Fluorogenic probes

Abstract

The invention relates to new turn-on fluorogenic cyanine dimer probes and their use for detecting or visualizing a molecular target in vitro or in vivo. The invention also relates to chemical intermediates useful for preparing the probes. It is possible to obtain such fluorogenic probes by introducing a specific substituent R in a particular position of the Cy5.5 scaffold, namely in place of a methyl group, as shown in the Formula of Cy5.5 scaffold: (A) Cy5.5 scaffold functionalized with R group.

Description

[0001] FLUOROGENIC PROBES

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to novel fluorogenic probes for detection of biological targets in vivo or in vitro, such as G protein-coupled receptors.

[0004] BACKGROUND OF THE INVENTION

[0005] Fluorescence imaging is a powerful tool to study biological processes both in living cells and in vivo.

[0006] G protein-coupled receptors (GPCRs) are the most prominent class of membrane proteins, and have been identified as the prime target of more than 30% of drugs currently on the market. Therefore, understanding their signalling and imaging their distribution in cells and tissues is crucial in the search for new therapeutic approaches.

[0007] The use of GPCR ligands labelled with fluorescent dyes is a common strategy to assess expression levels of receptors on the cell surface.

[0008] However, classical GPCR fluorescent probes suffer from limitations due to their fluorescence both in aqueous media and after binding to their target, resulting in a low signal-to-background ratio during bioimaging and the necessity to perform tedious washing steps to remove the unbound fraction of the probe. Such GPCR fluorescent probes do have limitations and even precluding in vivo imaging.

[0009] Designing new fluorogenic probes able to switch on their fluorescence only when interacting with the selected biological target has become of major interest. Ideally, a fluorescent probe for the in vivo imaging of endogenous GPCRs should meet the following requirements: 1) high affinity and selectivity for its target; 2) absorption and emission in the near-infrared (NIR) region to minimize the light scattering in tissues and to enhance tissue penetration; 3) an ability to “turn on” its fluorescence after binding to the target receptor to ensure a high signal-to-noise ratio.

[0010] The Inventors developed various dimeric dyes based on flat aromatic scaffolds with a tendency to form poorly fluorescent intramolecular H-aggregates in aqueous media and to disassemble in apolar media to recover their fluorescent form, such as probes based on (i) squaraine dye dimers (Karpenko et al. J. Am. Chem. Soc. 2015, 137, 405-412) and (ii) cyanine dye dimers (Esteoulle et al., Chem. Sci. 2020, 11, 6824, International application WO2021 / 228939). Although the squaraine dimer was shown to be a powerful tool for receptor labelling in living cells, it displays absorption and emission in the far-red region which is not optimal for the in vivo imaging. On the other hands, the cyanine dimer probes are characterized by a near-infrared fluorescence and high brightness, which make them more suitable for in vivo imaging. The Inventors described a NIR-fluorogenic probe based on cyanine dimer (Cy5.5) and having oxytocin as ligand. This probe enabled, for the first time, the detection and the visualization of the endogenous oxytocin receptor (OTR), in living mice (Esteoulle et al., supra). However, such probes are not optimal: due to their high hydrophobicity, Cy5.5 dimers are prone to undergo non-specific interactions with hydrophobic domains of circulating plasma proteins such as albumin and with lipid bilayer of cell membranes resulting in some residual background noise in imaging. One option investigated by the Inventors was the use of asymmetric Cyanine C.5.25 dimers (Berthome, Bioconjugate Chem. 2024, 35, 1182-1189). The resulting probe was less hydrophobic, exhibited a high brightness, and was less prone to non-specific interactions. However, its low fluorogenicity and the blue shift of the emission and the excitation spectra as compared to Cy5.5 dimers limits its possible use for in vivo imaging.

[0011] There is thus still a need for improved fluorogenic probes for the in vitro or in vivo imaging of biological targets such as G protein-coupled receptors.

[0012] SUMMARY OF THE INVENTION

[0013] The Invention relates to a cyanine fluorogenic dimer probe for detecting a molecular target, which is represented by the following formula (I):

[0014] [Cy] [Cy]

[0015] [Linker]

[0016] [Ligand]

[0017] (i)

[0018] Wherein

[0019] [Linker] is a chemical entity having at least three branches and comprising up to 300 carbon atoms in its backbone and optionally one or more heteroatoms, wherein one branch is linked to [Ligand] by at least one covalent bond and each of the two other branches is linked to one [Cy] by at least one covalent bond,

[0020] [Ligand] is a ligand to the molecular target,

[0021] each [Cy] is a cyanine group independently selected from: n (Dl) with n is 1, 2 or 3; and

[0022]

[0023] wherein:

[0024] (G2) is of formula (G2a) or (G2b)

[0025]

[0026] Wherein:

[0027] - R is a moiety for modulating, preferably for limiting non-specific interaction of the cyanine fluorogenic dimer probe with cellular membrane and / or with blood plasma proteins, said moiety being preferably in the form of a saturated or unsaturated hydrocarbon chain having at least 3 and up to 50 backbone carbon atoms, preferably up to 30 backbone carbon atoms in length,

[0028] wherein said hydrocarbon chain has a terminal moiety selected from: o H, -OH, -NH2, -NRsRs, C1-C3 hydroxyalkyl, C1-C3 alkoxy, C1-C3 halogenoalkyl, a halogen, -CN, C1-C3 cyanoalkyl, -NO2

[0029] o a cationic group preferably selected from -NR.5R.6R7, and

[0030] o an anionic group preferably selected from a phosphate, a phosphonate, a sulfonate, a carboxylate

[0031] wherein said hydrocarbon chain is optionally substituted by one or more substituents selected from C1-C3 hydroxyalkyl, C1-C3 alkyl, C1-C3 alkoxy and halogen, wherein said hydrocarbon chain is optionally interrupted by one or more connecting groups selected from -NRsCO-, NRs, N+R5R6, -OC(O)-, -NH(CO)O-, NH, S, O, CO, NH(C=O)NH, O(C=O)NH, -O-N=CRs-, -CRs=N-O-, -NH-N=CRs-, CRs=N-NH-, amino acid residues such as alanine, citrulline or valine and / or 3- to 20-, preferably 3- to 10- membered cyclic or heterocyclic groups such as pyrazole or triazole, optionally substituted,

[0032] wherein

[0033] o Each Rs, Re and R7 are independently Ci-Ce alkyl, Ci-Ce hydroxyalkyl or Ci-Ce alkyl alkoxy

[0034] o Each Rs is independently H, Ci-Ce alkyl, Ci-Ce hydroxyalkyl or Ci-Ce alkyl alkoxy

[0035] - Li is a chemical group connecting the [Cy] group to a branch of the [Linker] and Li is an unsaturated or saturated C1-C20 hydrocarbon chain, more preferably a C1-C10 alkylene, optionally substituted by one or more halogens, C1-C3 haloalkyl or C1-C3 alkyl,

[0036] - R2 is H, or a hydrophilic group of formula -(R9)z-(Arl)ziC(=O)R9’ wherein

[0037] R9 is a Ci-Ce alkylene, NR9”, S, or O, wherein R9” is H or C1-C3 alkyl

[0038] z is 0 or 1

[0039] Ari is an aryl group, in particular a Ce-Cis aryl group, preferably a phenyl, or a 6- membered heteroaryl,

[0040] zl is 0 or 1

[0041] R9’ is NR10R11, wherein Rio and R11 are independently an hydrogen, a (Ci-Ce) alkyl, a di(Ci-C5)alkylamino(Ci-C5)alkyl, a Ci-Ce hydroxyalkyl, including Ci-Ce di- or -tri- hydroxyalkyl, (C2-Ce)carboxyalkyl, polyethylene glycol represented by the formula - (CH2CH2O)n-R’; or a polypropylene glycol represented by the formula - (CH2CH2(CH3)O)n-R’, wherein n is an integer from 1 to 40 and R’ is a C1-C12 alkyl group, or, alternatively, Rio and R11 form with the nitrogen to which they are attached an heterocycle (such as piperazine) optionally substituted by a (Ci-C4)alkyl,

[0042] - R3 is H or a (C1-C20) alkyl, preferably a (Ci-Ce) alkyl

[0043] - R1 is of formula (a) -L1a-R12; (b) -L1b-NR13C(=O)-L1cR12 or (c) -L1bC(=O)NR13L1cR12 wherein:

[0044] o Lia is a saturated or unsaturated (C2-C40) hydrocarbon chain, preferably a C2- C20 alkylene, or a poly(C2-Cs oxyalkylene) chain comprising from 2 to 20 monomers, such as a polyethylene or polypropylene chain comprising from 2 to 20 or from 2 to 10 monomers,

[0045] o Lib and Lie are independently a saturated or unsaturated (C2-C20), preferably a (C2-C10) hydrocarbon chain, preferably a C2-C10 alkylene

[0046] o R13 is H, a Ci-Ce alkyl, a C2-C6 hydroxyalkyl, or a C2-C6 alkoxy

[0047] o R12 is -(CH2)PRi6 wherein p is an integer from 0 to 6 and Rie is selected from H, OH, halogen, C1-C3 alkoxy, and SO3H

[0048] X is a counter-anion, preferably a pharmaceutically acceptable counter-anion.

[0049] In some embodiments, the cyanine fluorogenic dimer probe is such that each [Cy] is of formula

[0050]

[0051] wherein n is 1, 2 or 3.

[0052] In some embodiments, Gi is of formula Gia or Gib, and G2 is of formula G2a

[0053] In some embodiments, Gi is Gia and G2 is G2a.

[0054] In some embodiments, the hydrocarbon chain of the R moiety is interrupted by at least one triazole group, optionally substituted.

[0055] In some embodiments of the cyanine fluorogenic dimer probe, R is of formula (ao): -(CH2)o- [G3] -L2-R15 (ao)

[0056] wherein:

[0057] o is an integer from 0 to 6, for instance 1, 2 or 3,

[0058] [G3] is a connecting group preferably selected from -NH(C=O)-, -(C=O)-NH-, S, O, NH, NH(C=O)NH, O(C=O)NH, NH(C=O)O, -O-N=CR8-, -CR8=N-O-, -NH-N=CR8-, CR8=N-NH-, and 3 to 10-membered cyclic or heterocyclic group, preferably [G3] is selected from triazole, pyrazole -O-N=CR8-, CR8=N-O-, -NH-N=CR8-, and -CR8=N- NH- - L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NR8(C=O)-, NR8, -N+RsRe, -OC(=O)-, -NH(C=O)O-, NH, S, O, and (C=O),

[0059] - R15 is selected from the group consisting of H, -OH, -NH2, -NR8Rs, C1-C3 alkoxy, halogen, CF3, -CN, -NRsReR?, a phosphate, a phosphonate, a sulfonate, a nitro, and a carboxylate

[0060] each R8, Rs, Re, and R7 are as defined herein, preferably each R8is H, C1-C3 alkyl or Ci- C3 hydroxyalkyl, and each Rs, Re, and R7 are independently C1-C3 alkyl or C1-C3 hydroxy alkyl.

[0061] In some embodiments, in the formula (ao), [G3] is a triazole group.

[0062] In some embodiments, in the formula (ao):

[0063] - o is 1, and / or

[0064] - [G3] is a triazole, and / or

[0065] - L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NR8(C=O)-, NR8, -N+RsRe, NH, O, and (C=O), and / or

[0066] - Ris is selected from the group consisting of H, -OH, -NH2, -NR8Rs, C1-C3 alkoxy, -NR.5R.6R7, a phosphonate, and a sulfonate,

[0067] each R8, RS, Re and R7 being as herein.

[0068] In some embodiments, R is of formula (ai):

[0069] 0

[0070]

[0071] o, L2and R15 being as defined herein.

[0072] In some embodiments, R is selected from the group consisting of:

[0073] N=N(a)wherein t is an integer from 2 to 10, preferably from 3 to 8 and • - R19 is H, -PO3H-, SO3-, -N(Me)3+, -N(Et)3+, -N(Me)2, -N(Et)2, -N(CH2CH2OH)2, -OH, -OMe, -OEt, -NH2, -C(O)NHC(CH2OH)3, -N(Me)2+(CH2)PSO3-, C(O)N[(CH2)pCH3][(CH2)PSO3-], -C(O)N[(CH2)pCH3][(CH2)PPO3H], N(Me)2+(CH2)pPO3H-, with p an integer from 2 to 10,

[0074]

[0075] and w an integer from 0 to 10.

[0076] In some embodiments, the cyanine fluorogenic dimer probe of the invention is such that [Ligand] is a ligand which specifically binds to a membrane protein, such as a membrane receptor, e.g. a G protein-coupled receptors (GPCR), and / or which specific binds to a cell type, e.g. a cancer cell type, said ligand being an endogenous ligand or a synthetic ligand.

[0077] and / or

[0078] [Linker] is a three-branch hydrocarbon group having up to 200, preferably up to 100 carbon atoms in its backbone, said hydrocarbon group comprising one or more unsaturated or saturated C2-C2o hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, - O(C=O)-, -O-N=CR4- -NHN=CR4, and / or amino acid residues such as lysine, citrulline, valine, alanine, and / or saturated or unsaturated, 3- to 20-membered cyclic or heterocyclic groups such as triazole or pyrazole, optionally substituted and / or fused, each R4 being independently H, or a C1-C3 alkyl and said three-branch hydrocarbon group being optionally substituted by one or more groups preferably selected from Ci- Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce aminoalkyl, -OH, -CN, -NH2 and halogen.

[0079] In some embodiments, the cyanine fluorogenic dimer probe of the invention is such that [Ligand] is selected from the group consisting of synthetic small chemical molecules, hormones, vitamins such as acid folic, oligosaccharides such as those comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine (GalNac), bridged GalNac and sialic acid and derivatives thereof (such as Neu5Ac, Neu5Aca2-6Gal, Neu5Aca2-8Neu5Ac), proteins or fragments thereof, peptides such as oxytocin, apelin, RGD peptide, Angiopep-2, or muscle targeting peptides, an aptamer, an antibody including heavy-chain antibody and bispecific antibody, and fragments thereof such as Fab, Fab’, and VHH (also called nanobody), a ScFv, a spiegelmer, and a peptide aptamer.

[0080] In some embodiments, the cyanine fluorogenic dimer probe is of Formula (la)

[0081]

[0082] (Ib) Wherein:

[0083] [Cy] is as defined herein,

[0084] - L3 is a chain comprising up to 70 carbon atoms, and comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, - amino acid residues and / or saturated or unsaturated C3-C6-membered cyclic or heterocyclic group such as triazole or pyrazole,

[0085] - L4is a unsaturated or saturated C2-C30 hydrocarbon chain which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,

[0086] - Each R4is independently H, or a C1-C3 alkyl

[0087] r is an integer from 0 to 3,

[0088] s is an integer from 0 to 6,

[0089] [Ligand] is as defined above,

[0090] In some embodiments, the cyanine fluorogenic dimer probe is such that the [Ligand] is a polypeptide comprising at least one cysteine residue and wherein [Ligand] is linked to the [Linker] by one of the following moi eties:

[0091] O

[0092]

[0093] (4')

[0094]

[0095] atom from the lateral chain of a cysteine residue in [Ligand],

[0096] The invention also relates to a chemical intermediate for the preparation of a cyanine fluorogenic dimer probe as described herein which is of formula:

[0097] [Cyi] [Cyi] [Cyi] [Cyi] [Cy] [Cy]

[0098] \ / \ / X /

[0099]

[0100] [Linker] [Linker] [Linker]

[0101] [Ligand] [CONNECT] [CONNECT]

[0102] (Ila), (Uh) or (lie)

[0103] Wherein:

[0104] [Linker] is as defined herein,

[0105] [Ligand] is as defined herein,

[0106] [Cy] is as defined herein,

[0107] Each [Cyi] is selected from the group consisting of

[0108] n (D1i) with n is 1, 2 or 3; and

[0109]

[0110] Wherein Gi, R2, and R3 are as defined in Claim 1 G2i is of formula

[0111]

[0112] Wherein:

[0113] - Li and X are as defined herein and

[0114] - Ri is a chemical reactive group able to form a covalent bond by click reaction or bioconjugation reaction preferably selected from alkyne-azide cycloaddition / sydnone and iminosydnone-alkyne cycloaddition, tetrazole / alkene reaction, reaction of aldehyde / ketone with alkoxyamine or hydrazine, amidation, reductive amination, diels- alder, and thia-diels-alder and

[0115] [CONNECT] is a reactive group able to create at least one covalent bond by click reaction or a reactive group able to create at least one covalent bond by bioconjugation with an amino acid preferably selected from cysteine, tyrosine and lysine,

[0116] and wherein [Ri] and [CONNECT] are selected so as to be orthogonal when the chemical intermediate is of formula (lib).

[0117] In some embodiments, the chemical intermediate is such that

[0118] - Ri is a chemical reactive group able to form a covalent bond by alkyne-azide cycloaddition, and / or

[0119] [CONNECT] is a reactive group able to create a covalent bond by bioconjugation with an amino acid residue, said reactive group being selected from aryldiazonium, phenyltriazolinedione (PTAD), 3 -arylpropionitrile (APN), diAPN, arylsulfonamide, maleimide, benzoyl acrylamide, iodoacetamide, isothiocyanate, isocyanate, benzoyl fluoride, and an activated carboxylic acid group, e.g. NHS-ester.

[0120] In some embodiments, Ri is -CH2-CNCH.

[0121] In some embodiments, the chemical intermediate is of formula

[0122]

[0123] Wherein:

[0124] - R17 is selected from the group consisting of OH, NH2, an activated carboxylic group, and [CONNECT]-L5- - Ris is selected from the group consisting of OH, NH2, an activated carboxylic group, [LIGAND]-L5- and [CONNECT]-L5-,

[0125] - L4 is a C2-C30 unsaturated or saturated hydrocarbon chain, which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR4(C=0)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,

[0126] - Ls is a hydrocarbon chain comprising up to 70 carbon atoms, and which comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, and / or amino acid residues and / or saturated or unsaturated CL-CL-membered cyclic or heterocyclic group such as triazole or pyrazole - Each R4 is independently H, or a C1-C3 alkyl

[0127] r is 0 or 1

[0128] [CONNECT], [LIGAND], [Cyi] and [Cy] being as defined herein. In some embodiments, the chemical intermediate comprises a [CONNECT] moiety which is a diAPN moiety, and wherein the chemical intermediate is preferably for the preparation of a cyanine fluorogenic dimer probe having an antibody as [ligand].

[0129] In some embodiments, the diAPN moiety is of formula:

[0130]

[0131] The invention also relates to a kit for the preparation of a cyanine fluorogenic dimer probe for the labelling of a molecular target as described herein, which comprises:

[0132] - a chemical intermediate as described herein,

[0133] - optionally one or more chemical reagents, and

[0134] -optionally instructions for preparing the cyanine fluorogenic dimer probe from the chemical intermediate.

[0135] The invention further relates to the use of a cyanine fluorogenic dimer probe as described herein for detecting a molecular target, in vitro or ex vivo, for instance in a tissue, a cell or an organoid The invention also relates to a cyanine fluorogenic dimer probe as described herein for use as a diagnostic agent in vivo. The invention also relates to a method of labelling a molecular target in vitro, ex vivo or in vivo, comprising a step of contacting the molecular target with a cyanine fluorogenic dimer probe as described herein. The invention further relates to the use of a cyanine fluorogenic dimer probe as described herein in the manufacture of a diagnostic agent in vivo.

[0136] FIGURES

[0137] Figure 1: Synthesis of cyanine dyes and their corresponding dimers, (i) Cy-A was synthesized as reported previously.10(ii) The synthesis of Cy-B is described in the SI. (iii) Cy-C - L were synthesized by CuAAC click reaction from the appropriate azide derivative X-N3 and Cy-B, CuSCh, sodium ascorbate, DMF / H2O, 25°C, 15min. (vi) d-Cy-A - L were synthesized from P2, 2 equivalents of corresponding Cy-A - L, PyBOP, DIPEA, DMF, 25°C, 2h (except for d- Cy-C). (v) d-Cy-C was synthesized by CuAAC click reaction from 2 equivalents of (3-azidopropyl)phosphonic acid, d-Cy-B, CuSCh, sodium ascorbate, DMF / H2O, 25°C, 15 min.

[0138] Figure 2: Fluorescence integral ratios (Flo) for evaluation of nonspecific interactions of the dimers with 1% BSA (A) and 200 pM liposomes (B) in HEPES buffer. Mean ± SEM from three independent experiments.

[0139] Figure 3: Confocal microscopy imaging with fluorescent conjugates. HEK293 cells overexpressing the ApelinR fused to EGFP (left) or wild-type HEK293 (right) cells were stained with dimeric Ap-d-Cy-A, E (200 nM) or with monomeric Ap-m-Cy-E (400 nM) in HBS supplemented with 0.1% BSA under no-wash conditions for 15 min at 37°C. Fluorescence of EGFP is shown in green, fluorescence of the cyanines is shown in red, nuclei stained with Hoechst are shown in blue. Scale bars, 20 pm (A).

[0140] Evaluation of the non-specific interactions on wild-type HEK293 by measurement of the normalized variance (B). Evaluation of the signal -to -noise ratio on HEK293 cells overexpressing the ApelinR (C).

[0141] DETAILED DESCRIPTION OF THE INVENTION

[0142] The Inventors sought to conceive improved “turn-on” fluorogenic probes, suitable for both in vitro and in vivo imaging. The fluorogenic probes should be poorly fluorescent in aqueous media but adopt a high fluorescent form when binding to their target. The fluorogenic probes should also have (i) limited non-specific interactions with circulating plasma proteins and cell membranes and (ii) near-infrared fluorescence properties: such properties are particularly required to limit background noises and / or to perform in vivo imaging.

