EP2178834A1 - Three-functional pseudo-peptidic reagent, and uses and applications thereof - Google Patents

Abstract

The invention relates to a three-functional pseudo-peptidic reagent, to the various uses thereof, in particular for preparing bioluminescent reagents or optionally luminescent bio-conjugates, to the use of said reagents and bio-conjugates for functionalising solid substrates, and to the use of solid substrates thus functionalised in the detection of molecules of interest.

Description

 TRIFUNCTIONAL PEPTIDE PSEUDO REAGENT, ITS USES

AND APPLICATIONS

The present invention relates to a trifunctional pseudopeptide reagent, for its various uses, in particular for the preparation of luminescent reagents or bioconjugates, optionally luminescent, the use of these reagents and bioconjugates for the functionalization of solid supports, as well as for the use of the solid supports thus functionalized for the detection of molecules of interest.

Many applications involving bioconjugates derived from biopolymers (nucleic acids, proteins, polysaccharides, etc.) involve being able to covalently (reversibly or non-reversibly) attaching these bioconjugates to a second molecular architecture (biopolymer, solid support,. ..) and to detect and / or quantify them accurately (optical detection, radioactive, ...). Thus, it is essential to have tools for effectively combining a biopolymer with other (macro) molecules without significantly altering the properties of each of the partners involved in the resulting and targeted molecular architecture.

For this purpose, many small bifunctional molecules (better known as "bifunctional cross-linking reagents") have been developed. A large number are marketed by Pierce. However, it is interesting to note that several academic groups continue to work on the development of new bifunctional reagents that are increasingly sophisticated and allow the development of ever more complex bioconjugates.

As mentioned above, it is sometimes necessary to have not two but three orthogonal functions (i.e., each function is able to react selectively with a targeted partner). Thus, the development of trifunctional reagents is required.

Thus it has already been described, for example in the articles by Alley, SC et al., J. Am. Chem. Soc., 2000, 122, 6126-6127 and Sinz, A. et al., J. Am. Soc. Mass Spectrom., 2005, 16, 1921-1931, the use of various types of trifunctional reagents, including, in particular, sulfo-SBED available from the company Pierce (Rockford, IL, USA), that is to say sulfosuccinimidyl-2- [6 (biotinamido) -2- (p-azidobenzimidazo) -hexanoamido] ethyl-1,3'-dithiopropionate, for the study of protein / protein interactions. This trifunctional reagent is composed of a reactive amine and a photoreactive site. The sulfo-SBED has the following structure:

Photoreactive site However, the sulfo-SBED is not entirely satisfactory inasmuch as it is not possible to modulate the hydrophobic / hydrophilic nature and especially the third functional unit (ie biotin) allows only strong interaction (quasi -covalent) with partners previously conjugated to avidin (glycoprotein) or streptavidin, which greatly limits its use.

It has also already been described, especially in International Application WO 00/02050, trifunctional reagents for preparing bioconjugates consisting of a trifunctional central unit selected from triaminobenzene, tricarboxybenzene, dicarboxyaniline and diaminobenzoic acid, on which three affinity ligands, a biomolecule-reactive group and an effector agent are attached via three distinct linker arms. This reagent, however, has the disadvantage of having a trifunctional central unit consisting of at least two identical chemical functions (ie, carboxylic acid or amine functions) which restricts the choice of the complementary functions carried by the connecting arms which may have a limiting effect.

In other applications, particularly for the detection of analytes by immunofluorescence in the heterogeneous phase, it has already been proposed to use trifunctional reagents (tripods) comprising three distinct functional poles, namely a luminescent group (L), a molecule (B) selected from the analyte to be detected, an analogue or a fragment thereof and finally a function ensuring the attachment of said trifunctional reagent to the surface of a solid support (see in particular French patent application published under No. FR 2,847,984 and the corresponding article by Volland, H. et al., Anal. Chem., 2005, 77, 1986-1904). These reagents are however not usable in any type of application.

The development of ever more advanced technologies requires the creation and use of ever more sophisticated bioconjugates that it is not easy (impossible to achieve) to achieve with the bi functional or trifunctional reagents currently available on the market.

