ¹ represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to

15) nucleotide residues.

It will be appreciated that the first reactants or compounds of the present disclosure may exist in different stereoisomeric forms. The present disclosure includes within its scope the use of all stereoisomeric forms, or the use of a mixture of stereoisomers of the first reactants or compounds. By way of example, where the first reactant or compound comprises one or more chiral centres, the present disclosure encompasses each individual enantiomer of the first reactant or compound as well as mixtures of enantiomers including racemic mixtures of such enantiomers. By way of further example, where the first reactant or compound comprises two or more chiral centres, the present disclosure encompasses each individual diastereomer of the first reactant or compound, as well as mixtures of the various diastereomers.

By way of further example, the first reactant may be represented as: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10).

By way of further example, the first reactant may be represented as:

The second reactant comprising an azide moiety may be any organic molecule comprising an azide group.

In some examples, the second reactant comprising an azide moiety may be represented as formula (II):

R ⁴ — C1-C4 alkyl — N ₃ (II) wherein R ⁴ may be optionally substituted aryl, optionally substituted heteroaryl, or -XR ⁵ ; wherein X may be selected from NR ⁹ , O and S;

R ⁵ may be selected from H, Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, and SO ₂ R ¹⁰ ;

R ⁹ may be selected from H and Ci-C ₆ alkyl; and

R ¹⁰ may be selected from optionally substituted aryl, optionally substituted heteroaryl and Ci-C ₆ alkyl.

In some examples, R ⁴ may be -OH.

In some examples, R ⁴ may be a six- to ten-membered aryl ring optionally substituted with from one to five substituents selected from halo, Ci-C ₆ alkyl, Ci-C ₆ alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR ¹¹ R ¹² , wherein R ¹¹ and R ¹² are each independently selected from H and Ci-C ₆ alkyl.

In some examples, R ⁴ may be a five- to ten-membered heteroaryl ring containing from one to three ring heteroatoms each independently selected from N, O and S, and being optionally substituted with from one to five substituents selected from halo, Ci-C ₆ alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR ¹¹ R ¹² , wherein R ¹¹ and R ¹² are each independently selected from H and Ci-C ₆ alkyl.

In some examples, R ⁴ may be NHSO2R ¹⁰ ’ wherein R ¹⁰ is six- to ten-membered aryl ring which is optionally substituted with from one to five substituents selected from halo, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, hydroxyl, amino and -NR ¹¹ R ¹² , wherein R ¹¹ and R ¹² are each independently selected from H and Ci-C ₆ alkyl.

In some cases, R ⁴ may be -OH, optionally substituted phenyl or optionally substituted pyridyl.

In some cases, R ⁴ — C1-C4 alkyl may be selected from any of the structures a, b, c, d or shown below. a b e d e

In some examples, the second reactant may be selected from the group consisting of benzyl azide, azidoethanol and picolyl azide.

As described in more detail below, the azide moiety may sometimes be appended, conjugated or linked (e.g. covalently linked) to a biomolecule or a tag. In some examples, compounds of formula (II) may be covalently bonded to the biomolecule or tag moiety at any suitable position. For example, any hydrogen atom(s) on the second reactant may be replaced with the covalent bond to the biomolecule or tag moiety, providing valencies are satisfied.

In some examples, the second reactant may be a peptide (e.g. an oligopeptide or a cell penetrating peptide) which has been derivatised with one or more azide groups.

As stated previously, the reactions and/or catalytic systems described herein may show particular utility not only in the synthesis of compounds but also in bioconjugation reactions, such as the preparation of biomolecule-containing compounds and the efficient labelling and/or tagging of biomolecules. In particular, the reactions and/or catalytic systems described herein have been shown to be highly chemoselective and may find use in site-specific and/or sequential modification of organic compounds and biomolecules.

By way of example, the reactions and/or catalytic systems described herein may show particular utility in the preparation of heterobifunctional molecules (such as proteolysis targeting chimeric (PROTAC) molecules), peptide-oligonucleotide or peptide- antibody-drug conjugates. By way of further example, the reactions and/or catalytic systems described herein may show utility in the fluorescent- or biotin-tagging of azide- modified biomolecules (e.g. glycoproteins, RNA, DNA and the like).

As described previously, there is provided a method of forming a triazole moiety in a bioconjugation reaction to link (e.g. covalently link) a first and second reactant, wherein the method comprises: contacting a first reactant comprising an ynamine moiety with a second reactant comprising an azide moiety in the presence of a copper catalyst and a reducing agent, which may preferably be glutathione, wherein at least one of the first and second reactants is a biomolecule.

In some examples, the other one of the first and second reactants is a tagging moiety (such as a labelling or binding tag).

As such, in some examples, the first reactant comprising the ynamine moiety may be or comprise a biomolecule. In particular, the first reactant may be or comprise the ynamine moiety appended, conjugated or linked (e.g. covalently linked) to the biomolecule.

The second reactant comprising the azide moiety may be or comprise a tagging moiety (such as a labelling or binding tag). In particular, the second reactant may be or comprise the azide moiety appended, conjugated or linked (e.g. covalently linked) to the tagging moiety.

In those examples, where the ynamine is appended, conjugated or linked to a biomolecule and the azide moiety is appended, conjugated or linked to a tagging moiety, under the reaction conditions described herein, the ynamine may react with the azide group to form the triazole product and link (e.g. covalently link) the biomolecule to the tag.

In alternative examples, the first reactant comprising the ynamine moiety may be or comprise a tagging moiety (such as a labelling or binding tag). In particular, the first reactant may be or comprise the ynamine moiety appended, conjugated or linked (e.g. covalently linked) to the tagging moiety.

The second reactant comprising the azide moiety may be or comprise a biomolecule. In particular, the second reactant may be or comprise the azide moiety appended, conjugated or linked (e.g. covalently linked) to the biomolecule.

In those examples, where the ynamine is appended, conjugated or linked to a tagging moiety and the azide moiety is appended, conjugated or linked to a biomolecule, under the reaction conditions described herein, the ynamine may react with the azide group to form the triazole product and link (e.g. covalently link) the biomolecule to the tagging moiety.

In some examples, the method further comprises forming the triazole moiety in a site-specific and/or sequential manner. By way of example, the biomolecule may comprise at least two sites able to participate in the cycloaddition reaction (such as two azide groups, or an ynamine and alkyne group). Under the reaction conditions described herein (and in view of the conditional and/or tuneable reactivity of the ynamine-containing reactant), the present inventors have identified that it is possible to effect site-specific and/or sequential modification of the biomolecule. For example, the method may comprise forming (e.g. selectively and/or sequentially forming) a first triazole moiety and a second triazole moiety in the biomolecule.

By way of further example, where the biomolecule comprises at least two azide groups (e.g. a first azide moiety and a second azide moiety), the method may comprise reacting a first ynamine (under the reaction conditions described herein) preferentially (or specifically) with the first azide moiety. The method may then comprise reacting an alkyne reagent preferentially (or specifically) with the second azide moiety. In this way, the method may provide a dual-functionalized biomolecule with good selectivity.

By way of further example, the biomolecule may comprise an ynamine moiety and an alkyne moiety. In such examples, the method may comprise reacting a first azide- containing reactant preferentially (or specifically) with one or other of the ynamine and alkyne on the biomolecule. The method may then further comprise preferentially (or specifically) reacting a second azide-containing reactant with the other of the ynamine and alkyne on the biomolecule. In this way, the method may provide a dual-functionalized biomolecule with good selectivity.

As used herein, the biomolecule may be any biological molecule typically found in a living organism. In the context of the present disclosure, the term “biomolecules” may encompass at least proteins, carbohydrates, lipids and nucleic acids, and the like. By way of further example, the term “biomolecules” may also embrace polypeptides, peptides, amino acids, polysaccharides, oligosaccharides, monosaccharides, polynucleotides, oligonucleotides, nucleotides, antibodies and the like. In some examples, the biomolecule may be a peptide (such as a cell penetrating peptide) or an oligonucleotide.

As used herein, a bioconjugation reaction may refer to a method of forming a covalent bond between two molecules, one of which is a biomolecule.

Suitable tags may include those that are typically used in the art to label compounds (e.g. labelling tags) and also those tags that are used to bind compounds (e.g. binding tags).