[0143] Surprisingly, the Inventors showed that it is possible to obtain such fluorogenic probes by introducing a specific substituent R in a particular position of the Cy5.5 scaffold, namely in place of a methyl group, as shown in the below Formula of Cy5.5 scaffold:

[0144]

[0145] Cy5.5 scaffold functionalized with R group

[0146] The introduction of this new substituent R enables to specifically modulate the properties of Cy5.5 dimers, in terms of non-specific interactions in particular with plasma proteins and cell membranes while retaining the turn-on properties, namely the capacity of the C5.5 scaffolds in the probe to form intramolecular H-aggregates in aqueous media and to disassemble in apolar media. The Inventors showed that the functionalized cyanine dimers had significantly less nonspecific interactions with bovine serum albumin (BSA) and liposomes in vitro as compared to similar unfunctionalized cyanine dimers. Of note, the Inventors demonstrated that the properties of the functionalized dimer probes (with respect to both turn-on properties and non-specific interactions) can be finely modulated by the choice of the substituent R introduced in the cyanine scaffolds.

[0147] The introduction of a substituent in the cyanine scaffold was a real challenge. Indeed, the synthesis of functionalized Cy5.5 dyes requires multiple tedious steps, can be incompatible with various chemical moieties and are time-consuming. The Inventors overcame these difficulties thanks to a versatile method of synthesis enabling to introduce a large variety of possible substituents in in the cyanine scaffold. This method relies on click chemistry strategy, in particular on (i) the introduction of “clickable” reactive moiety such as an alkyne group in the cyanine scaffold and (ii) the introduction of the desired substituent through an appropriate click reaction, (e.g. Copper-catalyzed alkyne-azide cycloaddition (CuAAC)), once the cyanine core is obtained. This versatile strategy gives access to a large variety of functionalized cyanine scaffolds that can be used to prepare functionalized dimer cyanine probes.

[0148] As a proof of concept, the Inventors conceived the first turn-on fluorescent probe for Apelin receptor, by conjugating Apelin 17 to a functionalized Cy5.5 dimer through a three-branch-linker, the substituent R bearing a quaternary amine. The resulting fluorescent functionalized cyanine dimer probe (Ap-d-Cy-E, invention) was shown to have a good turn-on property in vitro (turn-on ratio of 57) while exhibiting significant decreases in non-specific interactions towards BSA and liposomes in vitro as compared to the corresponding unfunctionalized cyanine dimer probe (Ap-d-Cy-A, comparative) and cyanine monomer probe (Ap-m-Cy-E). The better properties of Ap-d-Cy-E over Ap-d-Cy-A and Ap-m-Cy-E were confirmed through imaging assays in living cells in which the functionalized cyanine dimer probe Ap-d-Cy-E enabled the visualization of the Apelin receptor under no-wash conditions with a better selectivity than the non-functionalized probe Ap-d-Cy-A and with higher signal-to-noise ratio than the monomeric probe Ap-m-Cy-E.

[0149] Without wishing to be bound by any theory, the Inventors believe that the effects resulting from the replacement of a methyl group in the cyanine 5-membered heterocycle by a substituent R can be extrapolated to other cyanine such as C3.3 or C7.7. Without wishing to be bound by any theory, the Inventors believe that the interactions of the cyanine dimer probes with lipid bilayers and plasma proteins such as albumin can be finely tuned depending on the substituent R introduced in the scaffold, paving the way of a new generation of fluorescent dimeric cyanine probes whose properties can be adapted depending on (i) the linked ligand and (ii) the targeted target through the nature of the substituent R.

[0150] Accordingly, in a first aspect, the Invention relates to a turn-on cyanine fluorogenic dimer probe for detecting a molecular target, which is represented by the following formula (I):

[0151]

[0152] [Linker]

[0153] [Ligand]

[0154] (i)

[0155] as described herein.

[0156] The Invention also relates to the use of a cyanine fluorogenic dimer probe of formula (I) as described herein for detecting a molecular target, in vitro or ex vivo, for instance in a tissue, a cell or an organoid.

[0157] The Invention also relates to a cyanine fluorogenic dimer probe as described herein for use as a diagnostic agent in vivo.

[0158] The Invention relates to the use of a cyanine fluorogenic dimer probe of formula (I) as described herein in the manufacture of a diagnostic agent in vivo.

[0159] In another aspect, the Invention relates to a method for detecting or quantifying a molecular target, in vitro, ex vivo or in vivo based on the use of a cyanine fluorogenic dimer probe of formula (I) as described herein. For instance, the in vitro or ex vivo method may comprise a step of contacting the cyanine fluorogenic dimer probe of formula (I) as described herein with a sample containing the molecular target. As another example, the in vivo method can comprise a step of administering a cyanine fluorogenic dimer probe as described herein to a subject, e.g an animal or a human subject, so as to visualize or quantify the molecular target in vivo.

[0160] The Invention also relates to chemical intermediates for the preparation of a cyanine fluorogenic dimer probe according to the Invention, in particular chemical intermediates of formula (Ila), (lib) or (lie) as described herein further below.

[0161] A further object of the Invention is a kit for the preparation of a cyanine fluorogenic dimer probe as described herein which comprises:

[0162] - a chemical intermediate as described herein,

[0163] - optionally one or more chemical reagents, and -optionally instructions for preparing the cyanine fluorogenic dimer probe from the chemical intermediate.

[0164] In another aspect, the Invention relates to a method for modulating the properties, in particular for modulating the interactions of a cyanine fluorogenic dimer probe with plasma proteins and / or lipid bilayers such as cell membranes, said method comprising introducing a substituent R, as defined herein, in place of a methyl group in the cyanine scaffolds of the probe.

[0165] - General definitions

[0166] As used herein, the term “ molecular target” refers to any kind of molecules to be recovered, detected and / or quantified in a sample. The molecular target is preferably a biomolecule, i.e. a molecule that is present in living organisms or in viruses. Examples of biomolecules include, but are not limited to, nucleic acids, e.g. DNA or RNA molecules, peptides, proteins such as antibodies, enzymes, receptors or growth factors, lipids such as fatty acids or glycolipids, vitamins, hormones, neurotransmitters, and carbohydrates, e.g., mono-, oligo- and polysaccharides.

[0167] In the context of the Invention, a “ molecular target” is preferably a biomolecule present in, or attached to, or interacting with, a cellular membrane bilayer (for example in an eukaryotic cell). In other embodiments, the molecular target can be present in, or attached to, or interacting with, the cell wall of a prokaryote (e.g. bacteria) or the capsid of a virus. For instance, the molecular target can be a protein in a viral capsid or a in a bacterial wall.

[0168] In some preferred embodiments, the molecular target is a membrane protein, namely a protein or a glycoprotein that is part of, or interact with, a biological membrane. The molecular target may be a peripheral membrane protein, or an integral membrane protein such as a transmembrane protein.

[0169] The membrane protein can have an extracellular domain.

[0170] Molecular targets of interest encompass, without being limited to, membrane transport proteins (e.g. protein channels or protein carriers), membrane enzymes e.g. receptor tyrosine kinase (RTKs), cell adhesion molecules (CAMs) such as IgCAMs, cadherins, integrins, proteoglycans membrane receptors such as MHC class I or II, immunoreceptors or G protein-coupled receptors (GPCR) and the like, and proteins expressed on the surface of bacteria or viruses. For instance, the molecular target may be a G protein-coupled receptors (GPCR). As another example, the molecular target may be an immunoreceptor (namely a receptor expressed on the surface of an immune cell such as T cell, B cell or NK cell). GPCRs encompass, without being limited to, oxytocin receptors, histamine receptors, apelin receptors, 5-hydroxytryptamine receptors, muscarinic receptors, adenosine receptors, adrenoceptors, angiotensin receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, dopamine receptors, endothelin receptors, ghrelin receptors, glycoprotein hormone receptors, gonadotrophin-releasing hormone receptors, hydroxycarboxylic acid receptors, leukotriene receptors, lysophospholipid receptors, melanocortin receptors, opioid receptors, opsin receptors, orexin receptors, somatostatin receptors, tachykinin receptors, vasopressin receptors.

[0171] In some embodiments, the “molecular target” is specifically expressed or overexpressed in a tumor cell, in particular in a cancer cell and can be selected among GPCRs, integrins, transporters, RTKs, and ErbB family (e.g. EGFR and Her-2). In some embodiments, the “molecular target” is a biomarker of cancer. For instance, its detection and / or its quantification in vitro or in vivo is useful for diagnosis or monitoring a cancer in a subject.

[0172] As used herein, a “bioconjugation reaction” refers to a reaction involving an amino acid such as lysine, cysteine, or tyrosine with reactive groups as detailed in Koniev, O., Wagner, A, Chem. Soc. Rev., 44, 5495 (2015), the entire contents of which are incorporated by reference herein. Examples of bioconjugation reactions are for instance depicted in the below table:

[0173] a) Amine conjugation c) Thiol conjugation

[0174] 0 »-NH2R2,0AR— R2= NHS, PFP. amine / activated ester

[0175] b) Bioconjugation via carbon-nitrogen double bonds.

[0176] thiol / iodoacetamide

[0177]

[0178] X = O, NH, NHC(O)

[0179] Other examples of bioconjugation of interest encompass:

[0180] - the reaction between a tyrosine residue and an aryldiazonium:

[0181]

[0182] Tyrosine / aryldiazonium N=N- Ar - the reaction between an amine group (e.g. lysine) with reagents such as isocyanate, isothiocyanate, NHS-ester and benzoyl fluoride:

[0183] NH2R-N=C=O - ►

[0184] amine / isocyanate

[0185] Q — NH2R-N=C=S - ►

[0186] amine / isothiocyanate

[0187] O

[0188]

[0189] amine / NHS-ester

[0190]

[0191] amine / benzoyl fluorides

[0192]

[0193] Other reactions of interest for bioconjugation encompass reactions of aldehyde / ketone with alkoxyamine / hydrazine, amidation and reductive amination.

[0194] As used herein, “Click-reaction” or “Click-chemistry” refers to a concept introduced by Sharpless in 2001. “Click chemistry” generally refers to chemical reactions characterized by high yields, high chemoselectivity, which are simple to conduct, and which generate inoffensive by-products. “Click reactions” can be typically conducted in complex media with high efficiency. Click reactions are typically used to create covalent heteroatom links (C-X-C) between two entities of interest. For review about click chemistry, one can refer to Kolb et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021 and to Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117, the entire contents of both of which references are incorporated herein by reference.

[0195] Examples of click reactions encompass, without being limited to, copper-catalyzed azidealkyne dipolar cycloadditions (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), Diels-Alder reactions with tetrazines and strained alkynes or alkenes, alkene / tetrazole photoclick reaction, thia-Diels-Alder reactions, tetrazine-isonitrile cycloadditions, thiol-alkene click reactions such as maleimide-cysteine cycloadditions, Staudinger azide-triarylphosphine conjugation, iminosydnone alkyne cycloaddition, and sydnone-alkyne cycloadditions. Thus, examples of click reactions encompass:

[0196] Approach 1: Azide / cycloalkyne reaction Approach 3: Tetrazole / Alkene reaction

[0197]

[0198] Approach 4: Chloro-oxime / Norbornene reaction

[0199] HO

[0200] Approach 2: Tetrazine / Cyclooctene reaction

[0201]

[0202] A click-reaction of interest is the azide / cycloalkyne addition in which an azido group (-N3) reacts with an alkyne group in the presence of a copper catalyst, or with a strained alkyne. Strained alkynes of interest encompass, without being limited to cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DBCO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa- dibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne). Preferred strained alkynes are BCN and DBCO.

[0203] As use herein, the term "amino acid" refers to organic compounds that contain both amino and carboxylic acid functional groups in alpha- (a-), beta- (P-), or gamma- (y-) amino acid configuration and in any diastereoisomeric or eniantomeric configurations. The term "amino acid" proteogenic alpha-amino acids such as alanine, valine, lysine, tyrosine, cysteine, arginine and non-proteogenic amino acid residues such as citrulline, or beta-alanine.

[0204] As used herein, the term "activated carboxylic acid" is intended to mean a chemical function derived from the "carboxylic acid" group capable of reacting with a nucleophile such as a primary amino group. " Activated carboxylic acid" groups are well known to those skilled in the art and encompass acyl chlorides, mixed anhydrides and esters.

[0205] The activated carboxylic acid group may be in the form of an ester. This ester may result from the reaction of a carboxylic acid group with a compound selected from 1 -hydroxybenzotriazole (HOBt), l-Hydroxy-7-azabenzotriazole (HOAt) and N-hydroxysuccinimide, or a derivative thereof, preferably N-hydroxysuccinimide, or a derivative thereof such as sulfo-NHS, N-bromosuccinimide or pentafluorophenol. For instance, the activated carboxylic acid group is a N-hydroxysuccinimidyl ester of the following formula

[0206]

[0207] As used herein, an “Alkyl” refers to a saturated hydrocarbon radical which can be a straight or branched hydrocarbon group. For instance, a Ci-Ce alkyl encompasses, without being limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. A C1-C4 alkyl encompasses for instance methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. A Ci-C3 alkyl encompasses for instance methyl, ethyl, propyl, and isopropyl.

[0208] As used herein, a “cycloalkyl” refers to a saturated cyclic hydrocarbon group. For instance, a “C3-C6 cycloalkyF encompasses, without being limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0209] “Halogen” denotes a halogen atom such as Cl, Br, I, or F. Preferred halogens are Br, Cl and F, more preferably F.

[0210] An “alkoxy” refers to a radical of formula R-O- wherein R represents an alkyl group. Examples of alkoxy (or Ci-Ce alkoxy) include, for instance, methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, and hexyloxy.

[0211] An “alkylcarbonyF or “alkanoyF refers to a radical of formula R-C(=O)- wherein R represents an alkyl group, preferably a C1-C5 alkyl group.

[0212] An “alkoxycarbonyF refers to a radical of formula R-O-C(=O)- wherein R represents an alkyl group, preferably a C1-C5 alkyl group.

[0213] An “alkoxyalkylene” refers to a radical of formula R-O-A- wherein R is an alkyl and A is alkylene. Preferably R and A contain together from 2 to 6 carbon atoms (C2-C6 alkoxyalkylene). In some embodiments, the “alkoxyalkylene” is of formula - (CH2)nO(CH2)mCH3 wherein n is an integer from 1 to 5, m is an integer from 0 to 4 and preferably n + m < 6. In some embodiments, m = 0 and n is an integer from 1 to 5.

[0214] A “hydroxy alkyl” refers to an alkyl group, preferably a Ci-Ce alkyl group, wherein one or more hydrogens is / are replaced by -OH. Preferably, one single H is replaced by -OH.

[0215] A “haloalkyl” refers to an alkyl group preferably a Ci-Ce alkyl group, wherein one or more hydrogens is / are replaced by halogen atom(s). Examples of haloalkyl are fluoroalkyl such as -CF3, -CHF2, -CH2F or -CH2CH2F.

[0216] A “cyanoalkyF refers to an alkyl, preferably a Ci-Ce alkyl, wherein a hydrogen is replaced by -CN. An “alkenyl” refers to an unsaturated hydrocarbon radical comprising one or more, preferably one double bond. Example of C2-C6 alkenyl encompasses ethenyl, propenyl, butenyl, pentenyl and hexenyl.

[0217] An “alkenyloxy” refers to R-0 wherein R is an alkenyl, preferably a C2-C6 alkenyl.

[0218] An “alkynyl” refers to an unsaturated hydrocarbon radical comprising one or more, preferably one triple bond. Example of C2-C6 alkenyl encompasses ethenyl, propenyl, butenyl, pentenyl and hexenyl.

[0219] The term “heterocycle” or “heterocyclic group” refers to a saturated or unsaturated, aliphatic or aromatic cyclic hydrocarbon group in which at least one ring carbon atom has been replaced with a heteroatom, preferably selected from nitrogen, oxygen, or sulfur atom. Advantageously, the heterocycle comprises from 3 to 20 ring atoms, preferably 3, 4, 5 or 6 ring atoms, wherein at least one of the ring atoms is a heteroatom such as nitrogen, oxygen or sulfur atom, preferably nitrogen (N or NH) in the context of the invention. The term “heterocycle” encompasses heteroaryl groups as well as aliphatic, saturated or unsaturated, heterocyclic groups. For instance, examples of heterocycles encompass cycloheteroalkyl groups, namely a cycloalkyl wherein one or several carbon atoms have been replaced by a heteroatom. The term “heterocycle” includes for instance aziridinyl, azepanyl, diazepanyl, dioxolanyl, benzo [1,3] dioxolyl, azetidinyl, oxetanyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolinyl, pyridyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, pyrimidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiooxetanyl, thiopyranyl, thiomorpholinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, dihydrofuranyl, dihydrothiopyranyl, pyrrolyl, thienyl, quinolyl, furanyl, isoquinolyl, dihydrothiophene, dihydropiperidinyl, tetrahydropiperidinyl, tetrahydrothiopyranyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiophene.

[0220] In some embodiments, the “heterocycle” is a “heterocycloalkyl group”, namely a saturated heterocycle, comprising from 3 to 6 carbon atoms and one or two heteroatoms as ring atoms, heterocycloalkyl groups (also called saturated heterocycles) include azetidinyl, piperidinyl, pyrrolidinyl, piperazinyl, thiomorpholinyl and morpholinyl.

[0221] In some embodiments, the “heterocycle” is unsaturated and preferably comprises from 1 to 3 unsaturations, such as from 1 to 3 double bonds. Preferred heterocycles are triazoles, such as 1,2,3-triazole.

[0222] It goes without saying that the heterocyclic group can be unsubstituted or substituted by one or more groups as described herein. The heterocyclic group can be fused to another cyclic group, e.g. a C3-C8 carbocycle or C3-C8 heterocarbocycle which can be saturated or unsaturated.

[0223] As used herein, the term "aryl" refers to monocyclic and bicyclic fused ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. Exemplary aryl groups are phenyl, biphenyl, naphthyl, anthracyl and the like, which optionally includes one or more substituents. In some embodiments, an aryl group has from 6 to 18 ring members, said ring member being carbon atoms. A preferred aryl group in the context of the invention is a phenyl group.

[0224] Also included within the scope of the term "aryl", as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. The terms "heteroaryl" refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" also include groups in which a hetero aromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin- 3(4H)-one. A heteroaryl group is optionally mono- or bicyclic or fused to another ring that can be saturate or unsaturated, carbocyclic or heterocyclic.

[0225] The term "saturated" as used herein, means that the chemical group contains single bonds only, namely no unsaturation.

[0226] The term "unsaturated", as used herein, means that a moiety has one or more units of unsaturation, i.e. one or more double or triple bonds. Preferably, an unsaturated moiety refers to a moiety comprising one or more, e.g. 1, 2 or 3 double bond(s).

[0227] The term "optionally saturated" can be replaced with terms "saturated or unsaturated" throughout this application. The term "substituted" means that one or more hydrogen atoms on the designated atom or group is / are replaced by non-hydrogen substituent (s), provided that the designated atom's normal valency under the existing circumstances is not exceeded.

[0228] The term "optionally substituted" can be replaced with terms "substituted or unsubstituted" throughout this application.

[0229] In the context of the invention, preferred substituents encompass OH, NH2, CN, halogen, Ci-Ce alkyl, Ci-Ce cycloalkyl, Ci-Ce cyclo heteroalkyl, Ci-Ce alkoxy alkyl, Ci-Ce haloalkyl, Ci-Ce aminoalkyl, N-(Ci-C6)alkylamino, N, N-di(Ci-C6)alkylamino, and Ci-Ce alkylcarbonyl.

[0230] As used herein, the term "one or more" or “at least one" means 1 or more than 1, e.g. 1, 2, 3, 4 or 5, particularly 1, 2, 3 or 4, more particularly 1, 2 or 3.

[0231] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0232] “About”, “around" or “approximately” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods or compositions.

[0233] Cyanine fluorogenic dimer probe of the Invention

[0234] In a first aspect, the Invention relates to a cyanine dimer fluorogenic probe for detecting a molecular target, which is represented by the following formula (I):

[0235] [C^ [Cy]

[0236] [Linker]

[0237] [Ligand]

[0238] (I)

[0239] Wherein

[0240] [Linker] is a chemical group having at least three branches, wherein two of the branches are covalently bond to a [Cy] by at least one covalent bond, and a third branch is bound to [Ligand] by at least one covalent bond,

[0241] [Ligand] is a ligand able to bind to the molecular target,

[0242] Each [Cy] is a cyanine group in which one of the methyl groups borne by the indole is replaced by a R group, wherein R group is a moiety for modulating, preferably for decreasing non-specific interactions of the cyanine fluorogenic dimer probe with lipid bilayers of cell membranes and / or with a blood plasma protein.

