GB2598300A - Cross-linking method and applications in bioconjugation - Google Patents

Abstract

The invention relates to a vinyl disulfone compound of general formula I: I wherein X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Y is a fluorophore or biomolecule; R1 is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or R1 corresponds to X-Y as defined previously; R2 is a C1-C3 alkyl; and R3 and R4 are independently selected from H, an alkyl group or an aryl group. Preferably, R1 together with Y forms part of a cell-killing fluorophore, R2 is methyl, R3 and R4 are both H. The vinyl group can act as a Michael-like acceptor to react with a fluorophore or biomolecule comprising a nucleophilic group. Preferably the biomolecule is an oligopeptide which reacts with the vinyl group via the sulfur atom in a cysteine side chain of said oligopeptide. The invention also relates to methods of preparing bioconjugates using the compound of formula I, and bioconjugates prepared thereby.

Description

CROSS-LINKING METHOD AND APPLICATIONS IN BIOCONJUGATION

Field of the Invention

The present invention relates to a vinyl disulfone compound, as well as to methods of cross-linking in bioconjugation chemistry using the vinyl disulfone compound. In aspects, the invention relates to bioconjugates prepared by the methods of the invention.

Background of the Invention

Bioconjugation allows unique molecular systems to be prepared by linking one or more component biological molecules to other biological molecules or targeting or imaging agents to combine their characteristics. These resulting bioconjugates are useful in diverse areas such as targeting, imaging, detection, biosensing, bio-separation, and for use as probes and therapeutics. The diverse nature of biomolecules often requires comparatively diverse chemical processes to cross-link the biomolecules, resulting in increased complexity.

Current methodologies used to link biological molecules together suffer from a number of problems, including poor reactivity, poor stability, and/or a narrow range of functional group compatibility. Known methodologies also often require the use of Volatile Organic Compounds (VOCs), which are disadvantageous from an environmental perspective.

It is an aim of the invention to obviate or mitigate one or more of the disadvantages

associated with prior art methods.

Summary

In an aspect of the invention there is provided a vinyl disulfone compound of formula I In which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Visa fluorophore or biomolecule; 1:2" is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or 11" corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; and R3 and R4 are independently selected from H, an alkyl group or an aryl group.

The compound of formula I comprises a fluorophore or biomolecule linked to a vinyl sulfonyl species which can act as a Michael-like acceptor to react with a fluorophore or biomolecule comprising a nucleophilic group. This allows for versatile cross-linking of fluorophores and biomolecules to other entities, for example other fluorophores or biomolecules, to form bioconjugates for use in a host of applications, including imaging and therapeutic methods.

In an aspect of the invention there is provided a method of preparing a bioconjugate comprising: providing a compound of formula I: in which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Y is a fluorophore or biomolecule; and R1 is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or 131-corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; R3 and re are independently selected from H, an alkyl group or an aryl group; and cross-linking the compound of formula I with a fluorophore or biomolecule comprising a nucleophilic group, to form a bioconjugate.

The bioconjugate may have the general formula IV:

IV

In which X, V, R1 and R2 are as defined previously, and Nu is a nucleophilic group. The nucleophilic group may be attached either directly or via a spacer group to a fluorophore or biomolecule.

The bioconjugates may be used in diverse areas such as imaging, detection and therapy.

Further aspects and embodiments of the invention are as defined in the claims, and described in more detail below.

Brief Description of the Drawings

Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which: Figure 1 is a schematic illustration of a method of preparing a bioconjugate in accordance with embodiments of the invention; Figure 2 illustrates the preparation of a fluorophore-biomolecule bioconjugate in accordance with embodiments of the invention; Figure 3 illustrates the preparation of a biotargeting agent-fluorophore bioconjugate in accordance with embodiments of the invention; Figure 4A illustrates the preparation of compounds 4d, 4e and 41; Figure 4B of compounds 9d, 9e and 91 and Figure 4C of compounds 14a, 14b and 14c; and Figure 5 shows LCMS/MS data for M2' ion and fragmentation for the bioconjugate prepared in Example 1.5.2.

Detailed Description of the Invention

The invention relates to a vinyl disulfone compound of formula I; in which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Visa fluorophore or biomolecule; 1:1" is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or R' corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; 113 and 114 are independently selected from H, an alkyl group or an aryl group.

Throughout this application the term "biomolecule" is intended to refer to any molecule produced by a living organism, and also expressly includes synthetic molecules with a biological application. Examples of "biomolecules" include therapeutic molecules and biotargeting agents, such as, for instance, antibodies, affimers, aptamers, peptides and small molecule drugs.

A "fluorophore" is an organic molecule which can absorb light at a particular wavelength and re-emit light at a different, typically longer wavelength, upon excitation. Fluorophores are often characterised by the presence of several combined aromatic groups, or planar or cyclic molecules with several n bonds. Fluorophores would be known to a person skilled in the art and in the context of the present invention, the fluorophore may be of diagnostic, analytical, prognostic or therapeutic interest. Examples of fluorophores include naturally-occurring or endogenous fluorophores such as porphyrins, aromatic amino acids, Nicotine Adenine Dinucleotide (NADH), flavins, and derivatives of pyridoxyl, and exogenous fluorophores such as fluorescent dyes, derivatives of fluorescein, rhodamine, coumarins and cyanines.