Suitable labelling tags may include, but are not limited to, fluorophores, selflabelling protein tags (such as HaloTag or SNAP-tag®), sulfonyl(VI) fluorides (SuFEx reagents) or the like. SuFEx (sulfonyl (VI) fluoride reagents are electrophilic reactive groups that can modify nucleophilic side chains (e.g. lysine, tyrosine, serine, and the like). SuFEx reagents may therefore be considered as labelling tags. A representative example of a labelling tag includes Sulfo-Cy3 azide. Suitable binding tags may include, but are not limited to, those typically used in pull-down assays or the like. Representative examples include biotin and derivatives thereof e.g. d-desthiobiotin.

The present disclosure is further directed to any one or more novel compounds such as are described herein.

The present disclosure is also directed to a number of novel compounds that find particular applications in the methods described herein.

Thus, according to a further aspect there is provided a compound according to formula (lb): wherein R ⁶ and R ⁷ are each independently selected from the group consisting of H, optionally substituted aryl, and optionally substituted heteroaryl; or wherein R ⁶ and R ⁷ together form an optionally substituted aryl or an optionally substituted heteroaryl ring; and and wherein the compound is not: wherein R ⁸ is selected from H, Ci-C ₆ alkyl and silyl protecting group.

In some examples, R ⁶ and R ⁷ may be any of those described in relation to the first reactant as shown and described in relation to formula (la) above).

In some examples, R ⁸ may be any of those described in relation to the first reactant (e.g. as shown and described in relation to formula (la)). In other examples, R ⁸ may be a silyl protecting group. In other words, R ⁸ may be any silyl group that acts to protect the acetylenic hydrogen of the ynamine. By way of particular example, the silyl protecting group may be derived from an organosilane, e.g. an alkylsilane, which in some cases may be a trialkylsilane. Representative examples include, but are not limited to, triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), and thexyldimethylsilyl (TDS), and the like.

In some examples, the present disclosure provides the following compounds: wherein R ⁸ in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl).

Additional representative examples of compounds according to the present disclosure are set out below: wherein R ⁸ is H or silyl protecting group (e.g. triisopropylsilyl); n is 0 or any number between 1 and 10; p is 0, 1 , 2 or 3; and

R ¹³ is selected from Ci-C ₆ alkyl, halo, Ci-C ₆ haloalkyl, and Ci-C ₆ alkoxy.

Yet further representative examples of compounds according to the present disclosure are set out below: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ;

R ⁸ in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl); and

X ¹ represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues;

L is a covalent linker; p is 0, 1 , 2 or 3; and

R ¹³ is selected from Ci-C ₆ alkyl, halo, Ci-C ₆ haloalkyl, and Ci-C ₆ alkoxy.

By way of further example, novel compounds of the disclosure may be selected from one of the following examples. wherein R ⁸ in the example compounds above is H or silyl protecting group (e.g. triisopropylsilyl); and wherein X ¹ represents an oligonucleotide comprising from 1 to 30 (e.g. 1 to 20, or 1 to 15) nucleotide residues.

It will be appreciated that the compounds of the present disclosure may exist in different stereoisomeric forms. The present disclosure includes within its scope the use of all stereoisomeric forms, or the use of a mixture of stereoisomers of the compounds. By way of example, where the compound comprises one or more chiral centres, the present disclosure encompasses each individual enantiomer of the compound as well as mixtures of enantiomers including racemic mixtures of such enantiomers. By way of further example, where the compound comprises two or more chiral centres, the present disclosure encompasses each individual diastereomer of the compound, as well as mixtures of the various diastereomers.

By way of further example, the compounds provided by the present disclosure may be represented as: wherein n is 0 or any number between 1 and 10 (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) ; wherein R ⁸ in the example compound above is H or silyl protecting group (e.g. triisopropylsilyl).

By way of further example, a compound provided by the present disclosure may be represented as: wherein R ⁸ in the example compound above is H or silyl protecting group (e.g. triisopropylsilyl).

According to a further aspect, there is further provided a method of preparing any one or more of the novel compounds described herein.

The present disclosure further provide a kit for use in cycloaddition (e.g. Click) reactions. By way of example, the kit may be of particular use in bioconjugation reactions, such as the tagging and/or labelling of biomolecules.

The kit may comprise:

(i) one or more first reactants comprising an ynamine moiety (as described herein, such as those of formulae (la), (lb) and (Ic) and those example compounds described herein); and (ii) one or more second reactants comprising an azide moiety (as described herein, such as those of formula (II) and those example compounds described herein).

The kit may further comprise one or more additional components selected from the following list:

(iii) a reducing agent (e.g. glutathione or sodium ascorbate);

(iv) a copper catalyst as described herein (e.g. Cu(OAc) ₂ );

(v) a solvent as described herein (such as an aqueous buffered solution);

(vi) a co-solvent as described herein (such as a protic fluorinated solvent);

(vii) one or more third reactants comprising an alkyne moiety; and

(viii) a magnesium salt.

Definitions

In the present disclosure, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds described herein, is intended to be in accordance with the rules of the International Union of Pure and Applied Chemistry (IUPAC) for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)” (see A. D. Jenkins et al., Pure & AppL Chem., 68, 2287-2311 (1996)). For the avoidance of doubt, if an IUPAC rule is contrary to a definition provided herein, the definition herein is to prevail.

As used herein, the term “alkyl” refers to a straight or branched chain hydrocarbyl group. The chain may be saturated or unsaturated, e.g. in some cases the chain may contain one or more double or triple bonds.

As used herein, “Ci-C _n alkyl” may be selected from straight or branched chain hydrocarbyl groups containing from 1 to n carbon atoms. For example, “Ci-Cealkyl” may be selected from straight or branched chain hydrocarbyl groups containing from 1 to 6 carbon atoms and Ci-C ₃ alkyl may be selected from straight or branched chain hydrocarbyl groups containing from 1 to 3 carbon atoms. Representative examples are methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, etc. When a Ci-C ₆ alkyl group is substituted, any hydrogen atom(s), CH ₃ , CH ₂ or CH group(s) may be replaced with the substituent(s), providing valencies are satisfied. Where the Ci-C ₆ alkyl comprises a divalent hydrocarbon radical (containing from 1 to 6 carbon atoms), this moiety may sometimes be referred to herein as a Ci-C ₆ alkylene. As used herein, the term "aryl" may be a single or fused ring system comprising one or more aromatic rings. The term “aryl” may refer to a mono- or polycyclic aromatic hydrocarbon system having 6 to 14 carbon ring atoms, in some cases 6 to 10 carbon ring atoms. Where the aryl is a fused ring system, at least one of the rings is aromatic and the other ring(s) may be aromatic or aliphatic. Representative examples of suitable "aryl" groups include, but are not limited to, phenyl, biphenyl, naphthyl, 1 -naphthyl, 2- naphthyl and anthracenyl. As used herein, “substituted aryl” refers to an aryl group as defined herein which comprises one or more substituents on the aromatic ring. When an aryl group is substituted, any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.

As used herein, heteroaryl may be a single or fused ring system comprising one or more aromatic rings, wherein the one or more aromatic rings comprise one or more O, N and/or S atoms. The term “heteroaryl” may refer to a mono- or polycyclic heteroaromatic system having 5 to 14 ring atoms, in some cases 5 to 10 ring atoms. Where the heteroaryl is a fused ring system, at least one of the rings is aromatic and the other ring(s) may be aromatic or aliphatic. Representative examples of heteroaryl groups may include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, benzofuranyl, benzothiazolyl, benzimidazolyl, indazolyl, benzoxazolyl, benzisoxazolyl, benzodioxanyl etc. As used herein, “substituted heteroaryl” refers to a heteroaryl group as defined herein which comprises one or more substituents on the heteroaromatic ring. When a heteroaryl is substituted, any hydrogen atom(s) may be replaced with the substituent(s), providing valencies are satisfied.

As used herein, the term “substituted” means that the moiety comprises one or more substituents. As used herein, the “optionally substituted” means that the moiety may comprise one or more substituents.