[0243] - The [Cy]

[0244] Each [Cy] represents a cyanine scaffold functionalized with a R group. Preferably, each [Cy] is a cyanine group (also called herein cyanine scaffold) selected from:

[0245]

[0246] Wherein

[0247] n is 1, 2 or 3,

[0248] - R3 is H or an apolar group i.e. a (C1-C20) alkyl, preferably a (C1-C6) alkyl

[0249] - R2 is H, or a polar group, e.g. a group of formula -(R9)z-(Arl)ziC(=O)R9’ wherein:

[0250] - R9 is a Ci-Ce alkylene, NR9”, S, or O, wherein R9” is H or C1-C3 alkyl, z is 0 or 1,

[0251] Ar1 is an aryl having from 6 to 18 carbon atoms, preferably a phenyl, or a 6- membered heteroaryl,

[0252] zl is 0 or 1, and

[0253] - R9’ is NR10R11, wherein Rio and R11 are independently an hydrogen, a (Ci-Ce) alkyl, a di(Ci-C5)alkylamino(Ci-C5)alkyl, a Ci-Ce hydroxyalkyl, including Ci- Ce di- or -tri-hydroxyalkyl, (C2-Ce)carboxyalkyl, polyethylene glycol represented by the formula -(CH2CH2O)ni-R’; or a polypropylene glycol represented by the formula -(CH2CH2(CH3)O)ni-R’, wherein nl is an integer from 1 to 40 and R’ is a C1-C12 alkyl group, or, alternatively, Rio and R11 form with the nitrogen to which they are attached a heterocycle (such as a 4- to 6- membered heterocycles such as piperazine) optionally substituted by a (Ci- C4)alkyl,

[0254] In some embodiments, R2 is H or selected in the group consisting of:

[0255]

[0256] Wherein:

[0257] R31 is absent, O, S or NR”, and

[0258] R32 is -CH2- or -O-, and R33 is a polyethylene glycol represented by the formula -(CH2CH2O)n2-R’; or a polypropylene glycol represented by the formula -(CH2CH2(CH3)O)n2-R’, wherein n2 is an integer from 1 to 40 and R’ is an alkyl group in C1-C12.

[0259]

[0260] (G2) is of formula (G2a) or (G2b)

[0261]

[0262] wherein

[0263] - R is as defined further below

[0264] X' is a counter-anion, preferably a pharmaceutically acceptable counter-anion.

[0265] Examples of X' are provided further below.

[0266] - Li is a chemical group connecting the [Cy] group to a branch of the [Linker], preferably comprising from 1 to 30 carbon atoms in its backbone.

[0267] Preferably, Li is an unsaturated or saturated C1-C20 hydrocarbon chain, more preferably a C1-C10 alkylene, optionally substituted by one or more halogens, C1-C3 haloalkyl or C1-C3 alkyl.

[0268] - R1 is of formula (a) -L1a-R12; (b) -L1b-NR13C(=O)-L1cR12 or (c) -L1bC(=O)NR13L1cR12 wherein:

[0269] ■ L1a is a saturated or unsaturated (C2-C40) hydrocarbon chain, preferably a C2-C20 alkylene, or a poly(C2-C5 oxyalkylene) chain comprising from 2 to 20 monomers, such as a polyethylene or polypropylene chain comprising from 2 to 20 or from 2 to 10 monomers,

[0270] ■ L1b and L1c are independently a saturated or unsaturated (C2-C20), preferably a (C2-C10) hydrocarbon chain, preferably a C2-C10 alkylene ■ R13 is H, a C1-C6 alkyl, a C2-C6 hydroxyalkyl, or a C2-C6 alkoxy

[0271] ■ R12 is -(CH2)pR14 wherein p is an integer from 0 to 6 and R14 is selected from H, OH, halogen, C1-C3 alkoxy, and SO3H.

[0272] For instance, Ri can be a polyethylene chain comprising from 2 to 10 monomers and terminated by -(CH2)pR14 wherein p is an integer from 0 to 6 and R14 is selected from H, OH, halogen, C1-C3 alkoxy, and SO3H.

[0273] As another example, Ri can be of formula: -(CH2)3-CO-N(butyl)((CH2)3SO3H or C2-C10 alkyl.

[0274] In some embodiments, the two [Cy] are different.

[0275] In some embodiments, one [Cy] is of formula (D1), and the other [Cy] is of formula (D2) In some other embodiments, the two [Cy] are identical.

[0276] In some particular embodiments, each [Cy] is of formula (D2):

[0277]

[0278] In some preferred embodiments, each [Cy] is of formula

[0279]

[0280] n(D1)

[0281] In some embodiments, G1 is of formula (G1a) and (G2) is of formula (G2a).

[0282] In some embodiments, G1 is of formula (G1b) and (G2) is of formula (G2b).

[0283] In some embodiments, G1 is of formula (G1a) and (G2) is of formula (G2b).

[0284] In some embodiments, G1 is of formula (G1b) and (G2) is of formula (G2a).

[0285] In some embodiments, each [Cy] is of formula

[0286]

[0287] n(D1) wherein n is 1, 2 or 3, preferably 2 or 3, and

[0288] G1 is of formula (G1a) and (G2) is of formula (G2a), OR G1 is of formula (G1b) and (G2) is of formula (G2b).

[0289] For instance, each [Cy] can be of formula

[0290]

[0291] wherein Ri, Li, X' are as defined above and R is as defined below.

[0292] In some embodiments, - Li is a Ci-Cio alkylene, and / or

[0293] - R1 is a polyethylene chain comprising from 2 to 10 monomers and terminated by -(CH2)pR16 wherein p is an integer from 0 to 6 and R16 is selected from H, OH, halogen, C1-C3 alkoxy, and SO3H, or of formula -L1bC(=O)NR13L1cSO3H wherein L1b is a C2-C6 alkylene, R13 is a C1-C6 alkyl and L1c is a C2-C6 alkylene e.g. -(CH2)3-CO-N(butyl)((CH2)3SO3H

[0294] In some embodiments, the two [Cy] are identical and are of formula (D3) as described above.

[0295] As mentioned above, X’ is an anion which can be organic or inorganic.

[0296] According to an embodiment, X’ is an anion of acetic acid, 2,2-dichloroacetic acid, trifluoroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(15)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecyl sulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, a-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-l,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

[0297] In another embodiment, X~ is a fluoride (F‘), chloride (Cl’), bromide (Br), iodide (I’), acetate (CH3CO2’), trifluoroacetate (CF3CO2 ) (named as TFA), phosphate (PO4H2’, PCLH2’, or PO43’ ), or sulfate (HSOf or SCh2’).

[0298] In another embodiment, X’ is a chloride, a bromide, a iodide or a trifluoroacetate. The R group

[0299] The R group is a chemical group different from a methyl. This substituent may be added in the cyanine scaffold so as to modulate the non-specific interactions of the cyanine dimer fluorogenic probe with blood plasma proteins and / or cellular membrane lipid bilayers.

[0300] In some embodiments, R is selected so as to decrease said non-specific interactions.

[0301] In some embodiments, R is selected so that the turn-on properties and / or the NIR-fluorogenic properties of the probe are maintained.

[0302] In some embodiments, R is a moiety for modulating, preferably for limiting non-specific interactions of the cyanine fluorogenic dimer probe with cellular membrane and / or with blood plasma proteins

[0303] In some embodiments, R is a saturated or unsaturated hydrocarbon group having at least 3 carbon atoms and up to 50 carbon atoms in its backbone.

[0304] Preferably R is a hydrocarbon chain having from 3 to 50, preferably from 3 to 30 carbon atoms in length, e.g. 3 to 20 carbon atoms, wherein

[0305] said hydrocarbon chain has a terminal moiety selected from:

[0306] • H, -OH, -NH2, -NR8R5, C1-C3 hydroxyalkyl, C1-C3 alkoxy, C1-C3 halogenoalkyl, a halogen, -CN, C1-C3 cyanoalkyl, -NO2

[0307] • a cationic group preferably selected from -NR5R6R7+, and

[0308] • an anionic group preferably selected from a phosphate, a phosphonate, a sulfonate, a carboxylate

[0309] wherein said hydrocarbon chain is optionally substituted by one or more substituents selected from C1-C3 hydroxyalkyl, C1-C3 alkyl, C1-C3 alkoxy and halogen, wherein said hydrocarbon chain is optionally interrupted by one or more connecting groups selected from -NR8CO-, -NR8-,-N+R5R6, -OC(O)-, -NH(CO)O-, NH, S, O, CO, NH(C=O)NH, O(C=O)NH, -O-N=CR8-, -CR8=N-O-, -NH-N=CR8-, CR8=N-NH-, amino acid residues such as alanine, citrulline or valine and / or 3-to-20, preferably 3 to 10- membered cyclic or heterocyclic groups such as pyrazole or triazole, optionally substituted and / or fused,

[0310] wherein

[0311] • Each R5, R6 and R7 are independently C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 alkyl alkoxy

[0312] • Each R8is independently H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 alkyl alkoxy. In some embodiments, said hydrocarbon chain is interrupted by at least one triazole group, optionally substituted.

[0313] In some embodiments, the one or more optional substituent(s) present on the 3-to-10 membered cyclic or heterocyclic group can be selected from OH, NH2, CN, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce hydroxyalkyl, Ci-Ce alkoxy alkyl, Ci-Ce haloalkyl, Ci-Ce aminoalkyl, N-(Ci-Ce)alkylamino, N, N-di(Ci-C6)alkylamino, and Ci-Ce alkylcarbonyl.

[0314] In a particular embodiment, R is of formula (ao):

[0315] -(CH2)o-[G3]-L2-Ri5 (ao)

[0316] wherein:

[0317] o is an integer from 0 to 6, for instance 1, 2 or 3,

[0318] [G3] is a connecting group preferably selected from -NH(C=O)-, -(C=O)-NH-, S, O, NH(C=O)NH, O(C=O)NH, NH(C=O)O, -O-N=CRs-, -CRs=N-O-, -NH-N=CRs-, CRs=N-NH-, and 3- to 6-membered cyclic or heterocyclic groups optionally substituted. Preferably [G3] is selected from triazole, pyrazole -O-N=CR8-, CRs=N-O-, -NH-N=CR8-, and -CR8=N-NH-, more preferably [G3] is a triazole,

[0319] - L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NR8(C=O)-, -OC(=O)-, -NH(C=O)O-, -NRs-,-N+RsR6, NH, S, O, and C=O,

[0320] - Ris is selected from the group consisting of H, -OH, -NH2, -NRsRs, C1-C3 alkoxy, halogen, CF3, -CN, -NRsReR?, a phosphate, a phosphonate, a sulfonate, a nitro, and a carboxylate,

[0321] each Rs, RS, Re, and R7 are as defined above, preferably each Rs is independently H, Ci- Ce alkyl or Ci-Ce hydroxyalkyl, and each R7, Rs and Re are independently Ci-Ce alkyl or Ci-Ce hydroxy alkyl.

[0322] In a particular embodiment, [G3], in the formula (ao), is a triazole group.

[0323] In some embodiments, in the formula (ao):

[0324] - o is 1, and / or

[0325] - [G3] is a tri azole, and / or

[0326] - L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NR8(C=O)-, NRs. -N+RsRe, NH, O, and (C=O), and / or

[0327] - Ris is selected from the group consisting of H, -OH, -NH2, -NR.8 Rs, C1-C3 alkoxy, -NRsReR?, a phosphonate, and a sulfonate,

[0328] each Rs, Rs, Re and R7 being as defined above. In a particular embodiment, R is of formula (ai):

[0329]

[0330] wherein o, L2and R15 are as defined above. Preferably o is 1.

[0331] In a particular embodiment, in the formula (ai):

[0332] - o is 1, and / or

[0333] - L2is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NRs(C=O)-, NRs, -N⁺RsR6, NH, O, and (C=O), and / or

[0334] - R15 is selected from the group consisting of H, -OH, -NH2, -NRsRs, C1-C3 alkoxy, -NR.5R.6R7, a phosphonate, and a sulfonate,

[0335] each Rs, Rs, Re and R7 being as defined above.

[0336] For instance, R is selected from the group consisting of:

[0337]

[0338] wherein

[0339] • t is an integer from 2 to 30, preferably from 3 to 10 such as 3, 4, 5, 7 or 8 and

[0340] • R19 is H, -PO3H-, SO3-, -N(Me)3+, -N(Et)3+, -N(Me)2, -N(Et)2, -N(CH2CH2OH)2, -OH, -OMe, -OEt, -NH2, -C(O)NHC(CH2OH)3, -N(Me)2+(CH2)PSO3-, C(O)N[(CH2)pCH3][(CH2)PSO3-], -C(O)N[(CH2)pCH3][(CH2)PPO3H], N(Me)2+(CH2)pPO3H-, with p an integer from 2 to 10.

[0341] Preferably R19 is PO3H; SO3; -N(Me)3+, -N(Me)2+(CH2)3SO3-, -N(Me)2, OH, or - C(O)NHC(CH2OH)3, more preferably -N(Me)3+ or -N(Me)2+(CH2)3SO3-,

[0342]

[0343] and w is an integer from 0 to 10. In some embodiments, R is selected from the group consisting of:

[0344] N=N

[0345]

[0346] (a)

[0347] wherein

[0348] • t is an integer from 2 to 10 and

[0349] • R19 is -N(Me)3+, -N(Et)3+, -N(Me)2+(CH2)3SO3; and N(Me)2+(CH2)3PO3H’,

[0350]

[0351] (b) with u an integer from 2 to 10.

[0352] In a particular embodiment, each [Cy] in the probe is selected from

[0353]

[0354] wherein Li, X' and Ri are as defined above in formula (I) and L2and Ris are as defined above. In some embodiments, -L2-RIS corresponds to -(CH2)P-N+(CH3)3or -(CH2)tN+(CH3)2((CH2)PSO3H) with t is an integer from 2 to 6 and p an integer from 2 to 10, for instance both t and p are 3.

[0355] In some embodiments, the two [Cy] are identical and are of formula (D3a).

[0356] In some embodiments, the two [Cy] are of formula (D3a) wherein

[0357] - Li is a Ci-Cio alkylene, and / or

[0358] - Ri is a polyethylene chain comprising from 2 to 10 monomers and terminated by - (CH2)PRi6 wherein p is an integer from 0 to 6 and Ri6 is selected from H, OH, halogen, Ci-C3alkoxy, and SO3H, - L2 is a saturated or unsaturated, C2-C20, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NRs(C=O)-, -OC(=O)-, -NH(C=O)O-, NH, N+RsRe, S, O, and (C=O),

[0359] - R15 is selected from the group consisting of H, -OH, -NH2, -NRsRs, C1-C3 alkoxy, halogen, CF3, -CN, -NR.5R.6R7, a phosphate, a phosphonate, a sulfonate, a nitro, and a carboxylate

[0360] each Rs is independently H, Ci-Ce alkyl or Ci-Ce hydroxyalkyl, and each Rs, Re and R7 are independently Ci-Ce alkyl or Ci-Ce hydroxyalkyl. Preferably Rs, R7, Rs and Re have from 1 to 3 carbon atoms.

[0361] The [Ligand]

[0362] [Ligand] can be any molecule able to bind to the molecular target.

[0363] Preferably, the [Ligand] specifically binds to the molecular target.

[0364] The [Ligand] displays a high affinity for the target molecule. The dissociation constant (Kd) of a [Ligand] for its target molecule is typically from 10-3to 10-12M, preferably from 10-6to 10-12M. The term "specifically binding" is used herein to indicate that the [Ligand] has the capacity to recognize and interact specifically with its target molecule, while having relatively little detectable reactivity with other molecules which may be present in the sample. Preferably, the [Ligand] specifically binds to its target molecule if its affinity is significantly higher for the target molecule, as compared to other molecules, including molecules structurally close to the target molecule. For instance, the [Ligand] may have a Kd for the molecular target of at least 10-fold lower (e.g. at least 20, 50, 100-fold, 1000-fold) lower than for other molecules.

[0365] [Ligand] can be a naturally-occurring ligand, a recombinant ligand or a synthetic ligand.

[0366] [Ligand] can be also a suicide substrate or a suicide ligand.

[0367] [Ligand] can be of any chemical nature, for instance [Ligand] can be an oligosaccharide, a peptide, a polypeptide, a nucleic acid, a lipid or a small synthetic molecule.

[0368] In some embodiment, [Ligand] is an endogenous ligand of the molecular target.

[0369] For instance, the molecular target can be a G protein-coupled receptor (GPCR).

[0370] Endogenous ligands of GPCR (and thus of interest for [Ligand]) encompass, without being limited to, adenosine, bombesin, bradykinin, endothelin, y-aminobutyric acid (GABA), hepatocyte growth factor (HGF), melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, GH, tachykinins, members of the vasoactive intestinal peptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine, norepinephrine, histamine, serotonin, and melatonin); glutamate; glucagon; acetylcholine; chemokines; lipid mediators of inflammation (e.g., prostaglandins, platelet-activating factor, and leukotrienes); peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle-stimulating hormone [FSH], gonadotropin-releasing hormone [GnRH], neurokinin, thyrotropin-releasing hormone [TRH], and oxytocin); and endocannabinoids.

[0371] In some embodiments, [Ligand] can be selected from an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, VHH, and a ScFv.

[0372] In a particular embodiment, [Ligand] is an antibody including a full length antibody or an antigen-binding domain derived from an antibody.

[0373] As used herein, the term "antibody" refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding domain, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific (e.g. bispecific), humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments (e.g. VHH from single-chain antibody) that retain antigen-binding function, i.e., the ability to bind their target specifically. Typically, such fragments would comprise an antigenbinding domain. The terms "antigen-binding domain," or " antigen-binding fragment," refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. Where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant." An antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). However, it does not necessarily comprise both (see e.g. the antigen-binding domain of single chain antibodies and VHH fragments). Typically, an antigen-binding fragment or domain contains at least a portion of the variable regions (heavy and light) of the antibody sufficient to form an antigen binding site (e.g., one or more CDRs, and generally all CDRs) and thus retains the binding specificity and / or activity of the antibody.

[0374] As used herein, a "full-length antibody" (also called herein immunoglobulin of Ig) refers to a protein having the structure that constitutes the natural biological form of an antibody, including variable and constant regions. " Full length antibody" covers both monoclonal and polyclonal full-length antibodies and also encompasses wild-type full-length antibodies, chimeric full-length antibodies, humanized full-length antibodies, the list not being limitative. In most mammals, including humans and mice, the structure of full-length antibodies is generally a tetramer. Said tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). In the case of human immunoglobulins, light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the classes of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgMl, IgM2, IgD, and IgE.

[0375] In some embodiments, [Ligand] is a cell-type specific ligand. A cell-type specific ligand can derive from proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor βFGF, oligosaccharides comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine (GalNac), bridged GalNac and sialic acid, vitamins such as folic acid and targeting peptides such as muscle targeting peptide (MTP). In some embodiments [Ligand] is a cancer-type specific ligand, namely a ligand specifically binding to a molecule which is specifically expressed, or overexpressed in a cancer cell.

[0376] For instance, HER2 is overexpressed in multiple human cancer cells including breast and ovarian cancer.

[0377] For illustration, [Ligand] can be an anti-HER2 antibody such as trastuzumab, and the molecular target is HER2.

[0378] For illustration, [Ligand] is an endogenous ligand of a GPCR, e.g. oxytocin or apelin, or a non-endogenous ligand thereof (e.g. carbetocin for oxytocin receptor).

[0379] In some embodiments, [Ligand] is selected from the group consisting of synthetic small chemical molecules, hormones, vitamins such as acid folic, oligosaccharides such as those comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine (GalNac), bridged GalNac and sialic acid and derivatives thereof (such as Neu5 Ac, Neu5 Aca2-6Gal, Neu5Aca2-8Neu5Ac), proteins or fragments thereof, peptides such as oxytocin, apelin, RGD peptide, Angiopep-2, or muscle targeting peptides, an aptamer, an antibody including heavy-chain antibody and bispecific antibody, and fragments thereof such as Fab, Fab’, and VHH (also called nanobody), a ScFv, a spiegelmer, and a peptide aptamer. In a particular embodiment, [Ligand] is a peptide, preferably having at most 200, more preferably at most 150, for instance at most 100 amino acids. For illustration, [Ligand] can be a peptide having from 2 to 50 or from 5 to 40, amino acids.

[0380] The [Linker]

[0381] A linker is a molecular structure that connects two or more chemical entities together.

[0382] In the context of the Invention, [Linker] is thus a chemical entity that connects [Ligand] and the two [Cy],

[0383] Thus, [Linker] has at least three branches, one branch linked to [Ligand] and each remaining branch linked to one [Cy],

[0384] The branches are typically selected e.g. so as to limit the steric bulk on the [Ligand] (e.g. so as to enable its binding to the molecular target), and / or to promote solubility in aqueous medium and / or to enable the [Cy] moieties to form intramolecular H aggregates in aqueous medium, which disassemble in lipophilic medium. The connection between [Linker] and the [Ligand] can involve one or several (e.g. 2, 3 or 4) covalent bounds. The same applies between each [Cy] and [Linker],

[0385] Typically, [Linker] is a chemical entity having at least three branches and comprising up to 300, for instance up to 200 or 100 carbon atoms in its backbone. [Linker] may also contain heteroatoms such as O, S, N, Se, P, halogen and S, preferably S, O and N and more preferably at least O and N.

[0386] [Linker] may contain one or more connecting groups and / or one or more unsaturations.

[0387] As used herein, connecting groups encompass, without being limited to, heteroatoms such as O and S, amino acid residues including natural (proteogenic) and non-natural amino acid residues, carbocycles, heterocarbocycles, and chemical groups such as C(=O), C(=S), O, S, S(=O)2, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, -C(=O)S-, NR4C(=S), NR4C(=S)NR4, NR4C(=S)O, -O-N=CR4-, and -NHN=CR4and wherein each R4is independently H, Ci-Ce alkyl, Ci-Ce hydroxyalkyl or Ci-Ce alkyl alkoxy.