As used herein, the term "alkyl" refers to a fully saturated, branched, unbranched or cyclic hydrocarbon moiety, i.e. primary, secondary, or tertiary alkyl or, where appropriate, cycloalkyl or alkyl substituted by cycloalkyl. Where not otherwise indicated, an alkyl group comprises 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, or more preferably 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl and n-decyl.

As used herein the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system consisting only of hydrogen and carbon and, where not otherwise indicated, containing from 6 to 19 carbon atoms, preferably from 6 to 10 carbon atoms, wherein the ring system may be partially saturated. Aryl groups include, but are not limited to, groups such as fluorophenyl, phenyl, indenyl and naphthyl. The term "aryl" includes aryl radicals optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, cyano, nitro, amino, amidine, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl.

According to the invention, the compound of formula I comprises a biomolecule or fluorophore, and a vinyl sulfonyl moiety which allows the compound to be linked to another fluorophore or biomolecule, facilitating cross-linking. The incoming fluorophore or biomolecule may be of the same type as Y, i.e. where a fluorophore is being linked to another fluorophore, or may be of a different type, i.e. where a biomolecule is being linked to a fluorophore or vice versa.

X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms. This moiety can act as a spacer between the nitrogen atom and the fluorophore or biomolecule, Y. In an embodiment, X comprises from 1 to 12 carbon atoms. X may be a hydrocarbon moiety or may comprise one or more heteroatoms such as oxygen or nitrogen atoms. In an embodiment, X comprises one or more oxygen atoms.

In an embodiment, X is an ether or polyether moiety of formula: [(CH2)n-O]ni-(CH2)p in which n is an integer between 1 and 10, preferably between 2 and 4; m is an integer between 1 and 10, preferably between 1 and 6; and p is an integer between 0 and 10, preferably between 0 and 6.

X, when present, can be used to tune the physical properties of the compound of formula I, such as for instance, solubility properties of the compound.

In an embodiment, Xis absent.

In an embodiment, Y is a fluorophore. The fluorophore may be selected from porphyrins, aromatic amino acids, Nicotine Adenine Dinucleotide (NADH), flavins, derivatives of pyridoxyl, fluorescent dyes, derivatives of fluorescein, rhodamine, coumarins and cyanines.

In an embodiment, Y is a biomolecule. The biomolecule may be selected from antibodies, affimers, aptamers, peptides, and small molecule drugs.

In an embodiment, R1 is an organic moiety comprising from 1 to 16 carbon atoms. In this embodiment, R1 can be, for example, a hydrocarbon moiety or may comprise one or more heteroatoms such as oxygen or nitrogen atoms. In an embodiment, R1 is an alkyl group comprising from 1 to 16 carbon atoms, preferably from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. In an alternative embodiment, R1 can comprise one or more heteroatoms, such as oxygen atoms. R1 can be, for example, an ether or polyether moiety such as that defined in the context of X above. In some embodiments 111 can be used to tune properties of the compound, such as solubility properties.

In an embodiment, 111 is part of the fluorophore or biomolecule. In this embodiment, the fluorophore or biomolecule forms two bonds with the nitrogen atom.

In an embodiment, R1 corresponds to X-Y. In this embodiment, the amine can be attached to two fluorophores or biomolecules, which may be the same or different. This can be useful, for example, to increase the amount of fluorophore for use in specific applications such as to deliver greater fluorescence sensitivity/intensity per molecule or in photodynamic therapy (PDT) to delivery more intensity of emitted light to the treatment region. In embodiments, for example, two different fluorophores could be attached to the N atom, and different wavelengths used for both excitation and emission, with one fluorophore being attached for imaging purposes (e.g. to locate a tumour) and another to treat using PDT.

In an embodiment, R2 is a methyl group or an isopropyl group.

In the compound of formula I, 113 and 124 are independently selected from H, an alkyl group or an aryl group. When R3 and/or R4 are an alkyl group, the alkyl group is preferably a Ci-C4 alkyl.

When R3 and Fe are an aryl group, the aryl group is preferably a monocyclic ring system, preferably containing from 6 to 10 carbon atoms, and is preferably, phenyl.

In an aspect of the invention there is provided a method of preparing a bioconjugate comprising: providing a compound of formula I: in which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Y is a fluorophore or biomolecule; R1 is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or R1 corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; R3 and Fe are independently selected from H, an alkyl group, or an aryl group; and cross-linking the compound of formula I with a fluorophore or biomolecule comprising a nucleophilic group, to form a bioconjugate.

Throughout this specification, the term "bioconjugate" is understood to mean a complex formed from at least one "biomolecule", as defined herein. Examples of bioconjugates which can be prepared according to the invention include those in which a fluorophore is conjugated to a biota rgeting agent or vice versa. The bioconjugates may be used in imaging or photodynamic applications, amongst others.