As used herein, a “substituent” may include, but is not limited to, hydroxyl, thiol, carboxyl, cyano (CN), nitro (NO ₂ ), halo, haloalkyl (e.g. a Ci to C ₆ haloalkyl), an alkyl group (e.g. Ci to C or Ci to C ₆ ), aryl (e.g. phenyl and substituted phenyl for example benzyl or benzoyl), alkoxy group (e.g. Ci to C ₆ alkyl) or aryloxy (e.g. phenoxy and substituted phenoxy), thioether (e.g. Ci to C ₆ alkyl or aryl), keto (e.g. Ci to C ₆ keto), ester (e.g. Ci to C ₆ alkyl or aryl, which may be present as an oxyester or carbonylester on the substituted moiety), thioester (e.g. Ci to C ₆ alkyl or aryl), alkylene ester (such that attachment is on the alkylene group, rather than at the ester function which is optionally substituted with a Ci to C ₆ alkyl or aryl group), amine (including a five- or six-membered cyclic alkylene amine, further including a Ci to C ₆ alkyl amine or a Ci to C ₆ dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups), amido (e.g. which may be substituted with one or two Ci to C ₆ alkyl groups (including a carboxamide which is optionally substituted with one or two Ci to C ₆ alkyl groups), alkanol (e.g. Ci to C ₆ alkyl or aryl), or carboxylic acid (e.g. Ci to C ₆ alkyl or aryl), sulfoxide, sulfone, sulfonamide, and urethane (such as -O-C(O)-NR ₂ or-N(R)-C(0)-0-R, wherein each R in this context is independently selected from Ci to C ₆ alkyl or aryl).

In some examples, and unless the context indicates otherwise, a “substituent” may include, but is not limited to, halo, Ci to C ₆ alkyl, Ci to C ₆ haloalkyl and Ci to C ₆ alkoxy.

As used herein, a “halo” group may be F, Cl, Br, or I. In some examples, halo may be F.

As used herein, “haloalkyl” may be an alkyl group in which one or more hydrogen atoms thereon have been replaced with a halogen atom, e.g. a Ci-C ₆ haloalkyl may be a Ci to C ₆ alkyl in which one or more hydrogen atoms thereon have been replaced with a halogen atom. By way of a representative example, a Ci-C ₆ haloalkyl may be a fluoroalkyl, such as trifluoromethyl (-CF ₃ ) or 1 ,1 -difluoroethyl (-CH ₂ CHF ₂ ).

As used herein, alkoxy may refers to an alkyl group, as defined above, appended to the parent molecular moiety through an oxy group, -O-. As used herein, a Ci-C ₆ alkoxy refers to a Ci-C ₆ alkyl group (as defined above), appended to the parent molecular moiety through a oxy group, -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy etc. Similarly, Ci-C ₆ alcohol refers to a Ci-C ₆ alkyl group (as defined above) which is appended to an -OH group.

As used herein, a silyl protecting group may refer to any silyl group that acts to protect the acetylenic hydrogen of the ynamine. By way of particular example, the silyl protecting group may be derived from an organosilane, e.g. an alkylsilane, which in some cases may be a trialkylsilane. Representative examples include, but are not limited to, triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), thexyldimethylsilyl (TDS), benzyldimethylsilyl (BDMS), biphenyldimethylsilyl (BDMS), biphenyldiisopropylsilyl (BDIPS) and tris(biphenyl-4-yl)silyl (TBPS), and the like.

As used herein a “linker” or “covalently linked” may be a covalent linker. The linker acts to tether ynamine or azide moiety to a biomolecule or tagging moiety whilst also allowing the biomolecule to interact with the cellular environment, allowing the tagging moiety to perform its function (e.g. a labelling function), and allowing the ynamine or azide moiety to participate in the reaction to form a triazole. In many cases, a broad range of linkers will be tolerated. The selection of linker may depend upon the nature of the biomolecule the ynamine or azide-containing reactant is being tethered to. The linker may be selected to provide a particular length and/or flexibility. In some examples, the linker may be a covalent bond. In other examples, the linker may comprise any number of atoms between 1 and 30 or between 1 and 10. In some cases the linker may comprise any number of atoms in a single linear chain of between 1 and 30 or between 1 and 10. The ynamine or azide-containing reactant and the biomolecule may be covalently linked to L through any group which is appropriate and stable to the chemistry of the linker. By way of example only, the linker may be covalently bonded to these moeities via a carboncarbon bond, keto, amino, amide, ester or ether linkage.

It should be noted that the terms “comprise”, “comprising” and/or “comprises” is/are used to denote that aspects and embodiments of this disclosure “comprise” a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments which “consist essentially of” or “consist of” the relevant feature or features.

The disclosure also encompasses various deuterated forms of the compounds of any of the Formulae disclosed herein, including Formulae (I), and (II) (inc. corresponding subgeneric formulae defined herein), respectively, or a pharmaceutically acceptable salt and/or a corresponding tautomer form thereof (including subgeneric formulae, as defined above) of the present disclosure. Each available hydrogen atom attached to a carbon atom may be independently be present as a deuterium atom. A person of ordinary skill in the art will know how to synthesize deuterated forms of the compounds of any of the Formulae disclosed herein, including Formulae (I) and (II), (inc. corresponding subgeneric formulae defined herein), respectively, or a pharmaceutically acceptable salt and/or a corresponding tautomer form thereof (including subgeneric formulae, as defined above) of the present disclosure. For example, deuterated materials, such as alkyl groups may be prepared by conventional techniques (see for example: methyl-c/ ₃ -amine available from Aldrich Chemical Co., Milwaukee, Wl, Cat. No.489, 689-2).

The disclosure also includes isotopically-labelled compounds which are identical to those recited in any of the Formulae disclosed herein, including Formulae (I) and (II) (inc. corresponding subgeneric formulae defined herein), respectively, or a pharmaceutically acceptable salt and/or a corresponding tautomer form thereof (including subgeneric formulae, as defined above) of the present disclosure but for the fact that one or more atoms may be present as an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ¹²³ l or ¹²⁵ L Compounds of the present disclosure and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present disclosure.

DETAILED DESCRIPTION

The present invention will now be described in more detail with reference to the following non-limiting examples and figures which show.

Figure 1 shows (a) CuAAC as a bio-orthogonal tool in chemical biology; (b) GSH acts as a Cu ligand and redox mediator; and (c) Design concept: sequential labelling of biomolecules exploiting conditional ynamine reactivity mediated by GSH:Cu.

Figure 2 shows RP-HPLC susceptibility study of ynamine (1 a) in the presence of GSH. Reaction conditions: (a) 1a (200 pM), GSH (1 - 10 mM), room temperature (rt); and (b) 1a (200 pM), Cu(OAc) ₂ (350 pM),GSH (1 - 10 mM), room temperature (rt).

Figure 3a shows the influence of [GSH] on the formation of 5a; Reaction conditions: 1a (200 pM), 4a (500 pM), Cu(OAc) ₂ (350 pM), GSH (1 -4 mM), rt.

Figure 3b shows the influence of [GSH] (glutathione concentration) and sodium ascorbate (NaAsc) on the formation of 5a; Reaction conditions (all): 1a (200 pM), 4a (500 pM), Cu(OAc) ₂ (1.75 equiv., 350 pM), rt; black line (no GSH or NaAsc); red line GSH (1 mM), blue line NaAsc (1 mM).

Figure 4 shows reaction kinetics as a function of azide. Reaction conditions: lari (200 pM), 4a-c (500 pM), Cu(OAc) ₂ (350 pM), GSH (1 mM), 10% MeOH, DPBS, rt. (The asterix (*) indicates that addition of sodium ascorbate (NaAsc) (1 mM) was required).

Figure 5 shows investigation into the reaction conditions of the ynamine-azide [3+2]cycloaddition. (a) DoE analysis highlighting the influence of triazole (5a) conversion by the GSH:Cu(ll) ratio. Reaction conditions: 1a (200 pM), 4a (500 pM), Cu(GAc) ₂ (100- 400 pM), GSH (100 pM-1 mM), 10% MeOH in DPBS, rt; (b) The effect of organic cosolvent on the formation of triazole (5a). Reaction conditions: 1a (200 pM), 4a (500 pM) CU(OAC) ₂ (100 pM), GSH (100 pM), DPBS in 10% organic co-solvent.