[0388] Typically, the carbocycles and heterocarbocycles are selected from C3-C20 membered cyclic or heterocyclic groups that can be saturated or unsaturated (including aromatic), that can be unsubstituted or substituted and / or that can comprise fused cycles.

[0389] The cyclic or heterocyclic group(s) present in [Linker] can comprise one or more (e.g.l, 2, 3, or 4) substituents such as halogen, -CN, -OH, -NH2, -NO2, Ci-Ce alkyl, Ci-Ce hydroxyalkyl, Ci-Ce alkoxy alkyl, Ci-Ce amino alkyl and Ci-Ce haloalkyl. In some embodiments, [Linker] comprises one or several connecting groups resulting from a click-reaction or bioconjugation reaction, e.g. resulting from amidation reaction or from azidealkyne cycloaddition.

[0390] For instance, [Linker] can comprise one or several moieties as follows:

[0391]

[0392] (1): amide bond, (2): moiety resulting from the reaction of an azide with an alkyne, (3): moiety resulting from the reaction of an azide with DBCO, (4): moiety resulting from the reaction of an azide with BCN.

[0393] In some embodiments, [Linker] is binds to [Ligand] through a connecting group such as C(=O), C(=S), O, S, S(=O)2, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, -C(=O)S-, NR4C(=S), NR4C(=S)NR4, NR4C(=S)O, -O-N=CR4-, and -NHN=CR4(and herein each R4is independently H, C1-C6 alkyl, C1-C6 hydroxyalkyl or C1-C6 alkyl alkoxy) and moieties resulting from a click reaction such as azide-alkyne cycloaddition (e.g. moieties (2), (3) or (4) as shown above).

[0394] In some embodiments, [Linker] is binds to the [Ligand] through a moiety resulting from a bioconjugation reaction with an amino acid residue in the [Ligand],

[0395] For instance, [Ligand] is linked through a cysteine residue to the [Linker] by means of a moiety as followed:

[0396] O

[0397]

[0398]

[0399] For instance, [Ligand] is linked through a tyrosine residue to the [Linker] by means of a moiety as followed:

[0400]

[0401] For instance, [Ligand] is linked through a lysine residue to the [Linker] by means of a moiety as followed:

[0402] (6') (7')

[0403]

[0404] In some embodiments, [Linker] is a three-branch hydrocarbon group having up to 200, preferably up to 100 carbon atoms in its backbone, said hydrocarbon group comprising one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, NH, N=CR4- -NHN=CR4, and / or amino acid residues such as lysine, citrulline, valine, and / or alanine, and / or saturated or unsaturated 3-to 20-, preferably 3- to 10- or 3- to 6-membered cyclic or heterocyclic groups such as triazole or pyrazole, each R4being independently H, or a C1-C3 alkyl and

[0405] said three-branch hydrocarbon group being optionally substituted by one or more groups preferably selected from Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce aminoalkyl, -OH, -NH2, -CN and halogen. In some embodiments, [Linker] is a three-branch hydrocarbon group having up to 200, preferably up to 100 carbon atoms in its backbone and is made of one or more polyethylene moi eties (e.g. comprising from 2 to 10 monomers), one or more hydrocarbon chains, typically C2-C20 alkylene, and one or more connecting groups such as lysine, valine, citrulline, alanine, -CONH- and moieties (1), (2), (3) or (4) as shown above.

[0406] In some embodiments, [Linker] comprises a trifunctional connector on which the at least three branches are bound. Trifunctional connectors encompass, without being limited to, lysine, glycerol, 3, 5 -di (hydroxymethyl) phenol, 3,5-di(aminomethyl)phenol, 3,5-di(aminomethyl) phenylamine and tris(2-aminoethyl)amine.

[0407] In a particular embodiment, [Linker] comprises a lysine residue as trifunctional connector.

[0408] In some embodiments, [Linker] is of formula

[0409]

[0410] wherein:

[0411] - L3 is a chain comprising up to 80 carbon atoms, and comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-,, amino acid residues and / or saturated or unsaturated C₃-C₆-membered cyclic or heterocyclic group such as triazole or pyrazole,

[0412] - L₄ is a unsaturated or saturated C₂-C₃₀ hydrocarbon chain which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR₄(C=O)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,

[0413] - Each R₄ is independently H, or a C₁-C₃ alkyl

[0414] r is an integer from 0 to 3, preferably 0 or 1 and

[0415] s is an integer from 0 to 6.

[0416] Properties o f the fluorogenic probes o f the invention

[0417] The fluorogenic functionalized cyanine dimer probe of the invention has generally an emission wavelengths ranging from 580 to 900 nm, preferably from 640 to 850 nm and / or an absorption wavelength spectrum from 540 to 850.

[0418] In some embodiment, the emission maximum wavelength (em λmax) of the fluorogenic probe is generally in a range from 580 to 900 nm. In some embodiments, the emission maximum wavelength (em λmax) of the fluorogenic probe is in the range of 650 to 690 nm e.g. for a probe in which the cyanine core is Cy5.0. In some embodiments, em λmax is in a range from 680 to 720 nm, e.g. for a probe in which the cyanine core is Cy5.5. In some embodiments, em λmax is in a range from 800 to 850 nm, e.g. for a probe in which the cyanine core is Cy7.5.

[0419] In some embodiment, the absorption maximum wavelength (abs λmax) of the fluorogenic probe is in a range from 540 to 850 nm. In some embodiments, abs λmax is in a range from 590 to 670 nm, e.g. for a probe in which the cyanine core is Cy5.0. In some embodiments, abs λmax is an range from 620 to 700 nm, e.g. for a probe in which the cyanine core is Cy5.5. In some embodiments, abs λmax is a range from 750 to 800 nm, e.g. for a probe in which the cyanine core is Cy7.5.

[0420] The photophysical properties of the probes (or those of the dimers) can vary depending on the medium. In some embodiments, the above indicated photophysical properties refer to those in DMF.

[0421] In some embodiments, the probe of the invention has reduced non-specific interactions with BSA and / or liposomes, as compared to the corresponding “control” unfunctionalized probe (i.e. the probe having “methyl” groups in place of “R groups ” in the cyanine cores).

[0422] The reduction in non-specific interactions can be evidenced as shown in the example section (Example 2) by determining the fluorescence ratio (I / Io), (I) being the fluorescence intensity integral of the probe in the presence of BSA or liposomes and (Io) being the fluorescence intensity integral of the probe in the solvent alone (i.e. without liposome and BSA).

[0423] The non-specific interactions are reduced if (I / Io) of the probe of the invention is at least 1.1-fold lower (e.g. of at least 1.2-fold such as at least 1.5- or 2-, 3-, 4- or 5-fold) than the (I / Io) of the control probe.

[0424] Typically, I / Io can be determined as shown in the Examples section, e.g. in the presence of 1% of BSA in HEPES or 200 μm of DOPC / cholesterol (2:1) liposomes in HEPES.

[0425] In some embodiments, the I / Io ratio of the probe of the Invention determined in the presence of 1% of BSA in HEPES is at most 9.7, preferably lower than 6.0, e.g. from 1.5 to 5.5.

[0426] In some embodiments, the I / Io ratio of the probe of the Invention determined in the presence of 200 mM of DOPC / cholesterol (2:1) liposomes in HEPES is at most 37, preferably lower than 10, e.g. from 4.0 to 9.5.

[0427] It should be noted that that the nature of the Ligand can impact said values.

[0428] In some embodiments such (I / Io) ratios as shown just above correspond to those of the chemical intermediate (IIc) as shown further below.

[0429] As mentioned above, the probe of the Invention is preferably a turn-on fluorogenic probe, which means that the probe is able to switch on its fluorescence when interacting with the molecular target.

[0430] Indeed, the cyanin units of the probe are flat aromatic scaffolds with a tendency to 7t-stack into poorly fluorescent intramolecular H-aggregates in polar aqueous media and to disassemble in apolar media in the vicinity of membrane lipid bilayers to recover their fluorescent form. The turn-on properties of the probe of the invention can be quantified as shown in the Example section through the so-called turn-on “ratio” (see Example 2). The turn-on “ratio” corresponds to the ratio of the quantum yield (QY) of the probe in its highly fluorescent form in DMF (QYDMF) to the quantum yield of the probe in its non-fluorescent form in H₂O (QYH2O). Higher the ratio, higher the turn-on capacity of the probe.

[0431] The QY are typically measured by comparison with the QY of rhodamine 800 in 25% of EtOH as defined in Alessi, J. Lumin. 2013, 134, 385-389. https: / / doi.org / 10.1016 / j.jlumin.2012.08.017.

[0432] In some embodiments, the turn-on ratio of the probe of the Invention is higher than 10, preferably higher than 20, e.g. of at least 30, 40, or 50.

[0433] It should be noted that that the nature of the [Ligand] can impact said values. In some embodiments such turn-on ratio as shown just above correspond to those of the chemical intermediate of formula (lie) as shown further below.

[0434] Particular embodiments of the cyanine fluorogenic dimer probes of the invention.

[0435] In some embodiments, the cyanine fluorogenic dimer probe of formula (I) is characterized by one or several (e.g. 1, 2, 3, or all) of the following features:

[0436] each [

[0437]

[0438] Cy] isL Jn (DI) with n is 1, 2 or 3, preferably 2 or 3, and / or Gi is Gia and G2 is G2a, and / or

[0439] - R in G2 is of formula (a

[0440]

[0441] i):INwherein o, L2 and R15 are as defined herein and / or

[0442] - Ri in Gi is a polyethylene chain comprising from 2 to 10 monomers and terminated by -(CH2)PRi6 wherein p is an integer from 0 to 6 and Ri6 is selected from H, OH, halogen, C1-C3 alkoxy, and SO3H, and / or

[0443] - Li in G2 is a C1-C20 alkylene, and / or

[0444] [Ligand] is an antibody, a fragment thereof including Fab, Fab’, and VHH (also called nanobody), a ScFv, or an endogenous ligand of a GPCR such as oxytocin or apelin, and / or

[0445] [Linker] is of formula (Ea) and (Eb) as described above, and / or

[0446] [Ligand] is linked to [Linker] by a connecting group selected from moieties (1), (2), (3), (4), (1’), (2’), (3’), (4’), (5’), (6’), (7’), (8’) and (9’).

[0447] [Ligand] is an antibody and [Ligand] is linked to [Linker] through a moiety as shown in (1 ’), (2’), (3’), (4’), (5’), (6’), (7’), (8’) and (9’) as described above, in particular (4’)

[0448] In some embodiments, the cyanine fluorogenic dimer probe is or

[0449]

[0450] Wherein:

[0451] - Ls is a chain comprising up to 70 carbon atoms, and comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, - amino acid residues and / or saturated or unsaturated C3-C6-membered cyclic or heterocyclic group such as triazole or pyrazole,

[0452] - L4is a unsaturated or saturated C2-C30 hydrocarbon chain which is optionally interrupted by one or more groups selected from C(=O), O, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,

[0453] - Each R4is independently H, or a C1-C3 alkyl

[0454] - r is 0 or 1

[0455] s is an integer from 0 to 6

[0456] [Ligand] is as defined herein and

[0457] [Cy] is as defined herein.

[0458] In some embodiments, [Cy] is

[0459]

[0460] wherein Li, X' and Ri are as defined above in formula (I) and L2 and R15 are as defined above.

[0461] In some embodiments, [Ligand] is an endogenous ligand of a GPCR, an antibody or a fragment thereof.

[0462] As examples of fluorogenic cyanine dimer probe of the invention, one can cite:

[0463]

[0464] A fluorogenic cyanine dimer probe comprising a peptide, a polypeptide or a protein as ligand wherein [Ligand] is a peptide, a polypeptide, or a protein, preferably an antibody, and R is as defined herein.

[0465] [Ligand] is linked to [Linker] through the lateral chain of two cysteine residues as depicted by “S” atom.

[0466] - Method for preparing the cyanine fluorogenic dimer probes. Synthesis intermediates and kits

[0467] The cyanine fluorogenic dimer probes can be prepared by standard synthesis routes as illustrated in the example section. The synthesis of the molecules can involve different types of reactions depending on the chemical groups present in the probe. Typically, the method for preparing a cyanine fluorogenic dimer probe of the invention comprise several synthesis steps based on chemical reactions such as amidation reaction, peptide synthesis, bioconjugation reaction, click-chemistry reaction and the like as described herein. The skilled artisan will be able to adapt the chemical synthesis detailed in the example section so as to prepare any specific probe of the invention.

[0468] The method for preparing a probe according to the invention includes the synthesis of one or more chemical intermediates which are also an object of the invention per se.

[0469] Thus, in a further aspect, the Invention relates to a chemical intermediate

[0470] which is of formula:

[0471] [Cyi] [Cyi] [Cyi] [Cyi] [Cy] [Cy]

[0472] \y \y ' \y

[0473] [Linker] [Linker] [Linker]

[0474] [Ligand] [CONNECT] [CONNECT]

[0475] (Ila), (Uh) or (lie)

[0476] Wherein:

[0477] [Linker] is as defined herein for formula (I)

[0478] [Ligand] is as defined herein for formula (I)

[0479] [Cy] is as defined herein for formula (I)

[0480] - Each [Cyi] is selected from the group consisting ofn(Dli) with n is 1, 2 or 3; and

[0481]

[0482] G2i is of formula Wherein:

[0483] - Li and X are as defined in formula (I) and

[0484] - Ri is a chemical reactive group able to form a covalent bond by click reaction or bioconjugation reaction

[0485] - [CONNECT] is a reactive group able to create at least one covalent bond by click reaction or a reactive group able to create at least one covalent bond by bioconjugation with an amino acid,

[0486] and wherein [Ri] and [CONNECT] are selected so as to be orthogonal when the chemical intermediate is of formula (lib).

[0487] Click-chemistry reactions of interest are cited hereabove in the “general definition” section. For sake of clarity, one can consider that the click-reaction is performed between two reactive groups called “Q” and “M”. Examples of complementary click-chemistry groups and click chemistry reactions include, but are not limited to: azido-alkyne click-chemistry (M= azide and Q= alkyne (e.g. strained intra-cyclic alkyne)), Staudinger Ligation (M=azide and Q=phosphine), carbonyl condensation (M= aldehyde or ketone and Q= hydrazide or oxyamine), sydnone-alkyne cycloaddition (M=sydnone and Q=alkyne), tetrazole-ene reaction (M=tetrazole and Q=alkene), nitrile- oxide-ene click chemistry (M= nitrile oxide or aldehyde, oxime, or hydroxymoyl chloride or chlororoxime and Q= alkene or alkyne), nitrile imine-ene click chemistry (M= nitrile imine or aldehyde, hydrazone, hydrazonoyl chloride or chlorohydrazone and Q= alkene or alkyne), inverse electron demand Diels-Alder ligation (M= alkene and Q= tetrazine), thia-diels alder (M=phosphonodithioester Q = diene), isonitriletetrazine click chemistry (M= isonitrile and Q= tetrazine), Suzuki-Miyaura coupling (M= aryl halide and Q= aryl boronate).

[0488] A reactive group able to create a covalent bond by click-reaction may thus be selected among the M and Q couples as described above.

[0489] Other reactions of interest include reactions between aldehyde / ketone and alkoxyamine or hydrazine, and reductive amination.

[0490] Bioconjugation is as defined in the general definition and hereabove in the section dedicated to the [Linker] (with respect to the bond to [Ligand]).

[0491] Depending on the targeted amino acid in the [Ligand], the chemical reactive group may be:

[0492] - For reaction with amino group of amino acid residues (e.g. with epsilon-NFE in lysine), the reactive group may be an activated carboxylic acid e.g. NHS-ester, a benzoyl fluoride, an isocyanate, an isothiocyanate,

[0493] - For reaction with phenol group in a tyrosine residue: aryldiazonium, PTAD (4-Phenyl- 1,2,4-triazoline-3, 5 -di one)

[0494] - For reaction of -SH in a cysteine residue: maleimide, 3 -arylpropionitrile (APN), diAPN, iodoacetamide.

[0495] In the present application, it is understood that a “diAPN moiety” is a moiety comprising two 3-arylpropionitrile (APN) groups.

[0496] In some embodiments, the reactive group present in [Ri] is able to create a covalent bond through a click reaction selected from alkyne-azide cycloaddition, sydnone and iminosy dnone-alkyne cycloaddition, tetrazole / alkene reaction, reaction of aldehyde / ketone with alkoxyamine or hydrazine, amidation, reductive amination, Diels-Alder, and thia-Diels-Alder.

[0497] In some embodiments, the reactive group present in Ri is able to create a covalent bond through alkyne-azide cycloaddition. Preferably, said reactive group is selected from an alkyne, BCN, DBCO and an azide.

[0498] In some embodiments, Ri is of formula -CH₂-C≡CH.

[0499] In some embodiments, the reactive group present in [CONNECT] is able to create a covalent bond through a click reaction selected from alkyne-azide cycloaddition, sydnone and iminosydnone-alkyne cycloaddition, tetrazole / alkene reaction, reaction of aldehyde / ketone with alkoxyamine or hydrazine, amidation, reductive amination, Diels-Alder, and thia-Diels-Alder.

[0500] In some embodiments, the reactive group present in [CONNECT] is able to create a covalent bond through alkyne-azide cycloaddition. Preferably, said reactive group is selected from an alkyne, BCN, DBCO and an azide.

[0501] In some embodiments, [CONNECT] is able to react through a click reaction so as to provide one of the moieties (1), (2), (3), and (4) as described above.

[0502] In some embodiments, the reactive group present in [CONNECT] is able to create a covalent bond by bioconjugation with an amino acid residue selected from lysine, tyrosine and cysteine. In some embodiments, the reactive group present in [CONNECT] is selected from the group consisting of PTAD, APN, diAPN, arylsulfonamide, maleimide, benzoyl acrylamide, iodoacetamide, an activated ester such as NHS-ester, isothiocyanate, isocyanate, and benzoyl fluoride.

[0503] In some embodiments, [CONNECT] is selected from the group consisting of: -N₃, -CH₂C≡CH, a strained alkyne such as BCN and DBCO, NHS-ester,

[0504]

[0505] In the intermediate of formula (lib), both Ri and [CONNECT] are present. In that case, the reactive group present in Ri and that present in [CONNECT] are orthogonal, namely cannot cross-react or react in the same conditions. The skilled artisan clearly knows how to select the reactive groups so as to avoid any unwanted cross- or side-reactions. For instance, when Ri comprises an alkyne group (e.g. is -CH₂-C≡CH or a strained alkyne such as DBCO or BCN), [CONNECT] is selected from APN, -diAPN, maleimide, and N-hydroxysuccinimide (see above groups (1”), (2”), (3”) and (4”)).

[0506] In some embodiments, the Invention relates to a chemical intermediate which is of formula

[0507]

[0508] Wherein:

[0509] - R17is selected from the group consisting of OH, NH₂, an activated carboxylic acid, and [CONNECT]-L5- - R18is selected from the group consisting of OH, NH₂, an activated carboxylic acid, [LIGAND]-L5- and [CONNECT]-L5-,

[0510] - L4 is a C2-C30 unsaturated or saturated hydrocarbon chain, which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,

[0511] - Ls is a hydrocarbon chain comprising up to 70 carbon atoms, and which comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups preferably selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, and / or amino acid residues such as valine, citrulline, glycine and alanine, and / or saturated or unsaturated Cs-Ce-membered cyclic or heterocyclic group such as phenyl, triazole or pyrazole

[0512] - Each R4 is independently H, or a C1-C3 alkyl

[0513] r is an integer from 0 to 3, preferably 0 or 1

[0514] [CONNECT], [LIGAND], [Cyi] and [Cy] are as defined above.

[0515] In some embodiments, the chemical intermediate of formula (lib), (lie), (lid) or (lie) comprises a [CONNECT] moiety which is a diAPN moiety, preferably of formula:

[0516]

[0517] Such chemical intermediates are particularly suitable to create covalent bonds in cysteine residues initially engaged in a disulfide bridge in [Ligand], e.g. an antibody comprising an intrachain S-S bridge between two cysteines located in the hinge region. To allow the creation of the covalent bonds, the S-S bridge in the [Ligand] is firstly reduced by conventional means (e.g. by P-mercaptoethanol, dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)), preferably in mild condition so as to specifically reduce the sulphide bridges present in the hinge region. Then the [Ligand] is reacted with the chemical intermediate bearing the di-APN moiety. Such a reaction enables the formation of the following structures (depending on the diAPN moiety used - “S” depicting the lateral “S” present in a cysteine residue in the [Ligand])'.

[0518]

[0519] Such a strategy of coupling is of high interest when the ligand is an antibody. The Inventors showed that by this method of coupling enables specific coupling of the probe to cysteine residues in the hinge region of the antibody, giving a final probe / antibody ratio ranging from 0.6 to 1.2.

[0520] In a more general aspect, the invention relates to a method of covalent coupling a functional moiety on an antibody by using of a diAPN moiety preferably of formula (1”) or (4”) as shown hereabove.

[0521] More precisely, the Invention also relates to a method for chemically-modifying an antibody having a S-S bridge between two intramolecular cysteine, said method comprising the steps of a) Reducing a S-S bridge in the antibody so as to obtain two -SH group, preferably with TCEP so as to specifically reduced a S — S bridge in the hinge region, and

[0522] - b) Incubating said antibody with a chemical reagent bearing a diAPN group preferably of formula (1”) or (4”) in conditions conducive for reacting said diAPN group with the thiol groups formed in step a) so as to form covalent bounds.

[0523] The resulting structure are as shown above in (T) and (4’) (with LIGAND being an antibody).