The compound of formula I comprises a fluorophore or biomolecule armed with Michael-like acceptors which can react with another entity of interest, such as a fluorophore or biomolecule which comprises a nucleophilic group, to form a bioconjugate. For example, when Y is a fluorophore, the compound of formula I can be reacted with a biomolecule or biota rgeting function to facilitate selective cell-binding or incorporation into the cell for use in imaging applications. Examples of suitable biomolecules or biotargeting function include amino acids, peptides, proteins, antibodies, small molecule drugs etc. In the cross-linking step, the vinyl sulfonyl groups of the compound of formula I react with the nucleophilic group of the incoming fluorophore or biomolecule, e.g. in a Michael-type reaction, resulting in the formation of a chemical bond between the carbon atom of the vinyl sulfonyl group and a residue of the nucleophile, as represented by: In an embodiment, the nucleophilic group is a -OH, -SH or -NH2 group. In an embodiment, the nucleophilic group is an -SH or -NH2 group. In an embodiment, the nucleophilic group is an -SH group.

The nucleophilic group may be linked to the incoming fluorophore or biomolecule either directly or via a spacer group. Such a spacer unit may be, for example, an organic moiety comprising from 1 to 16 carbon atoms. The organic moiety may be a hydrocarbon moiety or may comprise one or more heteroatoms such as oxygen or nitrogen atoms. Suitable spacer groups include, for example, alkyl, ether or polyether moieties. In an embodiment, Xis an ether or polyether moiety of formula: [(CH2)n-O]m-(CH2)p in which n is an integer between 1 and 10, preferably between 2 and 4; m is an integer between 1 and 10, preferably between 1 and 6; and p is an integer between 0 and 10, preferably between 0 and 6.

In some embodiments the attachment of the nucleophilic group to the fluorophore or biomolecule via a spacer unit may be preferred, as the spacer group is highly tuneable, and can be manipulated to change physical properties of the bioconjugate such as solubility in different solvents, etc., depending on the intended application for the bioconjugate, as would be known to a person skilled in the art.

In an embodiment, the step of providing the compound of formula I comprises condensing a compound of formula II: In which R4, R2, X and Y are as defined for formula with an aldehyde or ketone of formula: in which R3 and R4 are independently selected from H, an alkyl or an aryl group; to form a mixture comprising the compound of formula I. In an embodiment, at least one of 113 and R4 is H. In an embodiment at least one of R3 and R4 is methyl.

In an embodiment, each of R3 and R4 is H, i.e. the aldehyde is formaldehyde. In an embodiment, R3 is H and R4 is methyl, i.e. the aldehyde is acetaldehyde. In an embodiment, each of R3 and R4 is methyl, i.e. the ketone is acetone. In an embodiment, R3 is H and R4 is phenyl, i.e. the aldehyde is benzaldehyde.

When the compound of formula II is condensed with an aldehyde or ketone, a mixture comprising the compound of formula land a hydrate (v) is formed as shown below: fik Hydrate (v) The compound of formula I can be isolated using known techniques.

The inventors have advantageously determined that formaldehyde is particularly useful in the condensation of the compound of formula II, since it shows high reactivity, enabling the facile formation of the vinyl sulfone, which in turn, shows high reactivity towards trapping nucleophiles for bioconjugation.

The compound of formula II can be formed by reacting a compound of formula III: se in which R2 is a Ca-C3 alkyl; with a fluorophore or biomolecule comprising a primary or secondary amine group, wherein the fluorophore or biomolecule comprising a primary or secondary amine group is represented by the formula -(X)YR1NH, to form the compound of formula II: in which X, Y, R1 and R2 are as defined previously.

In an embodiment, the compound of formula III is formed by dimerization of an a lkanesulfonyl chloride. In this method, the betaine is formed in solution with other species, and is reacted in situ with the primary or secondary amine group of the fluorophore or biomolecule. In an embodiment, the alkanesulfonyl chloride is preferably a Ca-C3 a lkanesulfonyl chloride, preferably methanesulfonylchloride.

In embodiments, the method can therefore comprise the following steps: Reacting a compound of formula III with a fluorophore or biomolecule comprising a primary or secondary amine group, wherein the fluorophore or biomolecule comprising a primary or secondary amine group is represented by the formula -(X)YR1NH, to form a compound of formula (II): condensing the compound of formula II with an aldehyde or ketone to form a mixture comprising a compound of formula (I): and cross-linking the compound of formula I with a fluorophore or biomolecule comprising a nucleophilic group to form a bioconjugate.

The compound of formula I can be isolated from the mixture using known techniques.

The bioconjugate can have the formula IV:

IV

In which Fe, Fe, X and Y are as previously defined, and Nu is a nucleophilic group attached to the molecule of interest, such as a fluorophore or biomolecule, either directly or via a spacer group.

This method is shown schematically in Figure 1.

In Figure 1, the first step is the dimerization reaction of a substituted sulfonyl chloride, in this case methane sulfonyl chloride (1) through the reactive intermediate sulfene 2. A stabilised betaine (3) is formed (amongst other species) which can be trapped by the primary or secondary amine of the fluorophore or biomolecule, to give the methylsulfonylmethylene sulphonamide (4). These reactive methylene compounds condense with aldehydes or ketones to give a mixture of hydroxymethyl adducts 5 and the vinyl sulfonyl compound of formula I (6). The compound of formula I is then cross-linked with a molecule of interest (fluorophore, biomolecule, etc) which comprises a nucleophile. The vinyl sulfonyl groups of the compound of formula I react with the nucleophilic groups resulting in the formation of a chemical bond between the carbon atom of the vinyl sulfonyl group and a residue of the nucleophile to form the bioconjugate, shown as (7) . In an embodiment of the invention, Y is a fluorophore and the compound of formula I is cross-linked with a biomolecule. This embodiment is illustrated schematically in Figure 2, using the exemplary cell-killing fluorophore shown below: The secondary amine group of the fluorophore reacts with the betaine moiety to form the sulphonyl sulphonamide (shown as 4), which is condensed with formaldehyde to give a mixture of hydroxymethyl adducts and the eliminated vinyl sulfonyl species. These two compounds exist in equilibrium in the presence, or otherwise, of water. The vinyl sulfonyl species reacts with a biomolecule with a nucleophilic function such as -OH, -SH or NH2, to give the fluorophore-biomolecule bioconjugate.