Figure 6 shows exemplary structures of azide-modified cell penetrating peptides (CPPs) used in the present disclosure. Figure 7a and 7b shows structures of triazole products. Figure 7c shows reaction profile of the formation of 20 (red) and 21 (grey). Reaction conditions: 10 (200 pM), 18 or 20 (200 pM), Cu(OAc) ₂ (100 pM), GSH (100 pM), 10% HFIP in 1X DPBS, rt, 1 - 4 h. Figure 7d shows comparative analyses of the reaction profile of the formation of 20 using different co-solvents (10% co-solvent) in 1 X DPFS.

Figure 8 shows: (a) Competition experiments highlighting the conditional reactivity of ynamine (10); (b) Reaction profile for the formation of 24a/b. Reaction conditions: 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) ₂ (500 pM),10% HFIP in 1 X DPBS, rt, 1 h; (c) Reaction profile for the formation of 23. Reaction conditions: 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) ₂ (500 pM), GSH (500 pM), 10% HFIP in 1 X DPS, rt, 1 h. Grey line (10), red line (23), blue line (22), green line (24a/b).

Figure 9 shows (a) Competition experiments highlighting the conditional reactivity of ynamine (10) with peptide (19); (b) Reaction profile for the formation of 26a/b. Reaction conditions: 10 (200 M), 22 (200 pM), 19 (200 pM), Cu(OAc) ₂ (500 pM),10% HFIP in 1 X DPBS, rt, 1 h; (c) Reaction profile for the formation of 25. Reaction conditions: 10 (200 M), 22 (200 pM), 18 (200 pM), Cu(OAc) ₂ (500 pM), GSH (500 pM), 10% HFIP in 1 X DPBS, rt, 1 h. Grey line (10), red line (25), blue line (22), green line (26a/b).

Figure 10 shows structure of bifunctional peptide (27) containing two azide functional groups.

Figure 1 1 shows: (a) Competition experiments highlighting the conditional reactivity of ynamine (10) and DBCO (22) with peptide (27); (b) Reaction profile for the formation of 28: Reaction conditions: (i) 10 (220 pM), 22 (220 pM), 27 (200 pM), CU(GAC) ₂ (500 pM), 10% HFIP in 1 X DPBS, rt, 2 h; (ii) EDTA (5 mM) shaken for 4 h. Black line (27), red line (monolabelled ynamine triazole), orange line (28), blue line (duallabelled ynamine triazole), green line (mono-labelled DBCO), purple line (dual-labelled DBCO); (c) HPL chromatogram formation occurs via ynamine (10) [3+2] cycloaddition at the picolyl azide site.

Figure 12 shows (a) Reaction of ODN-1 to form ODN-2. (b) Influence of the [Cu/GSH] on the formation of ODN-2. Conditions: ODN-1 (50 pM), 30 (100 pM) Cu(OAc) ₂ (50 (grey), 100 (red) or 500 (blue) pM), GSH (50 (grey), 100 (red) or 500 (blue) pM), 10% HFIP in 1 X DBPS, rt, 16 - 20 h.

Figure 13 shows (a) Sequential modification of a dual-tagged oligonucleotide 31 . (b) Reaction kinetics of the sequential, dual modification of 31 to produce 33 via the formation of mono-adduct (32). Reaction conditions: (i) 31 (20 pM), 30 (20 pM), 5% HFIP in 1X DPBS (20 mM MgCI ₂ ), rt, 4 h; (ii) 32 (30 pM), Cu(OAc) ₂ (50 pM), GSH (50 pM), rt, 2 h. (f) HPLC chromatogram of the crude reaction.

Figure 14 shows: (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) ₂ and GSH. (b) % Total area of triazole product ODN 4 after 2 hours as a function of cosolvent. (c) % Total area of triazole product ODN 4 after 2 hours as a function of buffer, (c) Time course of the reaction of ODN 3 with sulfo-Cy3-azide as a function of copper source. Conditions for Cu/THPTA/NaAsc: ODN 3 (10 pM), sulfo-Cy3-azide (20 pM), CU(OAC) ₂ (10 pM), THPTA (50 pM), NaAsc (1 mM) in H ₂ O (20 mM MgCI ₂ ) (10% MeOH as co-solvent for Cu(MeCN) ₄ OTf. (e) Observed peak areas of the triazole product when using Cu/THPTA/NaAsc conditions.

Figure 15 shows (a) Degradation of ODN 3 in the presence of Cu/GSH and Cu/THPTA/NaAsc. (b) Stability of ODN 3 in the presence of Cu/GSH. Conditions (Cu/GSH): ODN 3 (10 pM), Cu(OAc) ₂ (50 pM), GSH (50 pM) in buffer containing 10% MeOH unless otherwise indicated. Conditions (Cu/THPTA/NaAsc): ODN 3 (10 pM), CU(OAC) ₂ (10 pM), THPTA (50 pM), NaAsc (1 mM) in containing 10% MeOH unless otherwise indicated, (c) CuAAC reaction of ODN 3 with sulfo-Cy3-azide in different solvent systems. Conditions (Cu/THPTA/NaAsc): ODN 3 (10 pM), Cu(OAc) ₂ (10 pM), THPTA (50 pM), NaAsc (1 mM) in buffer containing 10% MeOH.

Figure 16 shows (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) ₂ , THPTA and NaAsc at different concentrations, (b) HPLC time courses of DoE results, (c) Further HPLC screening of THPTA and NaAsc concentrations to improve reactivity.

Figure 17 shows a) Reaction of ODN 3 with different azides using the optimised conditions for the Cu/THPTA/NaAsc system, (b) Percentage total area (HPLC) of triazole products after the indicated reaction time.

Figure 18 shows (a) Reaction of ODN 3 with sulfo-Cy3-azide using Cu(OAc) ₂ and GSH. (b) Increasing the Cu/GSH ratio increases the reaction rate. Conditions (Cu/GSH): ODN 3 (10 pM), CU(OAC) ₂ (25 or 50 pM), GSH (5, 10 or 25 pM) in H ₂ O (20 mM MgCI ₂ ) containing 10% MeOH.

RESULTS AND DISCUSSION

Profiling the influence of bio-orthogonal reaction classes in the presence of glutathione

The objective of this study was to determine the optimal conditions required to undergo a chemoselective ynamine-azide tagging of biomolecules within biological media. The present inventors hypothesized that the structural and mechanistic differences of ynamines relative to conventional alkynes would manifest in a divergence in their reactivity with an azide in the presence of biologically-relevant concentrations of GSH. Present in millimolar concentrations within a living cell, one of the primary functions of GSH is to mediate the cell’s oxidation state, and to deactivate electrophilic agents. ¹⁶¹ This poses a considerable challenge for the application of bio-orthogonal reagents as tools for biomolecule tagging and bioconjugation as many of the major classes e.g., SPAAC, iEDDA) are susceptible to side-reactions with GSH. ¹⁷¹

On the basis of this, the stability of ynamine (1a) across the physiological range of GSH (1 -10 mM) ^[8] in buffered solutions over 24 hr was investigated (Figure 2a).

In the presence of 1 and 5mM of GSH, only ~ 10% of ynamine (1a) formed GSH adducts 2 and 3 (Figure 2a). These adducts could arise from either a radical-based thiolyne addition to 1a to form 2, ^[91 or via a step-wise activation and subsequent nucleophilic attack of the thiol(ate) to form 3. ¹¹⁰¹ Although an increase in the formation of 2 was only observed at 10mM [GSH], at least 75% of 1a still remained after 24 hours, which confirmed the potential utility of ynamines as an alkyne surrogate for bio-orthogonal CuAAC ligations.

Next, the influence of the addition of a Cu(ll) source to a reaction mixture containing 1a and varying concentrations of GSH was explored (Figure 2b). In this case, exclusive formation of GSH adduct (2) was observed in the presence of Cu(OAc) ₂ (350 pM). The conversion to 2 increased from 3 % (1 mM GSH) up to 25 % and 27 % at 5 mM and 10 mM [GSH], respectively.

As a consequence of the lack of 3 formed and the known ability of Cu salts and GSH to form radical species, these studies suggest that the formation of 2 proceeds via a thio-yne-based mechanism.