[0524] In an additional aspect, the Invention relates to a kit for the preparation of a cyanine fluorogenic dimer probe for the labelling of a molecular target. The kit typically comprises:

[0525] a chemical intermediate as described herein

[0526] optionally one or more chemical reagents, so as to prepare the final probe (e.g. catalyst) and

[0527] optionally instructions for preparing the turn-on cyanine fluorogenic dimer probe from the chemical intermediate.

[0528] The kit of the invention may also comprise

[0529] one or several buffers for implementing the coupling reaction so as to obtain the final probe and / or

[0530] - the ligand to be coupled and / or

[0531] means for purification of the chemical intermediates and / or the final probe.

[0532] In some embodiments, the chemical intermediate is of formula (Ila). In such embodiments, the kit comprises one or more chemical agents so as to obtain the desired [Cy] moiety of the final probe, by reaction with [Cyi]. By virtue of the disclosure provided in the present application, one skilled in the art is able to determine the required chemical agent(s) so as to obtain the desired probe, specifically the desired [Cy] moiety. In some embodiments, the chemical intermediate is of formula (lib). In such embodiments, the kit comprises one or more chemical agents so as to obtain the [Cy] and [Ligand] moieties of the final probe, by reaction with [Cyi] and [Connect], By virtue of the disclosure provided in the present application, one skilled in the art is able to determine the required chemical agents so as to obtain the desired probe, specifically the desired [Cy] and [Ligand] moieties.

[0533] In particular, when the chemical intermediate is of formula (Ila) or (lib), the Rigroup of the [Cyi] moiety is typically of formula -CH2-C≡CH. In that case, the kit of the invention advantageously comprises one or more chemical agents so as to react with Ri via an azidoalkyne click-reaction, that is, the kit of the invention advantageously comprises at least one azide compound (-N=N+=N‘). For instance, the Rigroup of the [Cyi] moiety is of formula -CH2-C≡CH and the chemical agent(s) present in the kit is of formula R15-L2-N=N+=N-, R15 and L2 being as defined herein.

[0534] In some embodiments, the chemical intermediate is of formula (lie). In such embodiments, the kit comprises one or more chemical agents so as to obtain the [Ligand] moiety of the final probe, by reaction with [Connect], By virtue of the disclosure provided in the present application, one skilled in the art is able to determine the required chemical agent(s) so as to obtain the desired probe, specifically the desired [Ligand] moiety. In some embodiments, the chemical intermediate is of formula (lie).

[0535] In some embodiments, the Flo ratio of the chemical intermediate of formula (lie) determined in the presence of 1% of BSA in HEPES is at most 9.7, preferably lower than 6.0, e.g. from 1.5 to 5.5.

[0536] In some embodiments, the Flo ratio of the chemical intermediate of formula (lie) determined in the presence of 200 mM of DOPC / cholesterol (2:1) liposomes in HEPES is at most 37, preferably lower than 10, e.g. from 4.0 to 9.5.

[0537] In some embodiments, the turn-on ratio of the chemical intermediate of formula (lie) is higher than 10, preferably higher than 20, e.g. of at least 30, 40, or 50.

[0538] In some embodiments, the kit further comprises the [Ligand],

[0539] In an additional aspect, the Invention relates to a method for modulating the properties, in particular for modulating the interactions of a cyanine fluorogenic dimer probe with plasma proteins and / or lipid bilayers such as cell membranes, said method comprising introducing a substituent R, as defined herein, in place of a methyl group in the cyanine scaffolds of the probe In an additional aspect, the Invention relates to a method for selecting a probe of the invention, the method comprising: preparing at least two cyanine fluorogenic dimer probes from the kit as described above, each probes having a different substituent R,

[0540] evaluating at least one fluorescence ratio I / Io ratio and / or the turn-on ratio of the prepared cyanine fluorogenic dimer probes, and

[0541] selecting the cyanine fluorogenic dimer probe having the lower I / Io ratio and / or the higher turn-on ratio.

[0542] As defined above, in the fluorescence ratio (I / Io), (I) represents the fluorescence intensity integral of a probe of the invention in the presence of BSA or liposomes and (Io) represents the fluorescence intensity integral of said probe in the solvent alone (i.e. without liposome and BSA). I / Io can be determined as shown in the Examples section. For instance, I / Io may be determined in the presence of 1% of BSA in HEPES or 200 pm of DOPC / cholesterol (2:1) liposomes in HEPES. In some embodiments, the selected probe exhibits a I / Io ratio, determined in the presence of 1% of BSA in HEPES, of at most 9.7, preferably lower than 6.0, e.g. from 1.5 to 5.5. In some embodiments, the selected probe exhibits a I / Io ratio, determined in the presence of 200 mM of DOPC / cholesterol (2:1) liposomes in HEPES, of at most 37, preferably lower than 10, e.g. from 4.0 to 9.5.

[0543] As mentioned above, the turn-on ratio quantifies the turn-on properties of a probe of the invention. The turn-on ratio corresponds to the ratio of the quantum yield (QY) of the probe in its highly fluorescent form in DMF (QYDMF) to the quantum yield of the probe in its non-fluorescent form in H₂O (QYH2O). The turn-on ratio can be determined as shown in the Examples section. In some embodiments, the selected probe exhibits a turn-on ratio higher than 10, preferably higher than 20, e.g. of at least 30, 40, or 50.

[0544] - Method of use the cyanine fluorogenic dimer probes and kits thereof

[0545] In a particular aspect, the Invention relates to a method of labelling a molecular target in vitro, ex vivo or in vivo, comprising a step of contacting the molecular target with a cyanine fluorogenic dimer probe disclosed herein, e.g., a compound of Formula (I).

[0546] More generally, the Invention relates to the use of a cyanine fluorogenic dimer probe of the Invention as a labelling agent, e.g. in vitro, ex vivo or in vivo. The cyanine fluorogenic dimer probe of the Invention can be used as a research tool, but also as a diagnostic agent, e.g. in biological imaging, in pharmacological studies, clinical studies and / or medical diagnosis. For instance, the cyanine fluorogenic dimer probes can be used in the context of fluorescence image-guided tumor surgery, in drug-discovery to evaluate the efficacy of a treatment on tumor regression for instance or in assessing target engagement, in diagnostic e.g. to detect receptors expressed at the cell surface in pathological conditions (e.g. such as cancers or infections - e.g. to discriminate virus or bacteria).

[0547] It goes without saying that the [Ligand] present in the compound of Formula (I) has been selected so as to bind to the molecular target, preferably with both high affinity and specificity. As mentioned above, the molecular target is preferably a membrane protein, such as a membrane receptor, e.g. an endogenous GPCR.

[0548] The cyanine fluorogenic dimer probe of the Invention is particularly suitable for use in vitro or ex vivo, e.g. for labelling a molecular target present in a sample. The sample can be of any type, e.g. it may be a cell culture, a tissue or an organoid.

[0549] In some embodiments, the sample is an organoid or a cell culture.

[0550] In other embodiments, the sample is obtained from a subject, e.g. fluids such as blood, plasma, saliva, urine and seminal fluid samples, as well as, organ tissue, or cell samples or biopsies. Thanks to its low unspecific interactions and its turn-on properties, cyanine fluorogenic dimer probe of the Invention may enable to efficiently label the molecular target in the sample. A washing step prior to visualization can be unnecessary.

[0551] When used for in vitro or ex vivo labelling, the cyanine fluorogenic dimer probe of the Invention is dissolved in an appropriate medium, typically in culture medium in an appropriate concentration and then contacted with the sample.

[0552] The concentration to be used depends on the sample to analyze, the molecular target to label and the probe to use. Typically, concentration of the probe is from lOnM to IpM

[0553] The imaging of the molecular target can be performed by detecting the fluorescence at the appropriate wavelength emission. As illustrating in the Example, the visualization of the molecular target can be done by confocal microscopy. The excitation wavelength of a probe of the invention is typically in a range from 580 to 900 nm and the emission wavelength is typically in a range from 540 to 850.

[0554] Of course, any other methods disclosed in the prior art can be used.

[0555] As explained above, due to its particular properties, the probe of the Invention is particularly suitable for in vivo imaging of a molecular target. Accordingly, the Invention also relates to a probe as described herein for use as an imaging agent in vivo. The Invention also relates to the use of a cyanine fluorogenic dimer probe as described herein in the manufacture of a diagnostic agent in vivo.

[0556] The probe of the invention can be formulated with an appropriate pharmaceutical vehicle before being administered to a subject, e.g. in a physiological liquid. The administration route can be of any type and depends on the molecular target to visualize. For the instance, the administration route can be intravenous, intraperitoneal, oral, intracranial, intramuscular, subcutaneous, intradermal, intranasal, mucosal, pulmonary, intra-tumoral and the like.

[0557] In a particular embodiment, the Invention relates to a pharmaceutical composition comprising a probe of formula (I) as described herein and a pharmaceutically acceptable excipient. composition. The pharmaceutical composition may comprise (i) a compound according to the invention in combination with (ii) one or several pharmaceutically acceptable excipients. For instance, the pharmaceutical composition of the invention may comprise:

[0558] from 0.01% to 90% by weight of a compound of the invention, and

[0559] from 10% to 99.99% by weight of excipients,

[0560] the percentage being expressed as compared to the total weight of the composition.

[0561] The pharmaceutical composition of the invention may be formulated according to standard methods such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty first Edition, 2005).

[0562] Pharmaceutically acceptable excipients that may be used are, in particular, described in the Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; 6th revised edition, 2009). Typically, the pharmaceutical composition of the invention may be obtained by admixing the compound of the invention with at least one pharmaceutically acceptable excipient.

[0563] By pharmaceutically acceptable excipient is meant according to the present invention any ingredient commonly used in the formulation of a pharmaceutical composition, which is inactive and non-toxic, the purpose of which may be to impart a particular consistency, or other particular physical or taste characteristics to the finished product, while avoiding any chemical interaction with the therapeutically active compound. Examples of appropriate excipients include, but are not limited to, solvents such as water or water / ethanol mixtures, fillers, carriers, diluents, binders, anti-caking agents, plasticizers, disintegrants, lubricants, flavors, buffering agents, stabilizers, colorants, dyes, anti-oxidants, anti-adherents, softeners, preservatives, surfactants, wax, emulsifiers, wetting agents, and glidants. Examples of diluents include, without being limited to, microcrystalline cellulose, starch, modified starch, dibasic calcium phosphate dihydrate, calcium sulfate trihydrate, calcium sulfate dihydrate, calcium carbonate, mono- or disaccharides such as lactose, dextrose, sucrose, mannitol, galactose and sorbitol, xylitol and combinations thereof. Examples of binders include, without being limited to, starches, e.g., potato starch, wheat starch, corn starch; gums, such as gum tragacanth, acacia gum and gelatin; hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose; polyvinyl pyrrolidone, copovidone, polyethylene glycol and combinations thereof. Examples of lubricants include, without being limited to, fatty acids and derivatives thereof such as calcium stearate, glyceryl monostearate, glyceryle palmitostearate magnesium stearate, zinc stearate, or stearic acid, or polyalkyleneglycols such as PEG. The glidant may be selected among colloidal silica, dioxide silicon, talc and the like. Examples of disintegrants encompass, without being limited to, crospovidone, croscarmellose salts such as sodium croscarmellose, starches and derivatives thereof. Examples of surfactants encompass, without being limited to, simethicone, triethanolamine, polysorbates and derivatives thereof such as tween® 20 or tween® 40, poloxamers, fatty alcohol such as laurylic alcohol, cetylic alcohol and alkylsulfate such as sodium dodecyl sulfate (SDS). Examples of emulsifiers encompass for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, polyethyleneglycol and fatty acid esters of sorbitan or mixtures of these substances.

[0564] It goes without saying that the excipient(s) to be combined with the compound of the invention may vary upon (i) the pharmacokinetic profile sought for said active ingredient, (ii) the dosage form and (iii) the route of administration.

[0565] The pharmaceutical composition may be of any type.

[0566] In some embodiments, the pharmaceutical composition of the invention may be in the form of a liquid composition ready to be administered, a concentrated liquid composition to be diluted before administration, or a powder e.g. a freeze-dried powder which is to be dissolved or suspended in an appropriate vehicle just before being administered to the subject or used in vitro or ex vivo.

[0567] In addition to the kits for the preparation of a probe as described herein, the Invention also relates to a kit useful for implementing the method for labelling a molecular target as described herein, in vivo, in vitro or ex vivo.

[0568] Thus, in an additional aspect, the Invention relates to a kit which includes a container and at least one compound provided herein, e.g., a probe of Formula (I), e.g. in the form of a salt and / or a solvate such as a hydrate. The probe of formula (I) can be formulated with any appropriate pharmaceutical excipient, e.g. such as a carrier and the like. In other words, in some embodiments, the container contains a pharmaceutical composition of the invention.

[0569] The kit provided herein can further include a device that is used to administer the probe provided herein, e.g. by injection such as needles and syringes.

[0570] The kit provided herein can further include a pharmaceutically acceptable vehicle that can be used to administer one or more the compound provided herein. For example, if the probe provided herein is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the compound can be dissolved to form a sterile solution that is suitable for parenteral administration.

[0571] In some embodiments, the kit may further comprise

[0572] a control sample for the normalization of the biomarker quantified by the probe of the Invention,

[0573] a control sample, such as a positive or negative control sample, for validation.

[0574] Moreover, the kit according to the invention may comprise a notice providing its user with instructions for implementing the method according to the invention by means of the kit.

[0575] The following examples are given for purposes of illustration and not by way of limitation.

[0576] EXAMPLES EXAMPLE 1: Chemical synthesis

[0577] General Methods

[0578] Unless otherwise noted, reagents and anhydrous solvents were purchased from commercial sources (Sigma Aldrich, Fluorochem, TCI, etc.) and were used without any further purification. All compounds were named using the software ChemBioDraw Ultra 20.

[0579] Reactions were monitored by thin-layer chromatography (TLC) on Merck aluminium sheets silica gel 60 F254, which were visualized with UV light (254 nm, 365 nm) and KMnO4 stain or by analytical reverse-phase high-performance liquid chromatography (see methods below). VWR silica gel (40-63 pm) was used for chromatography columns. Semi-preparative reversephase HPLC purifications were performed on a Waters SunFire Cl 8 OBD Prep column (5 pm, 19 x 150 mm) on a Gilson PLC2020 system.

[0580] Low-resolution mass spectra (LRMS) and high-resolution mass spectra (HRMS) were obtained on an Agilent Technologic 6520 Accurare-Mass Q. Tof LC / MS apparatus equipped with a Zorbax SB C18 column (1.8 pm, 2.1× 50 mm) using electrospray ionization (ESI) and a time-of-fhght analyzer (TOF).

[0581] Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance III 400MHz, 500MHz or 700MHz BBFO+ probe spectrometer at 25°C. Deuterated solvents were purchased from Sigma-Aldrich. Chemical shifts are reported in ppm (5), relatively to residual solvent. Coupling constants values J are given in Hz. Abbreviations for NMR are as follows: br = broad, s = singulet, d = doublet, t = triplet, q =quartet, quint = quintet, m = multiplet.1H and13C NMR signals were assigned on the basis of 2D-NMR (COSY, NOESY, HMBC, HSQC) experiments. The analyses are indicated in the following manner:1H NMR (field MHz, solvent, Temperature) 5 (ppm) = chemical shift (multiplicity, coupling constant, integration value, attribution).13C NMR (field MHz, solvent) 5 (ppm) = chemical shift (attribution).

[0582] Analytical RP-HPLC: Analytical reverse-phase high-performance liquid chromatography was performed on a Cl 8 Kinetex column (5 pm, 4.6 mm × 150 mm) with a flow rate of 2.2 mL / min.

[0583] Method A. linear gradient 5% to 100% in 7 min, then 100% for 3 min of MeCN with 0.1% TFA (v / v) in H2O with 0.1% TFA (v / v) with detection at 220, 254, 320, 465, 530, and 630 nm.

[0584] Method B. linear gradient 30% to 100% in 10 min, then 100% for 3 min of MeCN with 0.1% TFA (v / v) in H2O with 0.1% TFA (v / v) with detection at 220, 254, 320, 465, 530, and 630 nm.

[0585] EXAMPLE 1A: Synthesis of a cyanine scaffold functionalized with an alkyne

[0586] [OF 0443] 1, 1,2-trimethyl-3-(2,5,8, 11,14, 17,20,23-octaoxapentacosan-25-yl)- 1H-benzo[e]indol-3-ium iodide

[0587] C32H50INO8 MW: 703.26 g mol’1Purple oil Yield: 56 %

[0588]

[0589] l,l,2-trimethyl-lH-benz[e]indole (101 mg, 0.483 mmol, 1.3 eq), 5-chloro-2,5,8,ll,14,17,20;23-9ctaoxapentacosanel (150 mg, 0.372 mmol, 1 eq) and sodium iodide (271 mg, 1.82 mmol, 5 eq) were dissolved in acetonitrile (1 mL). The mixture was heated at 120°C in a sealed tube for 16 h. The solvent was evaporated and the residue was purified by column chromatography on silica gel (CH2CI2 to CH2Ch / MeOH 85 / 15) to afford OF_0443 as a purple oil (145 mg, 56 %).

[0590] TLC monitoring: CH2Cl2 / MeOH (90 / 10) Rf = 0.45

[0591] ’H NMR (CDCh, 400 MHz, 300 K): 5 (ppm) 8.09 (q, J = 8.6, 8.0 Hz, 3H), 7.86 (d, J = 8.9 Hz, 1H), 7.79 - 7.64 (m, 2H), 4.97 (t, J = 4.9 Hz, 2H), 4.04 (t, J = 4.8 Hz, 2H), 3.66 - 3.44 (m, 28H), 3.36 (d, J = 0.8 Hz, 3H), 2.97 (s, 3H), 1.81 (s, 6H).

[0592] HPLC: tr = 3.553 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0593] [OF 0459] l,2-dimethyl-l-(prop-2-yn-l-yl)-lH-benzo[e]indole

[0594] C17H15N

[0595] MW: 233.31 g mol’1

[0596] Orange oil

[0597] Yield: 64 %

[0598]

[0599] (Naphthalen-2-yl)hydrazine hydrochloride (900 mg, 4.62 mmol, 1 eq) and 3-methylhex-5-yn-2-one2(509 mg, 4.62 mmol, 1 eq) were dissolved in acetic acid (18 mL) and the red mixture was stirred at 100°C for 16 h. The solvent was evaporated and the residue was purified by column chromatography on silica gel (Heptane / EtOAc 100 / 0 to 50 / 50) to afford OF_0459 as an orange oil (694 mg, 64 %).

[0600] Rf = 0.5 (Heptane / EtOAc 5 / 5)

[0601] ’H NMR (CDCh, 400 MHz, 300 K): 5 (ppm) 7.95 (d,3JHH = 9.7 Hz, 2H, CH^), 7.88 (d,3JHH = 8.5 Hz, 1H, CH^), 7.79 (d,3JHH = 8.5 Hz, 1H, CH^), 7.54 (m, 1H, CH^), 7.45 (m, 1H, CH^), 2.97 (dd,2JHH = 17.1 Hz,4JHH = 2.7 Hz, 1H, CH2A), 2.89 (dd,2JHH = 17.1 Hz,4JHH = 2.7 Hz, 1H, CH2B), 2.45 (s, 3H, CH3), 1.17 (t,4JHH = 2.7 Hz, 1H, CHakyne), 1.60 (s, 3H, CH3)

[0602] 1Prepared as disclosed in Esteoulle, L.; Daubeuf, F.; Collot, M.; Riche, S.; Durroux, T.; Brasse, D.; Marchand, P.; Karpenko, J.; Klymchenko, A. S.; Bonnet, D. A Near-Infrared Fluorogenic Dimer Enables Background-Free Imaging of Endogenous GPCRs in Living Mice. Chem. Sci. 2020, 11 (26), 6824-6829. https: / / doi.org / 10.1039 / D0SC01018A.

[0603] 2Prepared as described in Zhang, W.-B.; Xiu, S.-D.; Li, C.-Y. Rhodium-Catalyzed Synthesis of Multi- Substituted Furans from N-Sulfonyl-1,2,3-Triazoles Bearing a Tethered Carbonyl Group. Org. Chem. Front. 2015, 2 (1), 47-50. https: / / doi.org / 10.1039 / C4Q000273C.13C NMR(CDCl3, 100 MHz, 300 K): 5 (ppm) 187.2, 151.9, 135.9, 132.5, 129.9, 129.5, 128.7, 126.6, 124.6, 122.1, 119.9, 78.9, 70.8, 58.1, 26.2, 20.9, 16.2

[0604] HPLC: tr = 3.616 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0605] LRMS (ESI): 234.1343 [M+H]+.

[0606] [OF 0503] 3-(5-carboxypentyl)-l,2-dimethyl-l-(prop-2-yn-l-yl)-lH-benzo[e]indol-3-ium iodide

[0607] C23H26INO2

[0608] MW: 475.37 g mol'1Yellow oil

[0609] Yield: 55 %

[0610]

[0611] OF_0459 (80 mg, 0.343 mmol, 1 eq), 6-bromohexanoic acid (134 mg, 0.686 mmol, 2 eq) and sodium iodide (257 mg, 1.71 mmol, 5 eq) were dissolved in acetonitrile (2.2 mL). The mixture was heated at 80°C in a sealed tube for 16h. The volatils were evaporated, and the residue was partitioned between water and chloroform. The organic phase was dried on MgSCh and evaporated. The crude was purified by semi-preparative reversed-phase HPLC chromatography on a SunFire C18 column (5 μm, 19 × 150 mm) using a linear gradient (5% to 95% in 40 min, flow-rate of 18 mL.min-1of solvent B (0.1% TFA in ACN, v / v) in solvent A (0.1% TFA in H2O, v / v). Detection was set at 220 and 254 nm. Fractions containing the desired product were freeze-dried to afford OF_0503 as a yellow oil (89 mg, 55 %).