In an alternative embodiment, Y is a biotargeting agent and the compound of formula I is cross-linked with a fluorophore. This embodiment is illustrated schematically in Figure 3.

This embodiment can be particularly useful when the biotargeting agent is a small molecule drug, or peptide for example that can withstand the subsequent sulfonylation conditions.

The invention will now be described by way of reference to the following examples, which are intended to be illustrative only.

Examples:

General methodology: All reactions were performed under air unless otherwise specified.

Deuterated chloroform (CDCI3) was used as solvent for routine NMR measurements. 11-1 NMR spectra were recorded on a Varian-Mercury 400 MHz spectrometer, operating at ambient probe temperature unless specified elsewhere. Coupling constants (1) are given in Hz, and the multiplicity of the NMR signals is described as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). 1-3C NMR spectra were recorded on Varian BrDker Avance 400 MHz. 1+1 NMR and '3C NMR chemical shifts are reported in ppm (6) relative to tetramethylsilane, references to the chemical shifts of residual solvent resonances.

The purification of the reaction crudes was performed using silica gel medium pressure column chromatography, which was carried out using different supports; Silica gel as supplied from Sigma-Aldrich (230-400 mesh, 40-63 pm, 60 A); Merck® aluminium TLC plate, silica gel coated with fluorescent indicator F254s (specific surface area 480 -540 m2/g) were used. In all cases the TLC plates visualisation was aided by dipping plates into an alkaline potassium permanganate solution.

Mass spectra were obtained using an LCT Premier XE mass spectrometer and an Acquity UPLC (Waters Ltd, UK) (low resolution ASAP, atmospheric pressure solids analysis probe ionisation) unless stated elsewhere. Accurate mass spectrometry was obtained on an LCT Premier XE mass spectrometer and an Acquity UPLC (Waters Ltd, UK) using the atmospheric pressure solids analysis probe ionisation ion mode, unless otherwise stated. IR spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer.

Elemental analysis was carried out on an Exeter CE-440 Elemental Analyser. Example 1.1: Preparation of sulfonyl sulfonamides 1.1.1 General procedure for 4d, 4e A solution of triethylamine (Et3N) (2.70 mL, 19.5 mmol) in dichloromethane (DCM) dry (10.0 mL) was cooled to -40°C and methanesulfonyl chloride (also called "mesyl chloride") (1.0 mL, 13.0 mmol) was added using a syringe pump (rate 2.50 mL/h). Afterwards the amine (6.5 mmol) was added to the pale yellow suspension. After addition, the suspension was left to warm to room temperature. The suspension was diluted with DCM (100 mL) and washed with 5% aqueous HCL(3 x 10 ml), NaHCO3 (3 x 10 ml) and brine (3 x 10 m1). The organic phase was separated, dried with magnesium sulfate (MgSO4) and concentrated under reduced pressure.

1.1.1.1: N,N-diethyl-1-methanesulfonylmethanesulfonamide, 4d Diethylamine was used as starting material (0.47 g, 6.50 mmol). Compound 4d was obtained as a pale-yellow solid which was crystallized in Et0H to give an off-white solid (1.16 g, 78%): m.p. 138.3 -139.8 °C; IR (neat) umax (inter alia) 2978 (w, CH), 2921 (w, CH), 1304 (s, 502), 1148 (m, S02), 1109 (m, CN); 1H NMR (400 MHz, Chloroform-d) 64.38 (s, 2H), 3.39 (q, J = 7.2 Hz, 4H), 3.22 (s, 3H), 1.25 (t, J= 7.2 Hz, 6H); 13C NMR (101 MHz, Chloroform-d) 669.6, 42.7, 42.0, 14.3; LRMS (ASAP) m/z [M+H]. 230.043 (100%); Anal. Calcd. (%) C, 31.43; H, 6.59; N, 6.11; Found (%) C, 31.13; H, 6.56; N, 5.97.

The preparation of N,N-diethyl-1-methanesulfonylmethanesulfonamide is shown in Figure 4A.