The influence of [GSH] on the reaction kinetics of the Cu-catalyzed ynamine- azide [3+2]cycloaddition was then explored using 1a and benzyl azide (4a) as a model reagent pair to form 5a using [Cu(ll)] of 350 pM (Figure 3a and 3b).

The reaction did proceed in the absence of GSH, and the conversion to 5a reached a maximum of 20% after 2 hr. A rate acceleration was observed at 1 mM [GSH], resulting in full conversion to 5a. However, a further increase in [GSH] resulted in no increase in conversion to 5a after at 2 hr. This suggests that the Cu:GSH may play an influential role in the reaction kinetics of triazole formation. Addition of NaAsc (1 mM) instead of GSH only increased the conversion to 5a to 35% after 2 h. Therefore, in contrast to other classes of alkyne, at certain concentrations, GSH was found to be a more effective reductant for the ynamine CuAAC reaction than NaAsc.

Using the CuAAC conditions identified for the formation of 5a, the reaction kinetics of a suite of alkynes (1a-d) were investigated using azides (4a-c) as the reagent partner (see Figure 4).

One striking observation was no reaction observed using the conditions for triazole formation using 1a when alkynes (1 b-d) were used. For these alkynes, an addition of a reductant (sodium ascorbate, 1 mM) was required in order to observe any triazole products.

When benzyl azide (4a) was used, the rate of triazole formation was fastest for the ynamine substrate (5a), and did not require the addition of the ascorbate reductant (Figure 4a).

The triazole derived from the propiolamide (5b) was considerably slower, reaching 80% conversion after 24 hr. The formation of triazoles (5c-d) was considerably poorer still, reaching 60% and 20% conversion when 1 c and 1d were used (Figure 4b).

Although the overall rate of triazole formation (5e-h) was considerably slower when 4b was used as the azide reagent, the reactivity trend was similar to that observed for the benzyl azide series with ynamine (1a) exhibiting faster reaction kinetics relative to the other alkyne substrates.

One potential explanation of this phenomenon is the differences in water solubility between azides 1a and 1 b, which could result in an enhancement in rate for less water soluble substrates via the “on water” effect, which has been observed for other [3+2]cycloadditions. ^[11]

In contrast to the other azides tested in this study, the Cu-chelating picolyl azide resulted in full conversion to triazole products (5i-l) across all alkyne substrates (Figure 4c). ^[12] In particular, the formation of triazole 5j derived from the propiolamide (1 b) is on par with 5i derived from ynamine (1a) when ascorbate is added to the reaction mixture.

The studies illustrated in Figures 4a, 4b and 4c highlight the divergence of Cu- catalyzed ynamine-azide [3+2]cycloadditions relative to conventional alkynes, and how the structure of the azide group can influence the reaction rate of triazole formation in physiologically relevant buffer systems.

Reaction optimization of the Cu-catalyzed ynamine-azide cycloaddition The GSH:Cu(ll) ratio and the organic co-solvent were also investigated to see how these factors could influence the formation of triazole (5a), with a view to identifying optimal conditions which could be used for chemoselective, sequential modification of biomolecules.

Design of Experiments (DoE) was used to determine the optimal conversion to 5a. A central composite face 2 factor design (3 levels) was chosen in which the [Cu(l I)] was set to 100 pM, 250 pM and 400 pM, with the [GSH] was set to 100 pM, 550 pM and 1 mM. Using the DoE approach, an optimal GSH:Cu(ll) ratio of 1 :1 resulted in maximal conversion to 5a without causing a measurable lag period (Figure 5a). Reactions with GSH:Cu(ll) > 2:1 increased the induction period until no reaction is observed at a GSH:Cu(ll) ratio >3:1.

Electron paramagnetic resonance (EPR) spectroscopy was then used to investigate the influence of the GSH:Cu ratio on the Cu oxidation state. Complete reduction of Cu(ll) to Cu(l) was observed when GSH:Cu >3:1 , whereas only partial reduction was observed when GSH:Cu <3:1 was used. At a 1 :1 GSH:Cu, only partial reduction occurred in the first 1 min of the reaction in both HFIP and MeOH solvents, which suggests a mixed Cu oxidation state may be favourable in some cases. The residual Cu(ll) signal after 60 min resembled the EPR spectrum of GSSG + Cu(OAc) ₂ suggesting formation of a Cu(ll)-GSSG complex.

With optimal conditions for the GSH:Cu(ll) established, the influence of the organic co-solvent on the formation of 5a was explored, with particular focus on the use of alternatives to DMSO.

The choice of organic (co)-solvent greatly influences the reaction kinetics of cycloadditions with protic fluorinated solvents such as HFIP and TFE enhancing the rate of Diels-Alder cycloadditions. ^[13] However, their application as a (co)solvent in CuAAC reactions has not been extensively explored.

A solvent screen of polar protic and non -protic co-solvents (10% co-solvent in 1X DPBS buffer) revealed a striking increase in the reaction rate and conversion to 5a using either TFE or HFIP as the co-solvent relative to other polar solvents, reaching maximum conversion after 10min relative to 2.5 hr required when DMSO was used as the corresponding co-solvent (Figure 5b).

A pertinent comparator is the observed differences in conversion to 5a using TFE (full conversion in 10 min) relative to EtOH (75 % conversion after 2.5 hr), thus highlighting the importance of the enhanced polarity and hydrogen-bond donating character of these fluorinated solvents on the CuAAC even in the presence of the GSH additive.

Diversification of ynamine scaffolds

The standard reaction conditions were used to diversify the substrate scope of ynamines (Scheme 1 ). Ullmann conditions used previously were applied to form TIPS- protected ynamines (8a-i) from the corresponding AZ-heterocycle (6a-i) and the TIPS- protected bromoacetylene (7). Deprotection of the TIPS (triisopropylsilane) group with TBAF (tetra-n-butylammonium fluoride) afforded terminal ynamines 9a-i.

Cui (5moi%) C CO 1 1 i

Scheme 1. Substrate scope of ynamines prepared in this study.

In addition to the diversification of the ynamine substrates, ynamine analogues tagged with a desthiobiotin (10) and phosphoramidite (17) were prepared. The desthiobiotin group is used extensively for pull-down assays, whereas the phosphoramidite is an important group for solid phase nucleic acid synthesis.

Scheme 2. (a) Synthesis of compounds 10. and (b) 17.

Application of GSH-mediated ynamine reactivity for the dual differential modification of peptides and oligonucleotides

To explore the scope of the conditional reactivity of ynamines in the presence of GSH, two azide-modified cell penetrating peptides (CPPs) 18 and 19 were prepared (Figure 6, peptide 18 is based on the cell-penetrating sequence derived from the third helix homeodomain of Antennapedia (i.e., penetratin while peptide 19 is derived from a previously identified spontaneous membrane translocating peptide). ¹¹⁴¹ The formation of triazoles 20-21 (Figure 7a-b) was then explored using ynamine (10). The most efficient labelling was observed using 10% HFIP in DPBS buffer as the solvent.

The importance of GSH as an ynamine reactivity modulator was demonstrated by a competition experiment with peptide (18) in the presence of DBCO analogue (22) and ynamine (10, Figure 8a). In the presence of GSH, exclusive formation of the triazole adduct (23) was observed, whereas in the absence of GSH the triazole adducts derived from the [3+2]cycloaddition between 22 and 18 (/.e., 24a/b) was formed (Figure 8b-c).

The same reactivity profile was observed using azide-modified peptide (19, Figure 9a). As per the azide-modified penetratin peptide (18), the use of GSH was critical for the reactivity of the ynamine i.e., in the absence of GSH exclusive formation of the DBCO adducts (26a/b) was observed (Figure 9b), whereas in the presence of GSH resulted in the formation of the ynamine triazole adduct (25, Figure 9c).

These experiments highlight the conditional reactivity of a free ynamine on a peptide containing a single azide modification. However, there is a need to dual functionalise peptides for the development of, for example, fluorescence-based FRET analyses for multi-colour imaging or to incorporate fluorophores and biotin tags for flexible tracking and pull-down assays.

To explore the chemoselective nature of ynamine click tagging, the dual-modified peptide (27) was prepared (Figure 10).