It is therefore in order to remedy all of these disadvantages and to provide in particular a new family of trifunctional reagents in which it is possible to vary very broadly the nature of each function to be able to effectively associate a biopolymer with other (macro) molecules without unduly altering the properties of each of the partners involved in the resulting and targeted molecular architecture, that the inventors have developed what is the subject of the present invention. More specifically, the inventors have set themselves the goal of providing a trifunctional reagent for covalently binding (reversible binding or not) a bioconjugate to a second molecular architecture such as a biopolymer or a solid support.

The present invention therefore firstly relates to a trifunctional pseudopeptide reagent, characterized in that it comprises at least the following three reactive structural units A, B and C:

(a) a unit A consisting of a hydrophilic chain of pseudopoly (ethylene glycol) (pseudo-PEG chain) interrupted by at least one amide function and having two ends E1 and E2, said E1 end being free and having a pattern terminal selected from amino group (-NH ₂ ), activated carbamates and activated esters, and said E2 end having a terminal carbon atom carrying a carbonyl function, said carbon atom being engaged in an amide function (-C (O) -NH-) formed with the nitrogen atom of an α-amine function carried by the unit B;

(b) a unit B consisting of an amino acid chosen from α-amino acids of the L, D series and their racemic mixtures, said amino acid having on its side chain at least one oxyamine function protected by a protecting group or at least one minus a masked aldehyde function; and (c) a unit C consisting of an amino acid selected from α-amino acids of the L, D series and their racemic mixtures, said amino acid having on its side chain at least one thiol, maleimide, iodoacetyl, azide unit, true alkyne, phosphane or cyclooctyne; said units B and C being linked to one another via an amide function formed between the carbon atom carrying the carbonyl function of the α-amino acid constituting the unit B and the nitrogen atom of the α-amine function of the α-amino acid constituting the unit C.

The main originality of these structures lies in the combination of amino acids (natural or modified) protected on their side chains by selected protective groups, and a functionalized pseudo-PEG chain whose length is easily adjustable. Even if the presence of several chemical functions (protected or not) necessarily entails problems during the preparation of these reagents (incompatibilities between functions, degradation and / or premature deprotection of certain protective groups, ...), the structure segmented into three Separate structural units (prepared independently of each other and then coupled together in the final steps of developing the trifunctional reagent) allows for a highly convergent synthesis strategy that minimizes the simultaneous presence of incompatible chemical motifs between them. Furthermore, a large structural diversity (modulation of geometry, length, physicochemical properties and solubility in particular, etc.) is accessible by modifying only one of the three structural units of the trifunctional reagent.

According to the invention, the term "pseudo" -PEG string, a string having great similarities of structure with the PEG chain: but which differs by the presence of one or more functional groups (ester, amide, carbamate, urea, ...) within it.

According to the invention, the term "true" alkyne is understood to mean an alkyne whose triple bond is monosubstituted by a R: R 3 group. The three reactive structural units constituting the trifunctional reagents according to the present invention make it possible to carry out reactions totally chemo-selective under mild conditions (especially in aqueous media in which biopolymers are soluble).

The activated carbamate or activated ester unit which may be present at the free E-terminus of the pseudo-PEG chain constituting unit A is reactive with respect to compounds having a complementary reactive unit such as an amine function (generally a primary amine aliphatic). Moreover, the primary amine function which may alternatively be present at the free E-terminus of the pseudo-PEG chain constituting unit A is reactive with respect to compounds having a complementary unit such as a carbamate or an activated ester . This end E1 allows in particular the fixation of biological macromolecules which comprise, naturally or not, said complementary reactive functions (antibodies, nucleic acids or analogs, polysaccharides, proteins, peptides, radionuclides, toxins, enzyme inhibitors, haptens, etc.). .).

The grouping of unit B, after possible activation, that is to say after deprotection in the case of the oxyamine function or after unmasking in the case of the aldehyde function, under mild conditions compatible with the stability of the various partners involved, is reactive vis-à-vis compounds or materials having one or more carbonyl groups (aldehyde, ...) or alternatively amine. Among such compounds, mention may in particular be made of macromolecules such as antibodies, nucleic acids or the like, liposomes, polysaccharides, proteins, peptides, active principles, radionuclides, toxins, fluorophores, enzyme inhibitors, haptens, etc.