1.1.1.2: N-Benzv1-1-methylsulfonylmethanesulfonamide, 4e Benzylamine was used as starting material (0.70g, 6.50 mmol). Compound 4e was obtained as a pale yellow solid which was recrystallised in Et0H to give an off-white solid (1.06g. 62%): m.p. 145.9 -147.0 °C; IR (neat) umax (inter alia) 3273 (m, NH), 2982(m, CH), 1305 (s, -502), 1150 (s, -S02), 855 (m, -NH) cm-1;1H NMR (400 MHz, Chloroform-d) 67.43-7.33 (m, 5H), 5.50 (s, 1H), 4.36 (d, J = 6.4 Hz, 2H), 4.20 (s, 2H), 3.20 (s, 3H) (addition of D20 caused the peak at 5 5.20 ppm to disappear and that at 6 4.36 ppm to simplify to a s, 2H); 13C NMR (100 MHz, Chloroform-d) 5 134.9, 128.9, 128.4, 128.2, 68.3, 47.8, 41.9; LRMS (ASAP+) m/z [m+Fi] 264.036 (3.31%), [2M+H] 527.060 (100%); HRMS (ASAP+) m/z calculated C91-114N04S2 [M+H] 264.0364, found 264.0356; Anal. Calcd. (%) C, 41.04; H, 4.98; N, 5.32; Found (%) C, 40.83; H, 4.91; N, 5.27.

The preparation of N-benzy1-1-methylsulfonylmethanesulfonamide, 4e is shown in Figure 4A. 1.1.2: General procedure for 4f A solution of trimethylamine (Me3N) 13% in acetonitrile (CH3CN) (4.28 ml, 6.18 mmol) was cooled to -40 °C and methanesulfonyl chloride (0.16 mL, 2.06 mmol) was added dropwise using a syringe pump (rate 2.50 mL/h). Compound 87 (0.40 g (1.03 mmol) was added and left to react at -40 °C for 15 minutes, and then left to warm up to room temperature. The suspension was then concentrated under reduced pressure and diluted in ethyl acetate (Et0Ac) or DCM for workup. The organic phase was washed with 5% aqueous hydrochloric acid (HCI) (3 x 10 ml), NaHCO3 (3 x 10 ml) and brine (3 x 10 ml), before the organic phase was separated, dried with magnesium sulfate (MgSO4) and concentrated under reduced pressure.

N HN,

Compound 87 Compound 4f was obtained as a pure yellow solid (0.428 g, 76%): m.p. 229-234 °C; IR (neat) I) max (inter alia) 2977, 1314, 1164, 951, 825, 767, 525, 467 cm-'; 1H NMR (600 MHz, DM50d6) 67.72 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 15.9 Hz, 1H), 7.47 (dd, J = 51.3, 8.6 Hz, 4H), 7.01 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 16.0 Hz, 1H), 5.33 (s, 2H), 3.37 (d, J = 6.2 Hz, 2H), 3.19 (s, 3H), 1.49 (s, 9H). '3C NMR (150 MHz, DMSO-d6) 5 165.9, 150.7, 143.1, 134.2, 133.0, 131.9, 128.9, 125.1, 121.0, 115.7, 112.0, 92.8, 88.2, 80.5, 67.3, 47.6, 45.4, 42.6, 28.3. LRMS (ASAP+) m/z [M+H] 545.2 (37%), 389.2 (100%); HRMS (ASAP+) m/z calculated C27H32N20652 [M+H] 554.1701, found 554.1702.

The preparation of compound 4f is shown in Figure 4A. Example 1.2: Preparation of reference vinyl sulfonamides 1.2.1: Compound 9d To a solution of formaldehyde 37% in water, formic acid (95%) was added until pH 4 was reached and this solution was used in the following reaction.

The acidified solution (3.60 mmol, 0.29 mL) prepared above was added to methanol (Me0H, 3.2 mL) at 0 °C, followed by piperidine (6.00 mmol, 0.59 mL). The sulfonyl sulfonamide, 4d prepared in Example 1.2.1 (1.20 mmol, 0.27 g) was slowly added and the reaction was followed by TLC. Cold water (2.0 mL) was added to the reaction mixture, which was left to stir for 10 minutes. The precipitate was filtered and washed with cold water to obtain a solid which was dissolved in DCM, (1 mL) and 1 M hydrochloric acid aqueous solution (1.80 mL). The reaction mixture was left to stir vigorously for 3 hours. Additional aqueous 1 M HCI (1.0 ml) was added to the mixture before separating the organic layer. The aqueous layer was extracted with DCM, the organic extracts were combined and dried over MgSO4 and the solvent evaporated under reduced pressure to give the vinyl sulfonamide.

Compound 9d was obtained as a white solid (0.27 g, 84%): IR (neat) un,a" (inter alio) 2983 (w, CH), 1310 (s, S02), 1134 (s, S02), 520; 11-1 NMR (400 MHz, Chloroform-d) 6 6.94 (dd, J = 7.2, 1.1 Hz, 2H), 3.36 (q, J = 7.2 Hz, 4H), 3.24 (s, 3H), 1.23 (t, J = 7.2 Hz, 6H); 13C NMR (101 MHz, Chloroform-d) 6 150.76, 136.8, 43.8, 43.0, 14.5. LRMS (ASAP+) m/z [M+H]. 242.046 (100%); Anal. Ca lcd (%) C, 34.84; H, 6.27; N, 5.8. Found (%) C, 35.01; H, 6.48; N. 5.83.

The preparation of the compound 9d is shown in Figure 4B. 1.2.2: Compound 9e To a solution of formaldehyde 37% in water, formic acid (95%) was added until pH 4 was reached and this solution was used in the following reaction.