[0612] Rf= 0.40 (CH2Cl2 / MeOH 9 / 1)

[0613] ’H NMR (CDCI3, 400 MHz, 300 K): 5 (ppm) 8.16 (d,3JHH = 8.7 Hz, 1H, CH^), 8.09 (d,3JHH = 6.7 Hz, 1H, CH^), 8.02 (d,3JHH = 8.4 Hz, 1H, CH^), 7.80-7.66 (m, 3H, CH^), 4.71-4.51 (m, 2H, N-CH2), 3.28 (dd,2JHH = 17.2 Hz,4JHH = 2.6 Hz, CH2A), 3.22 (dd,2JHH = 17.2 Hz,4JHH = 2.6 Hz, CH2B), 2.95 (s, 3H, CH3), 2.36 (t,3JHH = 6.8 Hz, 2H, CH2-COOH), 2.05-1.90 (m, 2H, CH2), 1.86-1.76 (m, 4H, CH3 and CHalkv,lc), 1.74-1.64 (m, 2H, CH2), 1.63-1.53 (m, 2H, CH2)13C NMR(CDCl3, 125 MHz, 300 K): δ (ppm) 194.0, 177.1, 139.3, 134.4, 134.1, 132.5, 130.5, 129.3, 128.1, 127.8, 122.3, 111.9, 75.6, 72.9, 59.1, 48.8, 33.5, 28.1, 26.9, 26.1, 24.0, 20.4, 14.2

[0614] HPLC: tr = 3.362 min (purity > 95% at [220.4 nm] and [254.4 nm], method A) LRMS (ESI): 348.2014 [M]+

[0615] [Cy-B] 3-(5-carboxypentyl)-2-((lE,3E,5E)-5-(l,l-dimethyl-3-(2, 5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l,3-dihydro-2H-benzo[e]indol-2-ylidene)penta-l,3-dien-l-yl)-l-methyl-l-(prop-2-yn-l-yl)-lH-benzo[e]indol-3-ium 2,2,2-iodide

[0616] C58H75IN2O10 MW: 1086.45 g mol'1Dark blue powder Yield: 59 %

[0617]

[0618] The reaction was carried out under an argon atmosphere. OF_0443 (105 mg, 0.149 mmol, 1 eq) and malonaldehyde dianilide hydrochloride (43 mg, 0.160 mmol, 1.1 eq) were dissolved in acetic anhydride (1.4 mL) and acetyl chloride (1 mL). The mixture was stirred at 100°C for 3 h. The volatils were evaporated and the residue was eluted through a short plug of silica gel (CH2Cl2 / MeOH 90 / 10). The orange residue was then dissolved in pyridine (2 mL) and OF_0503 (71 mg, 0.149 mmol, 1 eq) was added. The mixture was stirred at 60°C for 16 h. The volatils were evaporated, and the residue was partitioned between HC1 1 M and dichloromethane. The organic phase was washed with an aqueous solution of Nal (12 %), dried on MgSCh and evaporated. The residue was purified by column chromatography on silica gel (CH2Cl2100% to CH2Cl2 / MeOH 80 / 20) to afford Cy-B as a dark blue solid (96 mg, 59 %). Rf= 0.35 (CH2Cl2 / MeOH 9 / 1)

[0619] ’H NMR (CDCh, 400 MHz, 300 K): 5 (ppm) 8.71-8.57 (m, 2H, CHbridge), 8.18 (d,3JHH = 8.1 Hz, 1H, CH^), 8.10 (d,3JHH = 8.2 Hz, 1H, CH^), 7.93-7.80 (m, 4H, CH^), 7.58-7.50 (m, 2H, CH^), 7.49-7.36 (m, 3H, CH^), 7.31 (d,3JHH = 8.0 Hz, 1H, CH^), 6.78 (t,3JHH= 12.4 Hz, 1H, CHbridge), 6.39 (d,3JHH= 13.7 Hz, 1H, CHbridge), 6.23 (d,3JHH= 13.7 Hz, 1H, CHbridge), 4.44-4.68 (m, 2H, N-CH2PEG), 4.16-4.02 (m, 2H, N-CH2), 3.94 (t,3JHH = 5.4 Hz, 2H, O-CH2PEG), 3.80 (d,2JHH = 14.4 Hz, CH2A), 3.66-3.41 (m, 29H, O-CH2PEGand CH2B), 3.33 (s, 3H, CH3PEG), 2.37 (t,3JHH = 7.2 Hz, 2H, CH2), 2.17 (s, 3H, CH3), 2.12 (s, 6H, 2 * CH3), 1.90-1.80 (m, 2H, CH2), 1.74-1.64 (m, 2H, CH2), 1.61-1.50 (m, 2H, CH2 and CHalkyne)13C NMR(CDCl3, 125 MHz, 300 K): δ (ppm) 176.4, 175.8, 171.4, 153.8, 152.3, 140.7, 139.8, 134.4, 131.9, 131.7, 131.0, 131.0, 130.9, 130.2, 130.1, 129.9, 128.3, 128.1, 128.0, 127.7, 126.9, 125.2, 125.0, 124.5, 122.7, 121.9, 111.4, 110.1, 104.2, 103.3, 79.1, 71.9, 71.1, 70.7-70.4 (multiple signals, O-CH2PEG), 68.3, 59.1, 54.7, 51.7, 45.1, 44.3, 33.9, 31.3, 28.0, 27.9, 27.4, 26.5, 26.3, 24.5.

[0620] HPLC: tr = 5.479 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0621] HRMS (ESI): calculated for C58H75N2O10 [M]+959.5417, found 959.5411 (A = 1.1 ppm)

[0622] EXAMPLE IB: General proceeding for CuAAC on cyanine scaffold or dimer Click reacton of functionalized azide on cyanine-alkyne:

[0623] In a glass vial, the alkyne cyanine (1 eq) and the azide compound (1.5 eq) were dissolved in DMF (lmL / 50 pmol alkyne substrate). An aqueous solution of copper sulfate pentahydrate (2 eq) and sodium ascorbate (2 eq) was added to reach a total of 10% water in volume. The solution was stirred at room temperature for 15 minutes. The volatils were evaporated and the residue purified by semi-preparative reversed-phase HPLC chromatography on a SunFire C18 column (5 μm, 19 × 150 mm) using a linear gradient (10% to 95% in 40 min, flow-rate of 18 mL.min-1of solvent B (0.1% TFA in ACN, v / v) in solvent A (0.1% TFA in H2O, v / v). Detection was set at 220 and 630 nm. Fractions containing the desired product were freeze-dried to afford to desired compound.

[0624] EXAMPLE 1C: preparation of functionalized cyanine scaffolds

[0625]

[0626] The below functionalized cyanine scaffolds were prepared from Cy-B according to the general procedure described in Example IB by using the appropriate azide derivative. The reactions were performed at 46.0 pmol scale except for Cy-L (9.2 pmol scale). Ref. cyanine R” HRMS (ESI) Yield Cy-C -(CH2)3-PO3H 562.7898 [M+2H]2+72% Cy-D -(CH2)3-SO31124.5604 [M+H]+92% Cy-E -(CH2)3-N+(CH3)3551.3367 [M]2+86% Cy-F -(CH2)3-N+(CH3)2-(CH2)3-SO3605.3308 [M+H]2+62% Cy-G -(CH2)3-N(CH3)2544.3288 [M+H]2+82% Cy-H -(CH2)3-OH 1060.5995 [M]+93% Cy-I -(CH2)2-CO-NH-C(CH2-OH)31177.6412 [M]+31% Cy-J -(CH2)2-(OCH2CH2)3-OCH3596.8447 [M+H]2+81% Cy-K -(CH2)2-(OCH2CH2)7-OCH3684.8972 [M+H]2+73% Cy-L -(CH2)-(CH2CH2-NH-CO)4-CH2-O- 1344.7509 [M]+60%

[0627] CH3

[0628]

[0629] EXAMPLE ID: General procedure for the preparation of cyanine dimers

[0630] The linker

[0631]

[0632] was prepared as described in patent J. Karpenko, A. Klymchenko, D. Bonnet, M. Collot, 2023, US 2023 / 0174480, The cyanine dimer (d-Cy-X)

[0633]

[0634] was prepared by reacting the linker P2 (1 eq) with the corresponding cyanine scaffold (Cy-X, 2 eq) in anhydrous DMF (2 mL), in the presence of DIPEA (4 eq) and PYBOP (2 eq). The mixture was stirred at room temperature for Ih. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography Fractions containing the desired product were freeze-dried to afford the desired compound.

[0635] Alternatively, the cyanine dimer was obtained from alkyne functionalized dimer (d-Cy-B) by Cu-AAC as described in Example IB. d-Cy-B was prepared from Cy-B (2 eq) by reaction with the linker in the presence of DIPEA (4 eq) and PYBOP (2 eq).

[0636] d-Cy-B:

[0637]

[0638] C141H189F6N7O29

[0639] MW: 2560.07 g mol'1

[0640] Blue oil, Yield: 79 %

[0641] HPLC: tr = 6.199 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0642] HRMS (ESI): calculated for C137H189N7O25 [M]2+1166.6878, found 1166.6887 (A = 0.8 ppm)

[0643] Ref. cyanine HPLC purity

[0644] Cyanine HRMS (ESI) Yield dimers at 220 nm

[0645] d-Cy-C Cy-C 1331.7201 [M]2+> 95 % 87 % d-Cy-D Cy-D 1353.6860 [M+2Na]2+> 95 % 63 % d-Cy-E Cy-E 654.9086 [M]4+> 95 % 68 % d-Cy-F Cy-F 952.1911 [M+Na]3+> 95 % 46 % d-Cy-G Cy-G 654.9006 [M]4+> 95 % 29 % d-Cy-H Cy-H 1267.7457 [M]2+> 95 % 54 % d-Cy-I Cy-I 1384.7895 [M]2+> 95 % 29 % d-Cy-J Cy-J 1399.8260 [M]2+> 95 % 46 % d-Cy-K Cy-K 1058.2832 [M+Na]3+> 95 % 45 % d-Cy-L Cy-L 1035.2699 [M+H]3+> 95 % 40 %

[0646]

[0647] EXAMPLE IE: Proof-of Concept probe directed against Apelin receptor

[0648] [OF 0625] d-Cy-E-COOH

[0649] C153H211F12N15O33 MW: 3016.42 g mol'1Blue solid Yield: quant.

[0650]

[0651] To a solution of OF_0605 (d-Cy-E) (2.0 mg, 0.651 pmol, 1 eq) in dichloromethane (100 pL), was slowly added TFA (100 pL). The mixture was stirred at 25 °C for 1 h. After evaporation under vacuum, the crude was dissolved in water and freeze-dried to afford OF_0625 as a blue oil (2.0 mg, quantitative yield)

[0652] HPLC: tr = 3.927 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0653] HRMS (ESI): calculated for C145H211N15O25 [M]4+640.8928, found 640.8991 (A = 6.8 ppm)

[0654] [OF 0776] d-Cy-E-alcyne

[0655] C156H214F12N16O32 MW: 3053.49 g mol'1Blue solid Yield: 49%

[0656]

[0657] To a solution of OF_0625 (1.96 mg, 0.650 pmol, 1 eq) and propargylamine (0.06 pL, 0.975 pmol, 1.5 eq) in anhydrous DMF (100 pL), were added DIPEA (1.13 pL, 6.50 pmol, 10 eq) and PYBOP (0.68 mg, 1.30 pmol, 2 eq). The mixture was stirred at 25 °C for 1 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography on a SunFire C18 column (5 μm, 10 × 150 mm) using a linear gradient (0% to 95% in 40 min, flow-rate of 6 mL.min-1of solvent B (0.1% TFA in ACN, v / v) in solvent A (0.1% TFA in H2O, v / v). Detection was set at 220 and 630 nm. Fractions containing the desired product were freeze-dried to afford OF_0776 as a blue oil (0.98 mg, 49 %).

[0658] HPLC: tr = 3.942 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0659] HRMS (ESI): calculated for C148H214N16O24 [M]4+650.1507, found 650.1499 (A = -1.2 ppm)

[0660] Apl7-N3H-K(N3)FRRQRPRLSHKGPMPF-OH

[0661] C110H161F21N36O34S MW: 2962.74 g mol’1White solid Yield: 45%

[0662]

[0663] Fmoc-K(N3)FR(Pbf)R(Pbf)Q(Trt)R(Pbf)PR(Pbf)LSH(Trt)K(Boc)GPMPF-Wang sequence was synthesized according to the following method: automated solid-phase peptide synthesis (SPPS) was carried out using a Liberty Blue synthesizer (CEM, France) by standard Fmoc solid-phase chemistry on a preloaded Wang resin (0.67 mmol.g'1resin, 0.1 mmol scale), using DMF as solvent. The coupling of each amino acid (2 M) was carried out using DIC (0.5 M) and Oxyma (IM). Fmoc groups were removed using a 20% v / v solution of piperidine in DMF. Washing steps were performed using DMF. At the end of peptide synthesis, the resin was washed with DCM and MeOH and then dried in DCM. Finally, the peptide was cleaved (TFA / EEO / phenol / thioanisole / EDT 82.5 / 5 / 5 / 5 / 2.5 v / v, 1 mL per 0.1 mmol of resin, 3h at room temperature) and purified by semi-preparative reversed-phase HPLC chromatography on a SunFire C18 column (5 μm, 19 × 150 mm) using a linear gradient (5% to 95% in 40 min, flow-rate of 18 mL.min-1of solvent B (0.1% TFA in ACN, v / v) in solvent A (0.1% TFA in H2O, v / v). Detection was set at 220 and 254 nm. Fractions containing the desired product were freeze-dried to afford Apl7-N3as a white powder (67 mg, 45 %).

[0664] HPLC: tr = 2.489 min (purity > 99% at [220.4 nm] and [254.4 nm], method A) HRMS (ESI): calculated for C96H157N36O20S [M+3H]3+722.4038, found 722.4010 (A = -5.5 PPm)

[0665] [OF 0778] Ap-d-Cy-E

[0666]

[0667] C266H375F33N52O66S, MW: 6016.23 g mol’1, Blue oil, Yield: 24 %

[0668] To a solution of OF_0776 (0.98 mg, 321 nmol, 1 eq) and Apl7-N3 (1.14 mg, 385 nmol, 1.2 eq) in anhydrous DMF (41 gL), were added an aqueous solution of copper sulfate (10 mg / mL, 12 qL, 481 nmol, 1.5 eq) and an aqueous solution of sodium ascorbate (10 mg / mL, 29 qL, 1.44 qmol, 4.5 eq). The mixture was stirred at 37 °C for 1 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography on a SunFire C18 column (5 μm, 10 × 150 mm) using a linear gradient (0% to 95% in 40 min, flow-rate of 6 mL.min-1of solvent B (0.1% TFA in ACN, v / v) in solvent A (0.1% TFA in H2O, v / v). Detection was set at 220 and 630 nm. Fractions containing the desired product were freeze-dried to afford OF_0778 as a blue oil (0.46 mg, 24 %)

[0669] HPLC: tr = 3.005 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0670] HRMS (ESI): calculated for C244H370N52O44S [M+2H]6+794.4693, found 794.4678 (A = -1.9 PPm)

[0671] EXAMPL 1F: Preparation of comparative compounds

[0672] The following comparative compounds were prepared as described in Esteoulle et al, Chem. Sci. 2020, 11 (26), 6824-6829. https: / / doi.org / 10.1039 / D0SC01018A or in US 2023 / 0174480:

[0673] [Cy-A] 3-(5-carboxypentyl)-2-((lE,3E,5E)-5-(l,l-dimethyl-3-(2,5,8,ll,14,17,20,23- octaoxapentacosan-25-yl)- 1,3 -dihydro-2H-benzo[e]indol-2-ylidene)penta- 1,3 -dien- 1 -yl)- 1,1- dimethyl-lH-benzo[e]indol-3-ium 2,2,2-trifluoroacetate C58H75F3N2O12

[0674] MW: 1049.24 g mol-1Dark green powder Yield: 62 %

[0675] C137H189F6N7O29, MW: 2512.03 g mol’1, Blue oil, Yield: 38 % HPLC: tr = 6.258 min (purity > 95% at [220.4 nm] and [254.4 nm], method B) HRMS (ESI): calculated for C133H189N7O25 [M]2+1142.6878, found 1142.6916 (A = 3.3 ppm)

[0676] [OF 0779] [Ap-d-Cy-A] - Unfunctionalized dimer cyanine apelin probe

[0677]

[0678] C246H345F27N44O62S, MW: 5455.73 g mol’1, Blue oil, Yield: 65 %

[0679] OF_0779 [Ap-d-Cy-A] was prepared following the same procedure as previously used for OF_0778 [Ap-d-Cy-E] on a 321 nmol scale to afford OF_0779 as a blue oil (1.10 mg, 65%) HPLC: tr = 4.115 min (purity > 95% at [220.4 nm] and [254.4 nm], method B) HRMS (ESI): calculated for C228H340N44O44S [M+2H]4+1108.1372, found 1108.1396 (A = 2.2 PPm).

[0680] [OF 0779] [Ap-m-Cy-E] - functionalized monomer cyanine apelin probe

[0681] [OF 0796]

[0682] C75H104F3N3O17 MW: 1376.66 g mol-1Blue oil Yield: 81%

[0683]

[0684] OF_0796 was prepared following a similar procedure as previously used for d-Cy-X on a 18.6 pmol scale, by reacting OF 0647 Cy-B with tert-butyl l-amino-3,6,9,12-tetraoxapentadecan- 15-oate to afford OF_0796 as a blue oil (21 mg, 81%)

[0685] HPLC: tr = 5.306 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0686] HRMS (ESI): calculated for C73H104N3O15 [M]+1262,7462, found 1262.7464 (A = 0.1 ppm)

[0687] [OF 0797]

[0688] C71H96F3N3O17 MW: 1320.55 g mol'1Blue oil Yield: 95%

[0689]

[0690] OF_0797 was prepared following a similar procedure as previously used for OF_0625 on a 15.3 pmol scale, to afford OF_0797 as a blue oil (19 mg, 95%)

[0691] HPLC: tr = 4.476 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0692] HRMS (ESI): calculated for C69H96N3O15 [M]+1206,6836, found 1206.6811 (A = -2.6 ppm) [OF 0807]

[0693] C79H111F6N7O19 MW: 1576.78 g mol'1Blue oil Yield: 71%

[0694]

[0695] OF_0807 was obtained according to the general procedure previously described in example IB from OF_0797 and (3-azidopropyl)trimethylazanium iodide on a 6.07 pmol scale (6.8 mg, 71 %).

[0696] HPLC: tr = 4.363 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0697] HRMS (ESI): calculated for C75H111N7O15 [M]2+674.9064, found 674.9067 (A = 0.4 ppm) [OF O8O8]

[0698] C82H114F6N8O18 MW: 1613.84 g mol'1Blue oil Yield: 65%

[0699]

[0700] OF_07807was prepared following a similar procedure as previously used for OF_0776 on a 15.3 µmol scale, to afford OF_0808 as a blue oil (4.5 mg, 65 %).

[0701] HPLC: tr = 4.427 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0702] HRMS (ESI): calculated for C78H114N4O14 [M]2+693.4222, found 693.4239 (A = 2.4 ppm) [OF 0809] Ap-m-Cy-E

[0703]

[0704] C192H275F27N44O52S, MW: 4576.58 g mol'1, Blue oil, Yield: 41 % OF_0809 [Ap-m-Cy-E] was prepared following the same procedure as previously used for OF_0778 [Ap-d-Cy-E] on a 281 nmol scale to afford OF_0809 as a blue oil (0.53 mg, 41 %) HPLC: tr = 3.693 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0705] HRMS (ESI): calculated for C174H272N44O34S [M+4H]6+592.6775, found 592.6755 (A = -3.4 ppm)

[0706] EXAMPLE 2: Biological assessment

[0707] Material and method

[0708] General spectroscopy procedures

[0709] Absorption and fluorescence measurements were carried out in 114F-QS 10 mm quartz fluorescence cuvettes (Hellma Analytics). Solvents used were of spectroscopic or HPLC quality. HEPES buffer was prepared as followed: 137.5 mM NaCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 6 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES. Absorption spectra were recorded on a UV2700i spectrophotometer (Shimadzu) or a Cary 4000HP spectrophotometer (Varian). Fluorescence spectra were recorded on a FluoroMax 4 (Jobin Yvon, Horiba) equipped with a cuvette holder thermostated at 20°C. The molar absorption coefficient (e) was measured in MeOH at Xmax for each dye as an average of 3 independent experiments. Absorption and emission spectra of monomeric and dimeric cyanine dyes were recorded using 0.4 pM or 0.2 pM solutions respectively. Spectra were recorded in solvents of increasing polarity (DMF, EtOH, MeOH, H2O, HEPES buffer). The recorded absorbance range was 450-800 nm. The excitation wavelength was 630 nm and the recorded emission spectral range was 640-850 nm (excitation slit: 2 nm, emission slit: 2 nm). Unless specified, all fluorescence spectra were corrected for instrumental factors. The absorbance of sample solutions at the excitation wavelength were kept below 0.05 to avoid inner filter effects. Fluorescence intensity values Slc / Rl were used to calculate fluorescence quantum yields and turn-on ratios. Determination of relative fluorescence quantum yields was performed using rhodamine 800 in EtOH as a reference (QY = 25 %) (Alessi, J. Lumin. 2013, 134, 385-389. https: / / doi. Org / 10.1016 / j.jlumin.2012.08.017). Data treatment was performed using OriginPro 8.6.