The thiol unit defined as a possible side substituent for the α-amino acid constituting the unit C is reactive with respect to the compounds or materials comprising a maleimide or iodoacetyl unit. The maleimide and iodoacetyl units, defined as possible side substituents of the α-amino acid constituting the unit C, are reactive with respect to the compounds or materials having a thiol function or a cysteine after their possible deprotection. Finally, the azide unit defined as possible lateral substituent of the α-amino acid constituting the unit C is reactive with respect to the compounds or materials having a true alkyne unit, phosphane or cyclooctyne and alternatively the true alkyne units, phosphane or cyclooctyne defined as possible side substituents of the α-amino acid constituting unit C are reactive with respect to compounds or materials having an azide pattern. This reaction site can in particular be used for the attachment of a fluorophore group carrying a complementary functional group reactive with the thiol, maleimide iodoacetyl, azide, true alkyne, phosphane or cyclooctyne units present on the C unit.

According to a particular embodiment of the present invention, the pseudo-PEG chain of unit A is chosen from the following chains of formula (A-I):

(AI) in which:

Ri represents a primary amine, an activated carbamate unit or an activated ester unit,

- m and n, identical or different, are integers between 2 and 10 inclusive,

p is an integer from 1 to 10 inclusive,

the arrow represents the covalent bond connecting the amide function of the E2 end of the pseudo-PEG chain to the unit B or C.

According to a preferred embodiment of the invention, the pseudo-PEG chain is chosen from the pseudo-PEG chains of formula (A-I) above in which m = n = 2 and p = 1.

Among the activated carbamate groups that may be present at the E1 end of the pseudo-PEG chain of unit A, mention may in particular be made of N-hydroxysuccinimidyl carbamate, N-hydroxysuccinimidyl carbamate, N-carbamate and the like. hydroxyphthalimidyl, N-hydroxypiperidinyl carbamate, β-nitrophenyl carbamate and pentafluorophenyl carbamate; N-hydroxysuccinimidyl carbamate being very particularly preferred. Among the activated ester groups that may be present at the E1 end of the pseudo-PEG chain of unit A, there may be mentioned in particular the N-hydroxysuccinimidyl ester, the N-hydroxysuccinimidyl sulfo ester, the ester cyanomethyl, the N-hydroxyphthalimidyl ester, the N-hydroxypiperidinyl ester, the p-nitrophenyl ester, the pentafluorophenyl ester, the benzotriazole esters, the hydroxybenzotriazole esters and the hydroxyazabenzotriazole esters; the N-hydroxysuccinimidyl ester being particularly preferred.

Among the α-amino acids that can be used for unit B, there may be mentioned lysine, homolysine, ornithine, 2,4-diaminobutanoic acid (DABA) and 2,3-diaminopropanoic acid.

Among such α-amino acids, lysine is particularly preferred.

Among the α-amino acids which can be used for unit C, there may be mentioned in particular lysine, cysteine, homolysine, ornithine, 2,4-diaminobutanoic acid (DABA), 2,3-diaminobutanoic acid (DABA), diaminopropanoic acid, aminomercaptoacetic acid, Thomocysteine, 5-mercapto-norvaline, 6-mercapto-norleucine, 2-amino-7-mercapto-heptanoic acid, and 2-amino-8-mercaptoacetic acid; octanoic.

Among such amino acids, lysine and cysteine are particularly preferred.

The protective groups for the oxyamine function of the side chain of the α-amino acid constituting the unit B, are preferably chosen from labile protective groups under mild conditions such as 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Z ), allyloxycarbonyl (Alloc), trichloroethoxycarbonyl (Troc), trimethylsilylethoxycarbonyl (Teoc), pyridyldithioethoxycarbonyl (Pydec), 2- (2-nitrophenyl) -propyloxycarbonyl (ΝPPOC), azomethyloxycarbonyl (Azoc), 2 - (trimethylsilyl) ethanesulfonyl (Ses) and phthalimide.

Among such protective groups, the groups Fmoc and phthalimide are particularly preferred.

For the purpose of the present invention, the term "masked aldehyde function", any aldehyde or ketone function included in a molecule organic compound selected from serine or any organic molecule containing the 1,2-diol (-CH (OH) -CH (OH) -), 1,2-amino-alcohol or 1,2-hydroxythiol moiety. Among such organic molecules, there may be mentioned tartaric acid, glyceric acid, 2,3-dihydroxypropanoic acid, 3,4-dihydroxybutanoic acid, 4,5-dihydroxypentanoic acid and the acid. 3-amino-2-hydroxypropanoic acid.