The acidified solution (3.60 mmol, 0.29 mL) prepared above was added to methanol (Me0H, 3.2 mL) at 0°C, followed by piperidine (6.00 mmol, 0.59 mL). The sulfonyl sulfonamide, 9e prepared in Example 1.2.2 (1.20 mmol, 0.3 g) was slowly added and the reaction was followed by TLC. Cold water (2.0 mL) was added to the reaction mixture, which was left to stir for 10 minutes. The precipitate was filtered and washed with cold water to obtain a solid which was dissolved in (DCM, 1 mL) and 1M hydrochloric acid aqueous solution (1.80 mL). The reaction mixture was left to stir vigorously for 3 hours. Additional aqueous 1 M HCI (1.0 ml) was added to the mixture before separating the organic layer. The aqueous layer was extracted with DCM, the organic extracts were combined and dried over MgSO4 and the solvent evaporated under reduced pressure to give the vinyl sulfonamide.

Compound 9e was obtained as a white solid (0.31 g, 92%): m.p. 197.2 -198.3 °C; IR (neat) u,"a" (inter alio) 3256 (m, NH), 2035 (w, CH), 2929 (w, NH), 1302 (s, S02), 1164(s, S02), 521 (m, NH); 1H NMR (400 MHz, Chloroform-d) 5 7.37 -7.30 (m, 5H), 6.87 (dd, J = 5.5, 1.3 Hz, 2H), 5.30 (br t, 1H), 4.19 (d, J = 6.3 Hz, 2H), 3.25 (s, 3H) (Addition of D20 caused the peak at 5 5.30 ppm to disappear and that at 64.19 ppm to simplify to a s, 2H); 13C N MR (100 MHz, Chloroform-d) 5 148.7, 136.8, 135.1, 128.9, 128.5, 128.5, 48.1, 43.6; LRMS (ASAP') rniz [M+H] 276.0(100%); Anal. Calcd (%) C, 43.62; H, 4.76; N, 5.09. Found (%) C, 43.45; H, 4.85; N, 5.17.

The preparation of compound 9e is shown in Figure 4B.

Example 1.3: Preparation of exemplary vinyl sulfonamides 1.3.1: Compound 9f To a solution of formaldehyde 37% in water, formic acid (95%) was added until pH 4 was reached and this solution was used in the following reaction.

The acidified solution (1.11 mmol, 0.08 mL) prepared above was added to methanol (Me0H, 1.00 mL) at 0 °C, followed by piperidine (1.84 mmol, 0.18 mL). The sulfonyl sulfonamide 4f prepared in Example 1.2.3 (0.37 mmol, 0.2 g) was slowly added and the reaction was followed by TLC. Cold water (2.7 mL) was added to the reaction mixture, which was left to stir for 10 minutes. The yellow precipitate was filtered and washed with extra ice-cold water to obtain a solid which was dissolved in (DCM, 1.5 mL) and 1M hydrochloric acid aqueous solution (3.0 mL). The reaction mixture was left to stir vigorously for 3 hours. Additional aqueous 1 M HCI (1.5 mL) was added to the mixture before separating the organic layer. The aqueous layer was extracted with DCM, the organic extracts were combined and dried over MgSO4 and the solvent evaporated under reduced pressure to give the vinyl sulfonamide compound as a yellow solid (0.156 g, 76%): IR (neat) umax (inter alio) 1316, 1149, 953, 517 cm-1. 1H NMR (700 MHz, Chloroform-d) 5 7.56 (d, J = 15.9 Hz, 1H), 7.48 (q, J= 8.7, 8.6, 7.0 Hz, 4H), 7.43 (d, J=8.7, 2H), 6.98 (dd, J = 79.1, 1.4 Hz, 2H), 6.86 (d, .1= 9.1 Hz, 2H), 6.37 (d, J = 16.0 Hz, 1H), 3.50 (br t, J = 5.3, 4.8 Hz, 4H), 3.33 (br t, J = 5.2, 4.8 Hz, 4H), 3.25 (s, 3H), 1.53 (s, 10H). 13C NMR (176 MHz, Chloroform-d) 5 166.1, 150.2, 149.7, 142.7, 137.8, 134.0, 132.9, 131.7, 127.9, 125.3, 120.6, 116.1, 114.4, 91.7, 88.1, 80.6, 48.7, 45.8, 43.8, 28.2. LCMS (ESI+) [M+H] m/z 557.2; HRMS (ESI+) calculated for C28H33N206S2 m/z [M+H] 557.1780, found 557.1780.

The preparation of compound 9f is shown in Figure 413.

Example 1.4: Cross-linking by Nucleophilic Addition 1.4.1: Nucleophilic addition of methanol ethylene sulfonyl sulfonamide, 14a Compound 9e (0.50mmol, 0.14g) was left stirring in an excess of Me0H (used as a solvent, 0.25 M) and followed by TLC. Once the reaction was completed, Me0H was removed under reduced pressure to give the final pure product. Obtained as a white solid (0.15 g, 100%): M.p. 189.3 -191.9 °C; IR (neat) umax (inter alio) 2933 (w, CH), 2920 (w, NH), 1313 (s, SO2), 1135 (s, SO2), 737 (s, NH), 466; 1H NMR (400 MHz, Chloroform-d) 5 7.40 -7.34 (m, SH), 5.55 (t, J = 6.3 Hz, 1H), 4.39 (dd, J = 14.2, 6.5 Hz, 1H), 4.33 (dd, J = 14.2, 6.2 Hz, 1H), 4.18 (dd, J = 11.5, 3.8 Hz, 1H), 4.13 (dd, J = 11.2, 4.3 Hz, 1H), 3.95 (t, J = 3.9 Hz, 1H), 3.42 (s, 3H), 3.20 (d, J = 0.8 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) 5 148.7, 136.8, 135.1, 129.2, 128.9, 128.5, 128.4, 48.1, 43.6; LRMS (ASAP) m/z [M+H] 308.0628 (20%).