[0710] Evaluation of non-specific interactions with BSA.

[0711] The evaluations of non-specific interactions of the dimers with BSA were performed as follows.

[0712] 200 nM solutions of the studied probes were prepared by diluting 2 pL of the 100 pM DMSO stock solutions of dimers in 998 pL of HEPES buffer containing 0, 0.1 or 1% w / w of BSA. The solutions were carefully mixed before measuring the fluorescence of the dimers.

[0713] The non-specific interactions of the probes with BSA were evaluated by calculating the ratio of fluorescence intensity integral in HEPES containing 1% BSA (I)to the integral for the solution in HEPES without BSA (Io).

[0714] Evaluation of non-specific interactions with liposomes.

[0715] a) Preparation of 1 mM DOPC / cholesterol (2:1) liposomes.

[0716] To a solution of DOPC (3 mL, 5 mM) in CHCh was added a solution of cholesterol (300 pL, 25 mM) in CHCh. The mixture was agitated for 5 minutes and the solvent was slowly evaporated under reduced pressure. The obtained film was rehydrated with 1 mL of simplified HEPES buffer (50 mM HEPES, 5 mM MgCl2, pH = 7.4) and mixed for 10 min. The suspension was then sonicated (I s cycle every 3 s) during 1 h at room temperature under a continuous flow of argon, using a Vibra Cell 75041 ultrasonicator (750 W, 20 kHz, Fisher Bioblock Scientific) equipped with a 3 mm diameter tip probe (40% amplitude). The resulting small unilamellar vesicles (SUVs) preparation was centrifuged at 13 000 * g for 10 min to remove the titanium dust coming from sonication probes. The size and dispersion of the SUVs were determined at 298 K by the dynamic light scattering method using a Zetasizer Nano-ZS (Malvern Instruments, Orsay, France) with a scattering angle of 173°. Analysis showed a mean diameter for the SUVs of 103 nm with a PDI of 0.3 indicating a monodisperse suspension. The exact concentration of the obtained solution was assessed using a commercial kit (LabAssay™ Phospholipid, Wako Chemicals GmbH, Neuss, Germany) then appropriate dilution afforded the ImM solution of liposomes.

[0717] b) Measurement of fluorescence intensity

[0718] The 200 nM solutions of studied probe in simplified HEPES buffer (50 mM HEPES, 5 mM MgCl2, pH = 7.4) with increasing amounts of DOPC / cholesterol (2: 1) liposomes were prepared as follows. 0 pL, 20 pL, and 200 pL of 1 mM DOPC / cholesterol (2:1) liposomes were accordingly added to 998 pL, 978 pL, 798 pL of HEPES buffer. Then 2 pL of 100 pM DMSO stock solution of the studied probe were added. The solutions were carefully mixed before measuring the fluorescence of the dimers.

[0719] The non-specific interactions of the probes with liposomes were evaluated by calculating the ratio of fluorescence intensity integral in HEPES containing 200 pM of liposomes (I) to the integral for the solution in HEPES without liposomes (Io). Cell lines and culture conditions.

[0720] HEK293 cells stably overexpressing the human EGFP-ApelinR receptor (prepared at PCBIS, Strasbourg) were cultured in Eagle’s minimal essential medium (MEM, Invitrogen 21090) with 10% FBS, 100 U / mL of penicillin, 100 pg / mL of streptomycin, 2 mM of glutamine and 50 pg / mL hygromycin at 37 °C in a humidified 5% CO2 atmosphere. 70-80% cell confluence was maintained by the removal of a portion of the culture and replacement with fresh medium twice a week. Cells were seeded into 35 mm ibiTreat p-dish (IBiDi) at 80 000 cells per dish 3 days before imaging.

[0721] Cell culture and confocal microscopy studies:

[0722] Live-cell no-wash Apelin receptor imaging.

[0723] The culture medium was removed, the cells were washed either with HEPES buffer (137.5 mM NaCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 6 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES, supplemented with 0.1% BSA) or with Eagle’s minimal essential medium (MEM Invitrogen 21090 supplemented with 10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin, 2 mM glutamine, 50 pg / mL hygromycin). Cells were incubated with the ligand and a 1 pg / mL solution of Hoechst 33342 in HEPES buffer for 15 min at 37°C before the imaging. Fluorescence confocal microscopy experiments were performed on a Leica TCS SPE microscope equipped with an HCX PL APO CS2 63x / 1.40 objective. The imaging was performed at 22 °C. The probe was excited with a 635 nm 18 mW laser and detected at 645 -750 nm. EGFP was excited with a 488 nm lOmW laser and detected at 490 - 540 nm. Hoechst 33342 was excited with a 405 nm 25 mW laser at and detected at 430 - 480 nm. The pinhole was set to 1 airy unit. The images were acquired at 1.5x magnification as a mean of 2 scans in 1024x1024 size. All images were processed with Image J.

[0724] Results and discussions

[0725] To evaluate the impact of the functionalization introduced in the cyanine scaffold, the Inventors synthesized the novel cyanine Cy-B bearing a terminal alkyne on one of the benzoindole moiety (see Example 1). A series of ten azide derivatives bearing anionic, cationic, zwiterrionic, neutral or peptidic substituents were reacted with Cy-B by a copper-catalyzed alkyne-azide cycloaddition (CuAAC) using CuSO4 and sodium ascorbate in a mixture of DMF and water as a solvent. The click reactions were complete in 15 min at room temperature to give ten new click-functionalized cyanines, Cy-C to Cy-L, with yields ranging from 31 to 93 %.

[0726] Next, click-functionalized cyanines were dimerized using bifunctional tweezers composed of a lysine residue and a PEG4 linker. Such a design should offer the probes enough flexibility to allow the cyanine dimer to open in the apolar environment and at the same time limit the impact of the cyanine dimer on the pharmacological properties of the Apelin ligand.

[0727] The photophysical properties of the new fluorogenic dimers were assessed to select the optimal ones for imaging experiments. The introduction of ionic or bulky substituents on the cyanine dimer could have a strong impact on the ability of the cyanine dimer to aggregate or disassemble. To assess the fluorogenic character of the new dimers, their quantum yields (QY) were measured in solvents of increasing polarity. Being flat aromatic scaffolds, cyanine dyes have a strong tendency to 7t-stack into non-fluorescent H-aggregates in polar solvent (water) due to strong hydrophobic interactions. In an apolar solvent, such as DMF, these aggregates are disrupted which leads to opening of the dimer and generates a strong fluorescence signal. To quantify the fluorogenicity of the dimers, their relative “turn-on” values were calculated as the ratio between the Quantum Yield (QY) of the highly fluorescent form in DMF and the QY of the poorly fluorescent aggregate in water (Table 1). The non-functionalized dimer d-Cy-A, was characterized by a turn-on ratio of 88. New functionalized dimers d-Cy-C to d-Cy-L exhibited turn-on ratios ranging from 24 to 69, highlighting that they were still prone to aggregation-caused quenching despite the introduction of the triazole ring and of the functional groups on the cyanine scaffold.

[0728] QY water

[0729] Probe QY DMF (%) Turn-on ratio

[0730] (%)

[0731] d-Cy-A 30,9 0,35 88

[0732] d-Cy-C 24,8 0,46 54

[0733] d-Cy-D 26,5 0,52 51

[0734] d-Cy-E 32,9 0,48 69

[0735] d-Cy-F 33,6 0,58 58

[0736] d-Cy-G 33,3 1,0 33

[0737] d-Cy-H 31,8 0,55 58

[0738] d-Cy-I 23,8 0,99 24

[0739] d-Cy-J 33,4 0,75 45

[0740] d-Cy-K 33,3 0,58 57

[0741] d-Cy-L 34,8 1,2 29

[0742]

[0743] Table 1: Fluorogenic properties of the dimers in DMF and water. Relative quantum yields were measured by using rhodamine800 in EtOH as a standard (Alessi, M. Salvalaggio and G. Ruzzon, J. Lumin., 2013, 134, 385-389)Tum-on was calculated as a ratio of QY in DMF and water. Concentration of dimers 200 nM. After this insight into the fluorogenic behaviour of the dimers, the Inventors addressed possible non-specific interactions with plasma proteins and cell membranes. Such interactions must be avoided as they may cause disaggregation of the dimer and generate background noise in wash-free cell imaging experiments. The Inventors measured the increase of fluorescence of the dimers in the presence of bovine serum albumin, as a mimic of circulating proteins in the bloodstream, as well as with liposomes - lipid bilayer mimicking the cell membrane. Nonspecific interactions were evaluated by calculating the ratio of the integral fluorescence measured in HEPES buffer containing BSA (1 %) or DOPC / cholesterol liposomes (200 pM) (I) to the integral fluorescence measured in HEPES buffer alone (Io) under the same excitation.

[0744] Probe (I / Io) Liposomes (200 gm) (I / Io) BSA (1%)

[0745] d-Cy-A 37.75 ± 0.78 9.71 ± 0.05

[0746] d-Cy-C 9.57 ± 0.43 4.71 ± 0.61

[0747] d-Cy-D 26.22 ± 1.47 5.16 ± 0.65

[0748] d-Cy-E 6.27 ± 0.19 2.01 ± 0.06

[0749] d-Cy-F 4.78 ± 0.73 1.74 ± 0.12

[0750] d-Cy-G 21.27 ± 1.31 2.09 ± 0.12

[0751] d-Cy-H 12.88 ± 1.51 5.23 ± 0.42

[0752] d-Cy-I 26.38 ± 1.64 4.00 ± 0.54

[0753] d-Cy-J 9.26 ± 0.49 2.99 ± 0.13

[0754] d-Cy-K 6.53 ± 0.30 2.29 ± 0.11

[0755] d-Cy-L 4.15 ± 0.26 2.85 ± 0.37

[0756]

[0757] Table 2: Nonspecific interactions of the cyanine dimers with liposomes: fluorescence intensity ratios (I / Io) with 200 pM of liposomes or 1% of BSA in HEPES. Error bars represent mean ± SEM from three independent experiments.

[0758] The results shown in Figure 2 and in Table 2 show a strong increase of fluorescence intensity for the non-functionalized dimer d-Cy-A in the presence of both BSA and liposomes (respectively 10- and 38-fold increase). Overall, the increase of fluorescence appears lower for all click-functionalized dimers d-Cy-C to d-Cy-L, probably as a result of the introduction of the triazole ring on the dye scaffold. The presence of ammonium (d-Cy-E), zwitterion (d-Cy-F), tertiary amine (d-Cy-G), PEG (d-Cy-J and K) or peptide (d-Cy-L) on the cyanine dye had a tendency to limit the interactions with BSA and kept the increase of fluorescence below a 3-fold ratio. The dimers functionalized with ammonium (d-Cy-E), zwitterion (d-Cy-F), PEG8 (d-Cy-K) or peptide (d-Cy-L) groups exhibited the lowest increase of fluorescence ratio indicating that they were less prone to open in the lipid membrane of liposomes. After careful examination of these data, d-Cy-E was identified as the optimal dimer for the subsequent imaging studies as it was characterized by the highest turn-on ratio among all functionalized dimers and displayed a combination of good fluorogenic properties and limited non-specific interactions with albumin and liposomes.

[0759] Next, the Inventors demonstrated the possibility of using the optimized dimer d-Cy-E for confocal microscopy imaging of living cells under no-wash conditions. For the proof of concept, it was conjugated to the apelin-17 peptide, an endogenous agonist of the ApelinR receptor. A conjugate of the non-functionalized dimer d-Cy-A was synthesized for comparison (see Example 1).

[0760] Their turn on ratios were respectively 77, and 57 proving that they retain good fluorogenic properties even after binding to the peptide. In parallel, Ap-m-Cy-E -a monomeric analogue of Ap-d-Cy-E bearing a single fluorophore was synthesized following a similar. Due to its monomeric nature and the inability to form non-fluorescent intramolecular aggregates, its turnon ratio was only 3 (Table 3). Ap-d-Cy-E was also shown to have the lowest non-specific interactions with BSA and liposomes (see Table 4).

[0761] QY (%)

[0762] Probe Turn-On

[0763] DMF H2O

[0764] Ap-d-Cy-A 33.4 0.44 77

[0765] Ap-d-Cy-E 30.4 0.53 57

[0766] Ap-m-Cy-E 39.7 12.4 3

[0767]

[0768] Table 3: Fluorogenic properties of the conjugates (200 nM for dimer, 400 nM for monomer). Quantum yields measured by comparison with rhodamine800 QY = 25% in EtOH.

[0769] d / lo)

[0770] Probe

[0771] BSA 1% Liposomes

[0772] Ap-d-Cy-A 7.55 ± 0.17 16.44 ± 0.09

[0773] Ap-d-Cy-E 1.85 ± 0.10 2.75 ± 0.04

[0774] Ap-m-Cy-E 1.61 ± 0.30 1.26 ± 0.01

[0775]

[0776] Table 4: Nonspecific interactions of the conjugates with BSA and liposomes: fluorescence intensity ratios (I / Io) with 0.1 or 1% of BSA concentration in HEPES or with 20 or 200 pM of liposomes in HEPES. (200 nM for dimer, 400 nM for monomer). Error bars represent mean ± SEM from three independent experiments. To assess the ability of the conjugates to label the ApelinR in a specific manner in living cells, microscopy studies were performed on HEK cells overexpressing the ApelinR fused to EGFP (green fluorescent protein). Both conjugates Ap-d-Cy-A and Ap-d-Cy-E enabled visualization of the ApelinR under no-wash conditions with a high signal -to-noise ratio after 15 minutes of incubation of living cells with the probes at 37°C (Figure 3A). The agonist character of the conjugates was confirmed by the observation of intracellular clusters in the cyanine and in the EGFP channels caused by the internalization of labelled receptors. Non-specific binding was evaluated on wild-type HEK cells which do not express ApelinR under the same labeling conditions. Strong interactions with the cell membrane were observed for the nonfunctionalized conjugates Ap-d-Cy-A, indicating that the signal observed at the surface of ApelinR-expressing cells is partially due to non-specific binding of the conjugate to the membrane. Meanwhile, no membrane staining of wild-type HEK cells was observed for the functionalized conjugate Ap-d-Cy-E highlighting its high selectivity toward the ApelinR receptor. This result, visible to the naked eye, was confirmed after measurement of the normalized variance on wild-type HEK cells, showing the lowest signal variance for Ap-d-Cy-E (Figure 3B). Next, to demonstrate the importance of the fluorogenic character of ApelinR probes, a comparison study was performed with the monomeric analogue Ap-m-Cy-E under the same labelling and imaging conditions. In both ApelinR and wild-type HEK cells, the excess of the unbound monomeric probe generated a significant background noise visible to the naked eye. Such strong background noise is due to the poor fluorogenic character of the monomer Ap-m-Cy-E, whose turn-on ratio is 20-fold lower than for the dimeric analogue Ap-d-Cy-E (Table 3). Measurement of the signal-to-noise ratio highlighted a two-fold increase in contrast between Ap-d-Cy-E and Ap-m-Cy-E (Figure 3C), confirming the importance of the strong fluorogenic character of the dimeric functionalized conjugates for live-cell imaging under no-wash conditions.

[0777] In summary, a straightforward CuAAC reaction between functionalized azide reagents and a cyanine bearing a terminal alkyne gave easy access to a library of ten new Cy5.5 dyes. A series of fluorogenic dimers was synthesized from these click-functionalized cyanines and a rational structure-interaction relationship study enabled the identification of an optimal dimer for nowash imaging experiments. This probe, bearing the quaternary ammonium functional group, displayed a high fluorogenic character and exhibited low non-specific interactions with BSA and liposomes in in vitro fluorescence studies. To develop the first dimeric turn-on probe for the ApelinR receptor, we conjugated the selected optimized dimer to its peptide agonist apelin-17. The improved performance of the ammonium functionalized conjugate Ap-d-Cy-E was demonstrated in an imaging experiment on living cells expressing the ApelinR. The conjugate allowed the visualization of the ApelinR under no-wash conditions with a better selectivity than the non-functionalized analogue Ap-d-Cy-A and a higher signal-to-noise ratio than the monomeric analogue Ap-m-Cy-E.

[0778] EXAMPLE 3

[0779] Example 3 shows the preparation of unfunctionalized dimer cyanin probes comprising an antibody as ligand. Functionalized dimer cyanine probes of the invention (namely with a R group in place of a methyl in the cyanine core) can be obtained through similar synthesis pathway by using appropriate cyanine core intermediates (see Example 1).

[0780] 3,1. Synthesis of “P7” linker

[0781]

[0782]

[0783] [OF 0632]

[0784] C22H34N2O7

[0785] MW: 438.52 g mol’1Colorless oil

[0786]

[0787] Yield: 89 %

[0788] To a solution of Cbz-n-amido-peg2-acid (1 eq., 800 mg, 2.57 mmol) and 3-(tert-butoxy)-3-oxopropan-l-aminium chloride (1.2 eq., 182 mg, 3.08 mmol) in anhydrous DMF (20 mL), were added DIPEA (7 eq., 1.7mL, 10.3 mmol) and PYBOP (1.2 eq., 1.60 g, 3.08 mmol). The mixture was stirred at 25 °C for 16 h. After evaporation under vacuum, the crude was purified by semipreparative reversed-phase HPLC. Fractions containing the desired product were freeze-dried to afford OF_0632 as a colorless oil (1.01 g, 89 %).

[0789] HPLC: tr = 4.232 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0790] [OF 0634]

[0791] C18H26N2O7

[0792] MW: 382.41 g mol-1Colorless oil

[0793]

[0794] Yield: 96 % To a solution of OF_0632 (1 eq., 1.01 g, 2.30 mmol) in dichloromethane (10 mL), was slowly added TFA (10 mL). The mixture was stirred at 25 °C for 1 h. The crude was evaporated and dried under high vaccum overnight to afford OF_0634 as a colorless oil (879 mg, 96 %). HPLC: tr = 3.066 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0795] C46H70N6O14 MW: 931.09 g mol'1White solid Yield: 82 %

[0796]

[0797] To a solution of tert-butyl L-lysinate hydrochloride (1 eq., 200 mg, 0.838 mmol) and OF_0634 (2.2 eq., 705 mg, 1.84 mmol) in anhydrous DMF (25 mL), were added DIPEA (10 eq., 1.4 mL, 8.38 mmol) and PYBOP (2.2 eq., 959 mg, 1.84 mmol). The mixture was stirred at 25 °C for 2 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography to afford OF_0636 as a white solid (639 mg, 82 %).

[0798] HPLC: tr = 4.574 min (purity > 95% at [220.4 nm] and [254.4 nm], method A)

[0799] HRMS (ESI): calculated for C46H71N6O14[M+Na]+931.5023, found 931.5061 (Δ = 4.0 ppm)

[0800] [OF 0677]

[0801] C30H58N6O10MW: 662.83 g mol-1Colorless oil

[0802]

[0803] Yield: 88 % To a solution of OF_0636 (1 eq., 400 mg, 0.429 mmol) in anhydrous MeOH (3 mL) was added Pd / C (10 %, 0.05 eq., 23 mg, 21 pmol). The mixture was stirred under H2 (1 atm) at 25 °C for 20 h. The mixture was filtered through a hydrophobic PTFE syringe filter (pore: 0.22 pM, diam.: 13 mm) and rinsed with MeOH. MeOH was evaporated under vacuum to afford OF_0677 as a colorless oil (251 mg, 88 %).

[0804] LRMS (ESI): calculated for C30H59N6O10 [M+H]+663.43, found 663.43 [OF 0682]

[0805] C146H204F6N10O32 MW: 2725.27 g mol'1Blue oil Yield: 76 %

[0806]

[0807] To a solution of OF_0677 (1 eq., 127 mg, 0.191 mmol) and OF_0647 (2.2 eq., 457 mg, 0.420 mmol) in anhydrous DMF (12 mL), were added DIPEA (20 eq., 0.63 mL, 3.82 mmol) and PYBOP (2.2 eq., 218 mg, 0.420 mmol). The mixture was stirred at 25 °C for 2 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography to afford OF_0682 as a blue oil (396 mg, 76 %).

[0808] HPLC: tr = 6.279 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0809] HRMS (ESI): calculated for C142H204N10O28 [M]2+1249.2435, found 1249.2434 (A = -0.1 PPm)

[0810] C142H196F6N10O32 MW: 2669.16 g mol'1Blue oil Yield: 82 %

[0811]

[0812] To a solution of OF_0682 (1 eq., 396 mg, 1.45 mmol) in dichloromethane (3 mL), was slowly added TFA (3 mL). The mixture was stirred at 25 °C for 1 h. The crude was evaporated and dried under high vaccum overnight to afford OF_0683 as a colorless oil (319 mg, 82 %). HPLC: tr = 5.884 min (purity > 90% at [220.4 nm] and [254.4 nm], method B)

[0813] HRMS (ESI): calculated for C138H196N10O28 [M]2+1221.2122, found 1221.2119 (A = -0.2 ppm) 3,2 Synthesis of the di-APN probe with PEG12 spacer

[0814] [OF 0689]

[0815]

[0816] C173H257F6N11O45, MW: 3324.98 g mol’1, Blue oil, Yield: 78 %

[0817] To a solution of OF_0683 (1 eq., 265 mg, 99.3 qmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate (1.3 eq., 87 mg, 129 qmol) in anhydrous DMF (4 mL), were added DIPEA (10 eq., 173 qL, 993 qmol) and PYBOP (1.5 eq., 78 mg, 149 qmol). The mixture was stirred at 25 °C for 2 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography to afford OF_0689 as a blue oil (256 mg, 78 %).