For the purposes of the present invention, the term "mild conditions", the use of oxyamine function deprotection reagents or unmasking function of the aldehyde function at room temperature and at neutral pH or in a pH range between about 5 and 9 inclusive.

As a deprotection or unmasking reagent, mention may in particular be made of periodic acid (HIO ₄ ), metaperiodates such as sodium metaperiodate (NaIO ₄ ) and potassium metaperiodate (KIO ₄ ), as well as the periodates of tetraalkylammonium.

Among the trifunctional reagents according to the invention, mention may be made especially of those selected from compounds of formula (I) below:

(I) in which:

R 1, m, n and p have the same meanings as those indicated above for the unit of formula (A-I),

- X ₁ and X ₂ , which are identical or different and, together with the carbon atom to which they are bonded, represent the hydrocarbon chain of an α-amino acid,

Proc is a protecting group selected from 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Z), allyloxycarbonyl (Alloc), trichloroethoxycarbonyl (Troc), trimethylsilylethoxycarbonyl (Teoc), pyridyldithioethoxycarbonyl (Pydec), 2- ( 2-nitrophenyl) -propyloxycarbonyle (NPPOC), azomethyloxycarbonyl (Azoc), 2- (trimethylsilyl) ethanesulfonyl (Ses) and phthalimide;

- Func is a thiol, maleimide, iodoacetyl, azide, true alkyne, phosphane or cyclooctyne unit.

According to a particularly preferred embodiment of the invention, Xi is an n-butyl chain.

According to another particularly preferred embodiment of the invention, X ₂ is an ethyl or n-butyl chain.

According to a particularly preferred embodiment of the invention, the compounds of formula (I) above are chosen from the following compounds of formulas (I-1) to (1-6):

(M)

(1-2)

(1-5)

(1-6).

The trifunctional reagents according to the invention can be prepared according to convergent synthesis methods according to which each of the units A, B and C is prepared individually, these being then assembled to lead to the expected trifunctional reagent. These convergent synthesis methods implement conventional reactions well known to those skilled in the art and the details of which are given in the synthesis examples illustrating the present application.

Thanks to their trifunctionality, the reagents according to the present invention and as described above can have multiple uses and applications.

The trifunctional reagents according to the present invention can firstly be used for the preparation of bioconjugates.

Another object of the present invention is the use of at least one trifunctional reagent as defined above for the preparation of a bioconjugate.

For the purposes of the present invention, the term "bioconjugate" means any trifunctional reagent as described above on which at least one biological molecule of interest is attached. The fixation of the molecule (s) of interest may be carried out at the level of the primary amine function or the activated carbamate or activated ester unit present at the free end of the pseudo-PEG chain constituting the unit A which is reactive vis-a-vis the with respect to biological molecules having an acid function (or carbamate) or a reactive amine function (generally an aliphatic primary amine). The binding of the biological molecule of interest can also be carried out at unit B, after deprotection of the oxyamine function or unmasking of the aldehyde function, which then become reactive with respect to the biological molecules having one or more carbonyl groups (aldehyde, etc.) respectively oxyaminated. It is thus possible to set one or two biological molecules, identical or different, trifunctional reagent according to the present invention.

Among the biological molecules that can be attached to the trifunctional reagents according to the present invention, there may be mentioned in particular antibodies, nucleic acid molecules and their analogues, polysaccharides, proteins, peptides, radionuclides, toxins, enzyme inhibitors, haptens, ... etc. Another object of the invention is thus a bioconjugate characterized in that it consists of a trifunctional reagent as defined above in which the primary amine unit, activated carbamate or activated ester present at the free end of the pseudo-chain. PEG constituting the unit A and / or the oxyamine or aldehyde function carried by the unit B after deprotection or unmasking is (are) functionalized by a biological molecule of interest.