The preparation of 14a is shown in Figure 4C.

1.4.2: Nucleophilic addition of 14b Compound 9e (0.50 mmol, 0.14 g) was added to a solution of THF (0.1 M, 5 mL) and benzyl thiol (0.50mmol, 0.062 g). The reaction was followed by TLC until completion. Once the reaction was completed, THE was removed under reduced pressure to give the final product. Compound 14b was obtained as a crude (0.20 g) and purified by silica gel chromatography (Et0Ac/Hex, 1:4) to give a clear liquid (0.18 g, 95%). 1H NMR (700 MHz, Chloroform-d) 5 7.41 -7.37 (m, 2H), 7.36 -7.32 (m, 7H), 7.30 -7.26 (m, 1H), 5.46 (t, J = 6.3 Hz, 1H), 4.34 -4.25 (m, 2H), 3.90 (t, J = 6.1 Hz, 1H), 3.76 (d, J = 13.6, 1H), 3.74 (d, 1=13.6 Hz), 3.26 -3.17 (m, 2H), 3.08 (s, 3H).13C NMR (176 MHz, Chloroform-d) 137.3, 135.9, 129.3, 129.2, 129.0, 128.6, 128.5, 127.8, 79.9, 48.2, 41.5, 37.7, 26.7.

LRMS (ESI+) m/z [M+H] 400.223 (100%). HRMS (ASAP+): m/z calculated Ci7H22NO4S3 [M+H] 400.0707, found 366.0711.

The preparation of 14b is shown in Figure 4C.

1.4.3: Nucleophilic addition of Compound 14c Compound 9d (0.50 mmol, 0.12 g) was added to a solution of THE (0.1 M, 5 mL) and benzyl thiol (0.50mmol, 0.062 g). The reaction was followed by TLC until completion. Once the reaction was completed, THF was removed under reduced pressure to give the final product. Compound 14c was obtained as a crude (0.19 g) and purified by silica gel chromatography (Et0Ac/Hex, 1:4) to give a clear liquid (0.16 g, 90%). NMR (700 MHz, Chloroform-d) 5 7.38 -7.31 (m, 4H), 7.28 -7.26 (m, 1H), 4.10 (dd, = 6.8, 4.5 Hz, 1H), 3.83 (s, 2H), 3.35 -3.24 (m, 2H), 3.16 (dd, Jr 15.0, 6.6 Hz, 1H), 3.14 (d, Jr 0.7 Hz, 3H), 1.20 (t, J = 7.2 Hz, 6H). '3C NMR (176 MHz, Chloroform-d) 5 137.6, 129.3, 128.8, 127.6, 81.5, 43.3, 41.6, 37.9, 27.4, 14.9. LRMS (ESI+) m/z [M+H] 366.286 (100%). HRMS (ASAP+): m/z calculated Ci4H24N04S3 [M+H] 366.0863, found 366.0863.

The preparation of 14c is shown in Figure 4C.

Example 1.5: Nucleophilic Addition -exemplary bioconjugation 1.5.1 Thiol trapping with N-acetyl-L-cysteine methyl ester Compound 9f was cross-linked with N-acetyl-L-cysteine methyl ester according to the following procedure. N-acetyl-L-cysteine methyl ester is a biomolecule which is To a solution of 9f (30 mg, 8.98 x 10-2 mmol) in dry THF (1 mL) was added N-acetyl-Lcysteine methyl ester (9 mg, 8.98 x 10-2 mmol) and left it stirring until nucleophilic addition was completed. Solvent was removed under reduced pressure to obtain the crude compound. Compound 13 was obtained as a yellow solid which was purified with silica gel chromatography in DCM/Me0H 9.8:0.2 (15 mg of a 3:2.3 mixture of two diastereoisomers, 50%): IR (neat) un,ax (inter alio) 1519, 1314, 1148, 959, 826, 728, 513. 1H NMR (600 MHz, Chloroform-d, major diastereoisomer) 67.54 (d, J = 15.9 Hz, 1H), 7.50 -7.41 (m, 6H), 6.86 (d, 1= 9.3, 2H), 6.36 (d, J = 16.3, 1H), 6.32 (m, 1H), 4.97 -4.91 (m, 1H), 4.90 - 4.86 (m, 1H), 3.78 (s, 3H), 3.61 (m, 4H), 3.43 (dd, J = 15.5, 3.6 Hz, 1H), 3.32 (m, 4H), 3.28 (s, 3H), 3.21-3.16 (m, 1H), 2.87 (dd, J = 14.5 Hz, 1H), 2.05 (s, 3H), 1.52 (s, 9H); 1H NMR (600 MHz, Chloroform-d, minor diastereoisomer) 67.54 (d, J = 15.9 Hz, 1H), 7.50 -7.41 (m, 6H), 6.86 (d, J = 9.3, 4H), 6.36 (d, 1H), 6.32 (1H), 4.89 (m, 1H), 4.88 (m, 1H), 4.67 (t, 1= 5.5 Hz, 1H), 3.79 (s, 3H), 3.63 (m, 4H), 3.47 (dd, J = 15.1, 5.2 Hz, 1H), 3.33 (m, 4H), 3.25 (s, 3H), 3.21-3.16 (m, 1H), 2.96 (dd, 1= 14.6 Hz, 1H), 2.06 (s, 3H), 1.52 (s, 9H); 13C NMR (151 MHz, Chloroform-d, major diastereoisomer) 5 171.4, 170.4, 166.3, 150.5, 142.8, 134.2, 133.0, 131.9, 128.0, 125.4, 120.8, 116.2, 114.5, 91.8, 88.3, 81.2, 80.8, 53.1, 51.8, 49.3, 46.5, 41.9, 37.3, 28.3, 27.8, 23.3.13C NMR (150 MHz, Chloroform-d, minor diastereoisomer) 5 171.3, 170.4, 166.3, 150.5, 142.8, 134.2, 133.0, 131.9, 128.01, 125.4, 120.8, 116.2, 114.5, 91.8, 88.3, 81.2, 80.8, 53.1, 52.3, 49.3, 46.7, 42.0, 36.5, 28.3, 23.3; LRMS (ESI-) rniz [M-H]-732.3 (100%); HRMS (ESI-) rniz calculated C34H43N309S3 [M-H]-732.2056, found 732.2083.