[0818] HPLC: tr = 6.196 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0819] HRMS (ESI): calculated for C169H257N11O41 [M]2+1548.9193, found 1548.9220 (A= 1.7 ppm), [OF 0690]

[0820]

[0821] C169H249F6N11O45, MW: 3268.87 g mol’1, Blue oil, Yield: 90 % To a solution of OF_0689 (1 eq., 119 mg, 36.8 pmol) in di chloromethane (2 mL), was slowly added TFA (2 mL). The mixture was stirred at 25 °C for 1 h. The crude was evaporated and dried under high vaccum overnight to afford OF_0690 as a blue oil (105 mg, 90 %).

[0822] HPLC: tr = 5.751 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0823] HRMS (ESI): calculated for C165H249N11O41 [M]2+1521.3897, found 1521.3906 (A = 0.6ppm),

[0824] [OF 0685]

[0825]

[0826] C175H254F6N12O44, MW: 3343.99 g mol’1, Blue oil, Yield: 64 % To a solution of OF 0690 (1 eq., 26 mg, 7.95 pmol) and bis(prop-2-yn-l-yl)amine (1.5 eq., 1.1 mg, 11.9 pmol) in anhydrous DMF (0.5 mL), were added DIPEA (10 eq., 13.9 pL, 79.5 pmol) and PYBOP (1.5 eq., 6.2 mg, 11.9 pmol). The mixture was stirred at 25 °C for 2 h. After evaporation under vacuum, the crude was purified by semi-preparative reversed-phase HPLC chromatography to afford OF_0685 as a blue oil (17 mg, 64 %).

[0827] HPLC: tr = 6.224 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0828] HRMS (ESI): calculated for C171H254N12NaO40[M+Na]3+1046.9386, found 1046.9380 (Δ = -0.6 ppm)

[0829] [OF 0692]

[0830]

[0831] C193H262F6N20O44, MW: 3680.31 g mol’1, Blue oil, Yield: 44 % To a solution of OF_0685 (1 eq., 17 mg, 5.08 pmol) and 3-(4-azidophenyl)prop-2-ynenitrile (4 eq., 3.4 mg, 20.3 pmol) in anhydrous DMF (400 pL), were added an aqueous solution of copper sulfate (4 eq., 5.1 mg, 20.3 pmol) in water (50 pL) and an aqueous solution of sodium ascorbate (4 eq., 4.0 mg,, 20.3 pmol) in water (50 pL). The mixture was stirred at 25 °C for 1 h. The crude mixture was diluted with water and purified by semi-preparative reversed-phase HPLC chromatography to afford OF_0692 as a blue oil (8.3 mg, 44 %)

[0832] HPLC: tr = 6.576 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0833] HRMS (ESI): calculated for C189H262N20O40 [M]2+1726.9569, found 1726.9596 (A = 1.5 ppm)

[0834] 3,3 Site-specific conjugation to mutated trastuzumab antibody

[0835] OF 0692 was conjugated to mutated trastuzumab according to a protocol similar to that described in Koniev et al., Med. Chem. Commun., 2018, 9, 827 with a probe-to-antibody ratio of 0.6. 3.4. Synthesis of the NHS-ester activated probe with PEG12 spacer

[0836] [OF 0695]

[0837]

[0838] C173H255F6N12O47, MW: 3365.95 g mol-1, Blue oil, Yield: 61 % To a solution of OF_0690 (1 eq., 16 mg, 4.89 qmol) in anhydrous DCM (0.8 mL), were added DIPEA (1.5 eq., 1.3 qL, 7.34 qmol) and TSTU (1.5 eq., 2.2 mg, 7.34 qmol). The mixture was stirred at 25 °C for 6 h. DCM was added and the diluted solution was quenched with water, washed with citric acid 5%, brine, and dried over Na2SO4. The solvent was removed under reduced pressure to afford OF_0695 as a blue oil (10 mg, 61 %).

[0839] HPLC: tr = 5.847 min (purity > 95% at [220.4 nm] and [254.4 nm], method B)

[0840] HRMS (ESI): calculated for C169H252N12O43 [M]2+1569.3962, found 1569.3984 (A = 1.4 ppm)

[0841] 3,5 Conjugation to trastuzumab antibody

[0842] Conjugation experiments were carried out in Eppendorf DNA low-bind tube 1.5 mL at atmospheric pressure at room temperature unless otherwise stated. All buffer solutions were prepared with deionized water and filter-sterilized prior to use. PBS was 140 × 10-3M NaCl and 12 × 10-3M sodium phosphate at pH 7.4. Ultrapure DMSO (analytical reagent grade) was purchased from Fischer Chemical. Trastuzumab was obtained in its clinical formulation (Roche). Ultrafiltration for buffer exchange of antibodies was carried for was carried out in a vivaspin 500 polyethersulfone (PES) membrane concentrator with a molecular weight cut-off (MWCO) of 50 kDa. Centrifugation was carried out on an Eppendorf mini spin operating at 10 000 RPM at room temperature. A trastuzumab solution (17 × 10-6M) was prepared in PBS pH 7.4. Next, a solution of dimer-NHS ester probe OF_0695 was prepared in DMSO (5 × 10-3M) and added to the trastuzumab solution. The reaction was incubated at 24 °C overnight with rotary shaking at 300 rpm. Afterward, excess of the reagent was removed by ultrafiltration (50 kDa MWCO) and washed with PBS (pH 7.4) to afford the modified antibody in PBS (400 pL, 10 min, 10k RPM, 7 times). The final conjugate was obtained with a probe-to-antibody ratio of 1.9 and a yield of 34%.

Claims

CLAIMS1. A cyanine fluorogenic dimer probe for detecting a molecular target, which is represented by the following formula (I):[Linker][Ligand](I)wherein[Linker] is a chemical entity having at least three branches and comprising up to 300 carbon atoms in its backbone and optionally one or more heteroatoms, wherein one branch is linked to [Ligand] by at least one covalent bond and each of the two other branches is linked to one [Cy] by at least one covalent bond,[Ligand] is a ligand able to bind to the molecular target,each [Cy] is a cyanine group independently selected from:n (Dl) with n is 1, 2 or 3; andwherein:Gi is of formulaWherein:- R is a moiety for modulating, preferably for limiting non-specific interactions of the cyanine fluorogenic dimer probe with cellular membrane and / or with blood plasma proteins, said moiety being in the form of a saturated or unsaturated hydrocarbon chain having at least 3 and up to 50 backbone carbon atoms, preferably up to 30 backbone carbon atoms in length,wherein said hydrocarbon chain has a terminal moiety selected from:o H, -OH, -NH2, -NRsRs, C1-C3 hydroxyalkyl, C1-C3 alkoxy, C1-C3 halogenoalkyl, a halogen, -CN, C1-C3 cyanoalkyl, -NO2o a cationic group preferably selected from -NR.5R.6R7, ando an anionic group preferably selected from a phosphate, a phosphonate, a sulfonate, a carboxylatewherein said hydrocarbon chain is optionally substituted by one or more substituents selected from C1-C3 hydroxyalkyl, C1-C3 alkyl, C1-C3 alkoxy and halogen, wherein said hydrocarbon chain is optionally interrupted by one or more connecting groups selected from -NRsCO-, NRs, N+R5R6, -OC(O)-, -NH(CO)O-, NH, S, O, CO, NH(C=O)NH, O(C=O)NH, -O-N=CRs-, -CRs=N-O-, -NH-N=CRs-, CRs=N-NH-, amino acid residues such as alanine, citrulline or valine and / or 3- to 20-, preferably 3-to 10- membered cyclic or heterocyclic groups such as pyrazole or triazole, optionally substituted,whereino Each Rs, Re and R? are independently Ci-Ce alkyl, Ci-Ce hydroxyalkyl or Ci-Ce alkyl alkoxy, ando Each Rs is independently H, Ci-Ce alkyl, Ci-Ce hydroxyalkyl or Ci-Ce alkyl alkoxy,- Li is a chemical group connecting the [Cy] group to a branch of the [Linker] and Li is an unsaturated or saturated C1-C20 hydrocarbon chain, more preferably a C1-C10 alkylene, optionally substituted by one or more halogens, C1-C3 haloalkyl or C1-C3 alkyl,- R2 is H, or a hydrophilic group of formula -(R9)z-(Arl)ziC(=O)R9’ whereinR9 is a Ci-Ce alkylene, NR9”, S, or O, wherein R9” is H or C1-C3 alkylz is 0 or 1Ari is a Ce-Cis aryl group, or a 6-membered heteroaryl group,zl is 0 or 1R9’ is NR10R11, wherein Rio and R11 are independently an hydrogen, a (Ci-Ce) alkyl, a di(Ci-Cs)alkylamino(Ci-Cs)alkyl, a Ci-Ce hydroxyalkyl, including Ci-Ce di- or -tri- hydroxyalkyl, (C2-Ce)carboxyalkyl, polyethylene glycol represented by the formula - (CH2CH2O)n-R’; or a polypropylene glycol represented by the formula - (CH2CH2(CH3)O)n-R’, wherein n is an integer from 1 to 40 and R’ is a C1-C12 alkyl group, or, alternatively, Rio and R11 form with the nitrogen to which they are attached an heterocycle (such as piperazine) optionally substituted by a (Ci-C4)alkyl,- R3 is H or a (C1-C20) alkyl,- R1 is of formula (a) -L1a-R12; (b) -L1b-NR13C(=O)-L1cR12 or (c) -L1bC(=O)NR13L1cR12 wherein:o Lia is a saturated or unsaturated (C2-C40) hydrocarbon chain, preferably a C2- C20 alkylene, or a poly(C2-Cs oxyalkylene) chain comprising from 2 to 20 monomers, such as a polyethylene or polypropylene chain comprising from 2 to 20 or from 2 to 10 monomers,o Lib and Lie are independently a saturated or unsaturated (C2-C20), preferably a (C2-C10) hydrocarbon chain, preferably a C2-C10 alkyleneo R13 is H, a Ci-Ce alkyl, a C2-C6 hydroxyalkyl, or a C2-C6 alkoxyo R12 is -(CH2)PRi6 wherein p is an integer from 0 to 6 and Rie is selected from H, OH, halogen, C1-C3 alkoxy, and SO3HX is a counter-anion, preferably a pharmaceutically acceptable counter-anion.

2. The cyanine fluorogenic dimer probe of Claim 1, wherein each [Cy] is of formulanwherein n is 1, 2 or 3.

3. The cyanine fluorogenic dimer probe of Claim 1 or 2, wherein Gi is of formula Gia or Gib, and G2 is of formula G2a.

4. The cyanine fluorogenic dimer probe of any one of Claims 1 to 3, wherein Gi is Gia and G2 is G2a.

5. The cyanine fluorogenic dimer probe of any one of Claims 1 to 4, wherein the hydrocarbon chain of the R moiety is interrupted by at least one triazole group, optionally substituted.

6. The cyanine fluorogenic dimer probe of any one of Claims 1 to 4 wherein R is of formula (ao):-(CH2)O-[G3]-L2-R15 (ao)wherein:o is an integer from 0 to 6, for instance 1, 2 or 3,[G3] is a connecting group selected from -NH(C=O)-, -(C=O)-NH-, S, O, NH, NH(C=O)NH, O(C=O)NH, NH(C=O)O, -O-N=CR8-, -CR8=N-O-, -NH-N=CR8-, CR8=N-NH-, and 3 to 10-membered cyclic or heterocyclic group, preferably [G3] is selected from triazole, pyrazole -O-N=CR8-, CR8=N-O-, -NH-N=CR8-, and -CR8=N- NH-,- L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NRs(C=O)-, NRs, -N+RsR6, -OC(=O)-, -NH(C=O)O-, NH, S, O, and (C=O),- R15 is selected from the group consisting of H, -OH, -NH2, -NRsRs, C1-C3 alkoxy, halogen, CF3, -CN, -NRsReR?, a phosphate, a phosphonate, a sulfonate, a nitro, and a carboxylate,each Rs, Rs, Re, and R7 are as defined in Claim 1, preferably each Rs is H, C1-C3 alkyl or C1-C3 hydroxyalkyl, and each Rs, Re, and R7 are independently C1-C3 alkyl or C1-C3 hydroxy alkyl.

7. The cyanine fluorogenic dimer probe of Claim 6, wherein, in the formula (ao), [G3] is a tri azole group.

8. The cyanine fluorogenic dimer probe of Claim 6 or Claim 7, wherein, in formula (ao):- o is 1, and / or-[G3] is a triazole, and / or- L2 is a saturated or unsaturated, C2-C30, preferably C3-C10 hydrocarbon chain optionally interrupted by one or more -NRs(C=O)-, NRs, -N+RsRe, NH, O, and (C=O), and / or- Ris is selected from the group consisting of H, -OH, -NH2, -NRsRs, C1-C3 alkoxy, -NR.5R.6R7, a phosphonate, and a sulfonate,each Rs, Rs, Re and R7 being as defined in Claim 6.

9. The cyanine fluorogenic dimer probe of any one of Claims 6 to 8, wherein R is of formula (ai):A0 N Rl5(ai)wherein o, L2 and Ris are as defined in Claim 6, preferably as defined in Claim 8.

10. The cyanine fluorogenic dimer probe of any one of claims 1 to 9, wherein R is selected from the group consisting of:N=N(a)wherein t is an integer from 2 to 10, preferably from 3 to 8 and• - R19 is H, -PO3H-, SO3-, -N(Me)3+, -N(Et)3+, -N(Me)2, -N(Et)2, -N(CH2CH2OH)2, -OH, -OMe, -OEt, -NH2, -C(O)NHC(CH2OH)3, -N(Me)2+(CH2)PSO3-, C(O)N[(CH2)pCH3][(CH2)PSO3-], -C(O)N[(CH2)pCH3][(CH2)PPO3H], N(Me)2+(CH2)pPO3H-, with p an integer from 2 to 10,and w an integer from 0 to 10.

11. The cyanine fluorogenic dimer probe of any one of Claims 1 to 10, wherein[Ligand] is a ligand which specifically binds to a membrane protein, such as a membrane receptor, e.g. a G protein-coupled receptors (GPCR), and / or which specific binds to a cell type, e.g. a cancer cell type, said ligand being an endogenous ligand or a non-endogenous ligand, and / or[Linker] is a three-branch hydrocarbon group having up to 200carbon atoms in its backbone, said hydrocarbon group comprising one or more unsaturated or saturated C2- C2o hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, -O-N=CR4- -NHN=CR4, and / or amino acid residues such as lysine, citrulline, valine, alanine, and / or saturated or unsaturated, 3- to 20-membered cyclic or heterocyclic groups such as triazole or pyrazole, optionally substituted and / or fused, each R4being independently H, or a Ci- C3 alkyl and said three-branch hydrocarbon group being optionally substituted by one or more groups preferably selected from Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, Ci-Ce aminoalkyl, -OH, -CN, -NH2and halogen.

12. The cyanine fluorogenic dimer probe of any one of Claims 1 to 11, wherein [Ligand] is selected from the group consisting of synthetic small chemical molecules, hormones, vitamins such as acid folic, oligosaccharides such as those comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine (GalNac), bridged GalNac and sialic acid and derivatives thereof (such as Neu5Ac, Neu5Aca2-6Gal, Neu5Aca2-8Neu5Ac), proteins or fragments thereof, peptides such as oxytocin, apelin, RGD peptide, Angiopep-2, or muscle targeting peptides, an aptamer, an antibody including heavy-chain antibody and bispecific antibody, and fragments thereof such as Fab, Fab’, and VHH (also called nanobody), a ScFv, a spiegelmer, and a peptide aptamer.

13. The cyanine fluorogenic dimer probe of any one of Claims 1 to 12 which is of Formula (la)Wherein:[Cy] is as defined in any one of claims 1 to 5,- L3 is a chain comprising up to 70 carbon atoms, and comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, - amino acidresidues and / or saturated or unsaturated C3-C6-membered cyclic or heterocyclic group such as triazole or pyrazole,- L4 is a unsaturated or saturated C2-C30 hydrocarbon chain which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR4(C=0)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,- Each R4 is independently H, or a C1-C3 alkylr is an integer from 0 to 3,s is an integer from 0 to 6,[Ligand] is as defined in anyone of claims 1, 11 and 12.

14. The cyanine fluorogenic dimer probe of any one of Claims 1 to 13 wherein the [Ligand] is a polypeptide comprising at least one cysteine residue and wherein [Ligand] is linked to the [Linker] by one of the following moi eties:atom from the lateral chain of a cysteine residue in [Ligand],15. A chemical intermediate for the preparation of a cyanine fluorogenic dimer probe of any one of Claims 1-14 which is of formula:[Cyi] [Cyi] [Cyi] [Cyi] [Cy] [Cy][Cyi][Linker] [Linker] [Linker][Ligand] [CONNECT] [CONNECT](Ila), (lib) or (lie)Wherein:[Linker] is as defined in any one of claims 1 and 11[Ligand] is as defined in any one of Claim 1, 11 and 12[Cy] is as defined in any one of claims 1 to 5Each [Cyi] is selected from the group consisting ofn (D1i) with n is 1, 2 or 3; andWherein Gi, R2, and R3 are as defined in Claim 1Wherein:Li and X are as defined in Claim 1 and- Ri is a chemical reactive group able to form a covalent bond by click reaction or bioconjugation reaction preferably selected from alkyne-azide cycloaddition / sydnone and iminosydnone-alkyne cycloaddition, tetrazole / alkene reaction, reaction ofaldehyde / ketone with alkoxyamine or hydrazine, amidation, reductive amination, diels- alder, and thia-diels-alder and[CONNECT] is a reactive group able to create at least one covalent bond by click reaction or a reactive group able to create at least one covalent bond by bioconjugation with an amino acid preferably selected from cysteine, tyrosine and lysine,and wherein [Ri] and [CONNECT] are selected so as to be orthogonal when the chemical intermediate is of formula (lib).

16. The chemical intermediate of Claim 15, wherein- Ri is a chemical reactive group able to form a covalent bond by alkyne-azide cycloaddition, and / or[CONNECT] is a reactive group able to create a covalent bond by bioconjugation with an amino acid residue, said reactive group being selected from aryldiazonium, phenyltriazolinedione (PTAD), 3 -arylpropionitrile (APN), diAPN, arylsulfonamide, maleimide, benzoyl acrylamide, iodoacetamide, isothiocyanate, isocyanate, benzoyl fluoride, and an activated carboxylic acid group, e.g. NHS-ester.

17. The chemical intermediate of Claim 15 or Claim 16, wherein Ri is -CH₂-C≡CH.

18. The chemical intermediate of Claim 15 which is of formulaWherein:- R17 is selected from the group consisting of OH, NH2, an activated carboxylic group, and [CONNECT]-L5- - Ris is selected from the group consisting of OH, NH2, an activated carboxylic group, [LIGAND]-L5- and [CONNECT]-L5-,- L4 is a C2-C30 unsaturated or saturated hydrocarbon chain, which is optionally interrupted by one or more groups selected from C(=O), O, NH, NR4(C=0)-, NR4(C=O)NR4, NR4(C=O)O, and -O(C=O)-,- Ls is a hydrocarbon chain comprising up to 70 carbon atoms, and which comprises one or more unsaturated or saturated C2-C20 hydrocarbon chains and / or one or more poly(C2-Cs alkyleneoxy) chains, said chains being connected together through connecting groups selected from C(=O), O, NH, NR4(C=O)-, NR4(C=O)NR4, NR4(C=O)O, -O(C=O)-, and / or amino acid residues and / or saturated or unsaturated C3- Ce-membered cyclic or heterocyclic group such as triazole or pyrazole- Each R4 is independently H, or a C1-C3 alkylr is 0 or 1[CONNECT], [LIGAND], [Cyi] and [Cy] being as defined in Claim 15.

19. A chemical intermediate of any one of claims 15 to 18 which comprises a [CONNECT] moiety which is a diAPN moietyand wherein the chemical intermediate is preferably for the preparation of a cyanine fluorogenic dimer probe having an antibody as [Ligand],20. The chemical intermediate of Claim 19, wherein the diAPN moiety is of formula:

21. A kit for the preparation of a cyanine fluorogenic dimer probe for the labelling of a molecular target of any one of Claims 1 to 14, which comprises:- a chemical intermediate of any one of claims 15 to 20,- optionally one or more chemical reagents, and- optionally instructions for preparing the cyanine fluorogenic dimer probe from the chemical intermediate.

22. Use of a cyanine fluorogenic dimer probe of any one of Claims 1 to 14 for detecting a molecular target, in vitro or ex vivo, for instance in a tissue, a cell or an organoid.

23. A cyanine fluorogenic dimer probe of any one of Claims 1 to 14 for use as a diagnostic agent in vivo.

24. Method of labelling a molecular target in vitro, ex vivo or in vivo, comprising a step of contacting the molecular target with a cyanine fluorogenic dimer probe of any one of Claims 1 to 14.

25. Use of a cyanine fluorogenic dimer probe of any one of Claims 1 to 14 in the manufacture of a diagnostic agent in vivo.

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