Peptide (27) contained a regular aliphatic azide on the C-terminus and a picolyl azide on the AZ-terminus. The picolyl azide functionality is known to react faster in the presence of an aliphatic azide ^[3c ’ ¹²¹ by virtue of its capacity to chelate a Cu atom within proximity to the benzylic azide group. It was therefore hypothesised that a sequential click reaction, first at the picolyl azide site, then at the regular azide would be possible using 10 and 22. Furthermore, the modulation of ynamine (10) reactivity would enable positional and sequential control of each triazole formed.

Dual azide-labelled peptide (27) was prepared by solid phase synthesis (Figure 10) and used in competition experiments in the presence of equimolar amounts of ynamine (10) and DBCO analogue (22). Previous work by Hosoya et al. has shown that transient protection of the internal alkyne of DBCO occurs in the presence of Cu(l). ^[15] Thus, without being bound by theory, the inventors hypothesise that in the presence of a Cu(ll) source and GSH, Cu(l) can form and perform two roles: first, activate the ynamine-based [3+2]cycloaddition preferably at the picolyl azide site, and second, act as a transient protecting group on the DBCO, thereby suppressing competitive reactivity.

The reaction conditions used in the competition experiment were based on conditions used in previous peptide labelling experiments (Figures 8 and 9). The only change in the reaction conditions was the addition of the Cu -chelator EDTA after 2 hours (Figure 11 a-b), which deprotects DBCO. The addition of EDTA suppressed the formation of the dual-labelled ynamine triazole adduct. Using these conditions, the dual-labelled triazole peptide 28 was formed in 93% as determined by RP-HPLC (Figure 1 1 c). The sequential nature of each triazole formed was also determined by RP-HPLC (Figure 1 1 b). The formation of the mono-labelled ynamine adduct correlated to the reduction of the starting peptide (27). Upon addition of EDTA to the reaction mixture and further stirring for 4 hours, resulted in the reduction of the mono-labelled ynamine adduct and concomitant formation of the dual-labelled peptide (28).

The chemoselective nature of the sequential click labelling was confirmed by subjecting peptide (27) to ynamine (10) to form the mono-labelled triazole adduct followed by a tryptic digest. Analysis by LC-MS confirmed the identity of the monolabelled adduct being triazole formation at the picolyl azide site based on the formation of 29 (Figure 1 1 d).

Taken collectively, these results show the conditional reactivity of an ynamine as a bio-orthogonal handle in the presence of a redox modulator (/.e., GSH). The bio- orthogonal nature is maintained in the presence of other bio-orthogonal alkyne reagents e.g., DBCO) and enables both chemoselective and site-specific modification of dual azide-labelled peptides where the reactivity of the azide is modulated by a Cu -chelating group.

The application of ynamines as a bio-orthogonal tagging tool was extended to the tagging of oligodeoxyribonucleotides (ODNs). Incorporation of phosphoramidite (17) onto the 5'-end of a dodcamer sequence (ODN-1 ) was achieved by automated solid phase synthesis (Figure 12a). Using the fluorescent azide (30) and our GSH-based reaction conditions, full conversion of the ODN-1 into triazole product (ODN-2) was observed by RP-HPLC and MALDI-ToF analyses (Figure 12b; blue 500 pM, red 100 pM, grey 50 pM Cu(OAc) ₂ and GSH).

Chemoselective, dual modification was then explored using 31 , which contains an ynamine incorporated on the 5' end, and a DBCO group linked to an internal T nucleotide (Figure 13a). In the absence of Cu(ll) and GSH, exclusive formation of the mono-adduct 32, where the [3+2]cycloaddition takes place at the DBCO site, was observed using azide 30 after 1 h (Figure 13b). Subsequent addition of Cu(ll):GSH (1 :1 ) and azide 32 then provided the dual functionalized product 33 in -90% conversion; cycloaddition regioselectivity was confirmed by enzymatic digestion.

In summary, it has been shown that the ynamine functional group exhibits conditional reactivity in the presence of the GSH. This phenomenon is not observed with conventional terminal alkynes, thereby rendering the ynamine functionality as an environmentally sensitive bio-orthogonal reagent. The ability to alter the reactivity of ynamines expands the arsenal of the bio-orthogonal toolkit by providing step-efficient approaches to prepare discrete mono- and dual-functionalized biomolecules.

Investigations into Ynamines for Oligonucleotide Conjugation

Further studies were carried out to explore the use of aromatic ynamines for conjugation and modification of oligonucleotides. In the following studies (described below and shown in Figures 14 to 18), ODN-3 has the sequence shown in Figure 14a.

A range of fluorinated alcohols and common CuAAC reaction co -solvents (5 - 10%) in phosphate buffer (1X DPBS) were screened using ynamine-modified oligonucleotide (ODN) 3, Cu(OAc) ₂ , GSH and sulfo-Cy3-azide (sulfo-Cyanine3 azide - a commercially available water-soluble dye azide for click chemistry) (Figure 14b). Overall, the addition of co-solvent decreased conversion to the triazole ODN 4 when compared to 100% 1 X DPBS buffer with the only exception being PFH, as indicate by RP-HPLC analysis (Figure 14b). Notably, HFIP, DMSO and MeCN also resulted in poor conversions (< 25%), whereas MeOH was tolerated well. When decreasing the percentage of co-solvent from 10 to 5% conversions increased, especially for HFIP (from 17 to 41% after 2 hours). As a result of this screen, the co-solvent was eliminated for the following experiments and the azide concentration was doubled to increase conversions.

The influence of range of aqueous buffer systems on the reaction were tested next (Figure 14c). 1X DPBS phosphate buffer was compared to H ₂ O as well as the influence of different concentrations of MgCI ₂ . This magnesium salt was reported to increase conversions for CuAAC reactions on oligonucleotides, possibly by displacing bound copper catalyst from the phosphate backbone of the oligonucleotide (Kollaschinski et al, Bioconjug. Chem. 2020, 31 , 507-512).

In the case of 1 X DPBS, the magnesium concentration (0.5, 10 and 20 mM) had marginal influence on conversions (Figure 14c). However, the addition of a magnesium salt (e.g. MgCI ₂ ) increased the conversion from only 10 to -70% after 2 hours when H ₂ O was used as a solvent. Further addition of 10 mM NaCI did not change conversions significantly. H ₂ O with MgCI ₂ (20 mM) was used for the further experiments to simplify the protocol.

Next, the influence of the copper source for both the Cu/GSH and the Cu/THPTA/NaAsc system was investigated. For both systems, the observed influences of the copper source were marginal (Figure 14d). The solubility of CuOAc was extremely poor under these conditions which likely led to the low conversion observed. In general, the NaAsc system led to higher and faster conversions when compared to the GSH system, even though five times less Cu(OAc) ₂ was used. However, the final product peak areas were 10% to 50% lower than expected, depending on the copper source (Figure 14e). The missing peak area was attributed to copper redox induced degradation.

These findings prompted a new investigation into the stability of oligonucleotide 3 in both the GSH and the NaAsc system. Phosphate buffers and methanol have been identified as reagents which could reduce copper induced DNA degradation (Schweigert, et al, Environ. Mol. Mutagen. 2000, 36 (1 ), 5-12). This could explain why the highest peak area was observed for Cu(MeCN) ₄ OTf (Figure 14e) as the catalyst as this reaction contained 10% MeOH as the co-solvent due to the poor solubility of the copper source.

A set of experiments was designed to investigate the influence of different buffers, co-solvent and reductants on oligonucleotide degradation. As buffers H ₂ O (20 mM MgCI ₂ ), 1 X DPBS and 10X DPBS were chosen (with 10% MeOH in select cases). These conditions were screened against both the GSH and the NaAsc system (Figure 15).