1.5. 2: 5-(24(4-(44(4-((E)-3-(tert-Butoxy)-3-oxoprop-1-en-1-yl)phenypethynyl) phenyl) piperazin-1-yl)sulfony1)-2-(methylsulfonypethyl) -L-cysteinyl-L-asparaginyl-L-allothreonyl-L-tryptophyl-L-valyl-L-leucyl-L- alloisoleucyl-L-seryl-L-asparagine trapping Compound 9f (543 pM) in THE (176 pi, 95.4 nmol) was added to H2N-Cys-Asn-Thr-Trp-ValLeulle-Ser-Asn-COOH 0.477 p.M in THE (1 mL, 95.4 nmol) a nd stirred at RT for 30 min. Volatile solvents were removed at reduced pressure to product, a pale yellow solid (0.15 mg, 100%). LCMS (QToF) m/z 1605.7 [M-'-H], 804.3 [M+2H]2+ inter alio; HRMS (QToF) calcd. for C74H104N114020S3 m/z [M+H]k 1605.6792, found 1605.6687. LCMS/MS data for M2 ion and fragmentation is shown in Figure 5.

The experimental results exemplify the trapping of peptides and demonstrate the utility and versatility of the cross-linking process of the invention, with a myriad of potential applications in areas such as imaging and photodynamic therapy.

OH 0*

0=5=0 NH, 0 OH 0 0 OH NH2

Claims (10)

1. A vinyl disulfone compound of formula I: in which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Visa fluorophore or biomolecule; and 143 is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or R4 corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; 1:13 and 114 are independently selected from H, an alkyl group or an aryl group.

2. A vinyl disulfone compound according to claim 1, wherein Visa fluorophore.

3. A vinyl disulfone compound according to claim 1 or claim 2, wherein R4 is an alkyl group comprising from 1 to 6 carbon atoms.

4. A vinyl disulfone compound according to any preceding claim, wherein at least one of R3 and R4 is H.

5. A vinyl disulfone compound according to claim 4, wherein R3 and R4 are both H.

6. A method of preparing a bioconjugate comprising: providing a compound of formula I: in which X, which may be present or absent, is an organic moiety comprising from 1 to 16 carbon atoms; Visa fluorophore or biomolecule; and R1 is an organic moiety comprising from 1 to 16 carbon atoms, together with Y forms part of the fluorophore or biomolecule, or IV corresponds to X-Y as defined previously; R2 is a Ci-C3 alkyl; R3 and 114 are independently selected from H, an alkyl group or an aryl group; and cross-linking the compound of formula I with a fluorophore or biomolecule comprising a nucleophilic group, to form a bioconjugate.

7. A method of preparing a bioconjugate according to claim 6, wherein the nucleophilic group is -OH, -SH or -NH2, preferably -SH or -NH2, more preferably -SH.

8. A method of preparing a bioconjugate according to claim 6 or claim 7, wherein the step of providing the compound of formula I comprises condensing a compound of formula II:IIin which R1, R2 X and Y are as defined for formula I; with an aldehyde or ketone of formula: in which Wand re are independently selected from H, an alkyl or an aryl group.

9. A method of preparing a bioconjugate as claimed in claim 8, wherein the compound of formula II is condensed with an aldehyde, which is preferably formaldehyde.

10. A method of preparing a bioconjugate as claimed in claim 8 or claim 9, wherein the compound of formula II is prepared by reacting a compound of formula Ill, in which R2 is a Ca-C3 alkyl; with a fluorophore or biomolecule comprising a primary or secondary amine group, wherein the fluorophore or biomolecule comprising a primary or secondary amine group is represented by the formula -(X)YRiNH to form a compound of formula II:II11. A method of preparing a bioconjugate as claimed in claim 10, wherein the compound of formula Ill is formed by dimerization of an alkanesulfonyl chloride, preferably methanesulfonylchloride.