From these experiments several trends could be observed. First, the addition of 10% MeOH increased ODN 3 stability from -50% to -80% in H ₂ O (20 mM MgCI ₂ ) when the NaAsc was used as the reducing agent (Figure 15b). This effect was less pronounced when using GSH. Additionally, the degradation induced by the copper and GSH redox couple is less than NaAsc even though 5 times more copper was added in the GSH system. Second, the switch from H ₂ O to 1X DPBS indeed reduced the degradation in a concentration dependent manner. This effect is more pronounced for the NaAsc system, where the oligonucleotide is completely stable over the 6 hour duration of the experiment when using 10X DPBS. The third difference is the degradation profiles for the NaAsc and GSH systems. In the NaAsc system, degradation is coupled to NaAsc consumption and once the antioxidant depleted (-4 hours in H ₂ O) then degradation stopped. In contrast, the GSH system led to steady degradation over time, which is less influence by buffer and co-solvent parameters.

As the oligonucleotide in the NaAsc system had shown remarkable stability when 10X DPBS was used, this buffer was tested in the CuAAC reaction along with H ₂ O and 1 X DPBS (Figure 15c). All reactions were run with 10% MeOH as a co-solvent to minimise degradation. Perhaps unsurprisingly, the reactivity in 10X DPBS was poor reaching only -10% conversion after 6 hours. 1 X DBPS gave an intermediate conversion with -70% whereas in H ₂ O (20 mM MgCI ₂ ) the reaction reached -90% after 2 hours, However, the product (ODN 4) area steadily declined after that timepoint indicated degradation after the completion of the reaction. This highlighted that there may be a balance between reactivity and stability to be struck in some cases.

To find this balance in the NaAsc system, we designed a full factorial design of experiments (DoE) with one centre point (Figure 16). The copper concentration was fixed at 10 pM and the THPTA concentration varied between 10 - 50 pM and NaAsc between 100 - 1000 pM. The centre point was run in duplicate to test the repeatability of the experiments (Figure 16b). Two main trends can be observed from the DoE. First, a [NaAsc] of 100 pM was sufficient to reach -80% conversion but led to slightly more starting material remaining than 1000 pM. As before the excess of NaAsc led to degradation after 2.5 hours. This was not the case for experiment 1 as all the NaAsc was depleted after 2 hours. Second, higher [THPTA] led to a slightly faster reaction rate. These results further show that it can be important to tune the sodium ascorbate concentration (i.e. [NaAsc]) to control and/or limit degradation in some examples.

The following experiments investigated NaAsc concentrations of 100, 140 and 200 pM as well as further increasing [THPTA] to 100 pM (Figure 16c). A [NaAsc] of 200 pM led to almost full consumption of starting material while limiting degradation. Increasing THPTA to 10 equivalents yielded a further increase in reaction rate. However, the final conversion remained around -80%. A doubling of the copper concentration while keeping 10 equivalents of both THPTA and NaAsc increased the final conversion to -90% in 1 hour.

These optimised conditions were then tested on a range of azides (Figure 17). Fluorescent azides such as dansyl and NBD azide gave a very fast reaction to the corresponding triazoles (ODN 5 and 6) reaching -90% conversion in under 2 min (the first time point measured after setting up the reaction). Biotin -PEG-picolyl azide also led to rapid conversion to ODN 8 in 30 mins, while sulfo-Cy5-azide reached full conversion to ODN 7 within 1 hour.

Next the Cu/GSH reaction conditions were optimized using ODN 3 and sulfo- Cy3-azide (Figure 18a). To increase the reaction rate, the ratio of Cu/GSH was increased from the previously used 1 :1 to 2:1 (50/25 pM) and 5:1 (50/10 and 25/5 pM) (Figure 17b). The increase from 2:1 to 5:1 allowed the reaction to reach full conversion within 1.5 h (red line) instead of 2.5 h (black line). Halving the copper concentration to 25 pM while keeping the Cu/GSH ratio (blue line) decreased the reaction rate slightly but increased the final conversion when compared to 50 pM Cu(OAc) ₂ (red line). This indicates less oligonucleotide degradation when only 25 pM copper was used. Furthermore, the product ratios obtained with the Cu/GSH system were consistently higher than the Cu/NaAsc system (~95%+ vs -90%). It can be concluded that the advantage of the Cu/GSH system is less oligonucleotide degradation while the Cu/NaAsc gives faster reaction rates at the cost of oligonucleotide degradation.

General Protocol for Oligonucleotide Synthesis:

ODN-1 and ODN-3 were synthesized using standard solid phase oligonucleotide synthesis protocols on an ABI 392 synthesizer. Phosphoramidites and CPG supports loaded with standard nucleosides were purchased from LINK Technologies Ltd (Bellshill, UK). For the modified phosphoramidite 2-cyanoethyl (6-(1 -((triisopropylsilyl)ethynyl)-1 H- benzo[cf]imidazol-6-yl)hexyl) diisopropylphosphoramidite (compound 17, scheme 2) a longer coupling time of 5 min was used.

After the solid phase synthesis was finished the TIPS protecting group was removed on solid support. TBAF (50 pL, 1 M in THF) was mixed with 1.95 mL of acetonitrile and this mixture was taken up in a plastic syringe. The mixture was repeatedly passed over the column containing the CPG support for 2 min. The CPG support was washed with MeCN (3 x 5 mL) and dried with air.

Ammonia (DNA grade, 1 .5 ml/mmol) was added, and the suspension was shaken for 16 h at room temperature. The supernatant was removed, and the CPG support was then washed with water (2 x 1 .5 mL). The combined aqueous phase was concentrated under reduced pressure to obtain crude ODN-2 as a white solid. Oligonucleotides were purified by reverse-phase HPLC on a Dionex UltiMate 3000 System using a Phenomenex Clarity Oligo-RP column (250 x 10 mm, 5 pm, 5 mL/min, A = 0.1 M TEAA in water (pH 7), B = 0.1 M TEAA, 80% MeCN in water (pH 7)), lyophilised and then desalted using a NAP-25 Sephadex column.

1 -((2F?,4S,5F?)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methy l)-4- hydroxytetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1 /-/,3/-/)-dione

5'-lodo-2'-deoxyuridine (2.0 g, 5.6 mmol, 1 equiv.) was dissolved in anhydrous pyridine (56 ml). NEt ₃ (1.6 mL, 1 1.3 mmol, 2 equiv.) and DMTrCI (2.0 g, 5.9 mmol, 1.1 equiv.) were added and the reaction mixture stirred for 24 h at room temperature. The mixture was quenched with water (20 mL) and the organic phase was extracted with CHCI ₃ (3 x 100 mL). The combined organic phase was dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, DCM/MeOH, 1/0 to 96/4) to provide the desired product as a white solid (3.1 g, 83 %). ¹ H-NMR (CDCI ₃ , 500 MHz): d 8.13 (s, 1 H), 7.42-7.41 (m, 2H), 7.34-7.29 (m, 6H), 7.24- 7.22 (m, 1 H), 6.85 (d, J = 8.8 Hz, 4H), 6.31 (t, J = 6.6 Hz, 1 H), 4.55-4.53 (m, 1 H), 4.08 (br. s, 1 H), 3.79 (s, 6H), 3.43-3.36 (m, 2H), 2.50-2.48 (m, 1 H), 2.29 (app. quint, J= 6.7 Hz, 1 H).

¹³ C-NMR (CDCI ₃ , 125 MHz): 5 158.9, 144.4, 135.6, 135.5, 130.3, 130.2, 128.3, 128.2, 127.3, 1 13.6, 87.3, 86.6, 85.8, 72.7, 63.6, 55.4, 41.6. Two signals not observed/coincident.

7-(1 -((2F?,4S,5/ ^: ?)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxytetrahydrofuran-2-yl)-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-5-yl)hept-6-ynoic acid

Pd(PPh ₃ )2CI ₂ (0.1 g, 0.2 mmol, 0.09 equiv.), Cui (0.09 g, 0.3 mmol, 0.16 equiv.) and 1 - ((2fi,4S,5fi)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4- hydroxytetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1 /-/,3/-/)-dione (1.3 g, 2.0 mmol, 1 equiv.) were placed in a dried flask filled with argon. Anhydrous DMF (22 mL) was added followed by heptynoic acid (2.6 g, 11 .0 mmol, 5.1 equiv.). The mixture was degassed by freeze-pump-thaw cycles. NEt ₃ (0.8 mL, 5.8 mmol, 2.9 equiv.) was added and the solution stirred at room temperature for 3 h. The reaction mixture was quenched with water (20 mL) and the organic phase was extracted with EtOAc (3 x 100 mL). The combined organic phase was washed with 10% aq. solution of LiCI (2 x 25 mL), aq. solution of EDTA (10 mg/mL, 25 mL) and brine (25 mL), dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, CHCI ₃ /MeOH, 98/2 to 94/6) to provide the desired product as a brown solid (1 .0 g, 73%).

¹ H-NMR (CDCI ₃ , 500 MHz): d 8.00 (s, 1 H), 7.42 (d, J= 7.6 Hz, 2H), 7.33 (dd, J= 8.7, 1 .6 Hz, 4H), 7.29 (t, = 7.4 Hz, 2H), 7.23-7.20 (m, 1 H), 6.85-6.83 (m, 4H), 6.30 (t, J = 6.6 Hz, 1 H), 4.52-4.49 (m, 1 H), 4.06 (q, J = 3.2 Hz, 1 H), 3.79 (s, 6H), 3.42 (dd, J = 10.8, 3.2 Hz, 1 H), 3.32 (dd, J = 10.7, 3.7 Hz, 1 H), 2.49-2.44 (m, 1 H), 2.40-2.33 (m, 1 H), 2.31-

2.25 (m, 1 H), 2.21 (t, J = 7.4 Hz, 2H), 2.11 (t, J = 7.1 Hz, 2H), 1.60-1.54 (m, 3H), 1.33-

1.26 (m, 3H).

¹³ C-NMR (CDCI ₃ , 125 MHz): 5 177.4, 162.4, 158.8, 149.3, 144.6, 141.9, 135.7, 130.1 , 129.3, 128.2, 128.1 , 127.1 , 113.5, 101.0, 94.9, 87.2, 86.5, 85.6, 72.4, 71.3, 63.6, 55.4, 41.5, 33.3, 27.5, 24.1 , 19.3, 8.7.

7-(1 -((2/T4S,5fi)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl )-4- hydroxytetrahydrofuran-2-yl)-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-5-yl)-/V-(1 - ((triisopropylsilyl)ethynyl)-1 -/-benzo[c/]imidazol-6-yl)hept-6-ynamide

7-(1 -((2/ ^: ?,4S,5/ ^: ?)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxytetrahydro-furan-2-yl)-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-5-yl)hept-6-ynoic acid (0.5 g, 0.8 mmol, 1 equiv.) and 1 -((triisopropylsilyl)ethynyl)- 1 H-benzo[c/]imidazol-6- amine (0.4 g, 1.1 mmol, 1.5 equiv.) were dissolved in dry DMF (6 mL). EDC (0.2 g, 1.5 mmol, 2 equiv.), HOAt (0.2 g, 1.5 mmol, 2 equiv.) and DIPEA (0.3 mL, 1.7 mmol, 2.1 equiv.) were added and stirred under argon atmosphere for 16 h at room temperature. The reaction mixture was diluted with EtOAc (100 mL), washed with 10% aq. solution of LiCI (2 x 15 mL) and brine (15 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, CHCI ₃ /MeOH, 98/2 to 94/6) to provide the desired product as a brown solid (0.7 g, 95%). ¹ H-NMR (CDCI ₃ , 500 MHz): d 8.55 (br. s, 1 H), 8.33 (br. s, 1 H), 8.08 (s, 1 H), 8.01 (s, 1 H), 7.71 (d, J = 8.6 Hz, 1 H), 7.43 (d, J = 7.5 Hz, 2H), 7.36-7.34 (m, 5H), 7.29 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1 H), 6.85-6.83 (m, 4H), 6.39 (br. s, 1 H), 4.61 (br. s, 1 H), 4.12 (br. s, 1 H), 3.79-3.78 (m, 6H), 3.41-3.36 (m, 2H), 2.70 (br. s, 1 H), 2.53-2.50 (m, 1 H), 2.46 (t, J = 7.9 Hz, 2H), 2.34-2.28 (m, 1 H), 2.19-2.1 1 (m, 2H), 1 .88-1 .82 (m, 3H), 1 .49- 1.42 (m, 3H), 1.16-1.15 (m, 21 H).

¹³ C-NMR (CDCI ₃ , 125 MHz): 5 172.1 , 162.5, 158.6, 150.0, 144.5, 143.5, 141.7, 137.4, 136.6, 135.7, 135.5, 134.8, 130.0, 129.9, 128.0, 127.9, 127.0, 120.2, 1 17.4, 1 13.3, 102.6,

101.1 , 94.9, 89.9, 87.1 , 86.7, 85.7, 77.2, 73.4, 72.4, 72.0, 63.7, 55.3, 41.8, 37.2, 26.1 ,

25.1 , 18.7, 11.2.

HRMS (ESI): CssHesNsOsSi [M+Na] ⁺ calculated 972.4338, found 972.4335.

(2R,3S, 5fi)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2,4- dioxo-5-(7- oxo- 7-((1 -((triisopropylsilyl)ethynyl)- 1 /-/-benzo[c/]imidazol-6-yl)amino)hept-1 -yn-1 -yl)-3 ,4- dihydropyrimidin-1 (2/-/)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

A/,A/-Diisopropylethylamine (0.4 mL, 2.4 mmol, 6 equiv.) and 2-cyanoethyl diisopropylchlorophosphoramidite (0.1 mL, 0.5 mmol, 1.2 equiv.) were added to a solution of 7-(1 -((2fi,4S,5fi)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methy l)-4- hydroxytetrahydro-furan-2-yl)-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-5-yl)-/V-(1 - ((triisopropylsilyl)ethynyl)- 1 H-benzo[c]imidazol-6-yl)hept-6-ynamide (0.4 g, 0.4 mmol, 1 equiv.) in THF (2.7 mL). The reaction mixture was stirred at room temperature for 1 .5 h, and then partitioned between CHCI ₃ (50 mL) and aq. sat. solution of NaHCO ₃ (10 mL). The organic layer was washed with brine (2 x 50 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (silica gel, n-hexane/EtOAc, 1/1 to 0/1 +2% NEt ₃ ) to provide the desired product as a white solid (0.2 g, 47%).

¹ H-NMR (CDC , 500 MHz): d 8.32-8.30 (m, 1 H), 8.22 (s, 1 H), 8.12-8.07 (m, 1 H), 8.01 (s, 1 H), 7.67 (d, J = 7.8 Hz, 1 H), 7.43 (d, J = 7.6 Hz, 2H), 7.35-7.33 (m, 4H), 7.30-7.27 (m, 3H), 7.24-7.20 (m, 1 H), 6.86-6.83 (m, 4H), 6.32-6.27 (m, 1 H), 4.64-4.62 (m, 1 H), 4.2M.16 (m, 1 H), 3.85-3.83 (m, 1 H), 3.79-3.78 (m, 6H), 3.75-3.73 (m, 1 H), 3.60-3.54 (m, 2H), 3.47-3.39 (m, 1 H), 3.32-3.30 (m, 1 H), 2.62 (t, J = 6.2 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.33 (app. quint, J = 6.8 Hz, 1 H), 2.11 (t, J = 6.8 Hz, 2H), 1.79 (t, J = 7.3 Hz, 2H), 1.26-1.24 (m, 2H), 1.18- 1.16 (m, 28H), 1.78 (d, J = 6.8 Hz, 5H).

¹³ C-NMR (CDCI _3I 125 MHz): 5 162.3, 158.7, 149.1 , 144.5, 143.9, 142.0, 136.3, 135.8, 135.6, 135.1 , 130.2, 130.1 , 128.2, 128.1 , 127.1 , 120.7, 117.6, 1 17.0, 1 13.5, 102.7, 100.9, 95.3, 90.2, 87.2, 85.8, 73.8, 71.7, 63.3, 58.5, 58.4, 55.4, 43.5, 43.4, 40.9, 37.1 , 32.1 , 31.6, 30.5, 30.3, 29.8, 26.5, 25.3, 24.8, 24.7, 24.6, 22.8, 20.6, 20.5, 19.2, 18.8, 1 1.4. ³¹ P-NMR (CDCI3, 202 MHz): 5 149.1 , 148.7.

HRMS (ESI): Ce^soNyOgPSi [M+H] ⁺ calculated 1150.5597, found 1150.5598.

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