According to the invention, it is therefore possible to have the following three types of bioconjugates: i) a bioconjugate constituted by a trifunctional reagent in which only the amine function or only the activated carbamate or activated ester unit present at the free end of the pseudo chain -PEG constituting the unit A is functionalized by a biological molecule, ii) a bioconjugate constituted by a trifunctional reagent in which only the deprotected oxyamine or aldehyde function unmasked from the unit B is functionalized by a biological molecule, and iii) a bioconjugate comprising two biological molecules, constituted by a trifunctional reagent in which the primary amine function or the activated carbamate or activated ester unit present at the free end of the pseudo-PEG chain constituting the unit A and the deprotected oxyamine or aldehyde function unmasked from unit B are each functionalized by a biological molecule; in the latter case the bioconjugate comprises two biological molecules that may be identical or different from each other. It may in particular be two identical biological molecules but modified by two different markers.

The preparation of these bioconjugates can be made conventionally by reacting a trifunctional reagent according to the invention with the biological molecule or molecules of interest to be fixed, while using the methods well known in the technical state for reacting by example:

an activated carbamate or amino-activated ester (Hermanson, GT, Bioconjugate Techniques, 1996, Academy Press, Inc.) or an oxyamine function with a carbonyl group (Sing, Y. et al, Org Biomol Chem. , 2006, 4, 1413-1419 or Zatsepin, TS et al, Bioconjugate Chem., 2005, 16, 471-489. The tri functional reagents according to the present invention can also be used for the preparation of luminescent reagents, in particular fluorescent reagents.

Another object of the present invention is therefore the use of at least one trifunctional reagent as defined above, for the preparation of a luminescent reagent, in particular fluorescent reagents.

In this case, the grafting of a luminescent group (L) can be carried out: i) by reacting the thiol, maleimide, iodoacetyl, azide, true alkyne, phosphane or cyclooctyne unit carried by the unit C with a maleimide complementary function , iodoacetyl (if unit C has a thiol function), thiol (if unit C has a maleimide or iodoacetyl unit) or true alkyne, phosphane or cyclooctyne (if unit C has an azide function), or else azide (if the unit C comprises a true alkyne function, phosphane or cyclooctyne), said complementary function being carried, naturally or not, by said luminescent group, and / or ii) by reacting the deprotected oxyamine or aldehyde function unmasked from the unit B with a luminescent group carrying a carbonyl function such as an aldehyde or ketone function or with an oxyamine function, respectively.

In these two cases, it may be necessary to previously functionalize the luminescent group to react by a complementary pattern of the function with which it wants to react. The preparation of these luminescent reagents can be made conventionally according to the reactions known to those skilled in the art.

It is thus possible to obtain luminescent reagents comprising: i) a single luminescent group attached to the unit B via the deprotected oxyamine function or the unmasked aldehyde function or on the unit C via the thiol function or maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide units; ii) two luminescent groups, one being attached to the unit B via the deprotected oxyamine function or the unmasked aldehyde function and the other being attached to the unit C via the thiol function or maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide units.

These trifunctional luminescent reagents constitute another object of the invention.

The nature of the luminescent group or groups that can be used according to the invention is not critical from the moment when they naturally comprise or are functionalisable by a thiol or carbonyl function, or by a maleimide, iodoacetyl or alkyne unit. true, phosphane, cyclooctyne or even azide.

According to the invention, the term luminescent group any substance which, when excited at a given wavelength or by a given chemical compound, is capable of emitting a photon, for example fluorophore or rare earth.

Among the luminescent groups (including fluorophores) that can be used according to the invention, mention may be made in particular of the fluorophores with polymethine chains (ie polyene chain); fluorescent cyanines such as those sold under the references Cy3, Cy3.5, Cy3B, Cy5, Cy5.5 and Cy7 by the company GE Healthcare; fluorescein (sodium fluorescein) and its derivatives such as fluorescein isothiocyanate (FITC) and 6-carboxyfluorescein (6-Fam); rhodamine and its derivatives such as tetramethyl rhodamine isothiocyanate (TRITC); water-soluble derivatives of rhodamine in the form of N-hydroxysuccinimide ester, such as the products sold under the trade name Alexa Fluor® by Invitrogen, for example Alexa Fluor® 488, 500, 514, 532, 546, 555, 568, 594, 610-X, 633, 647, 660, 680, 700, 750 and 790; rhodols and their derivatives; coumarin derivatives such as 7-amino-coumarin; 9-aminoacridine and 9-acridinecarboxylic acid; reactive amine fluorescent dyes such as 6 - ((7-amino-4-methylcoumarin-3-acetyl) amino) hexanoic acid succinimidyl ester (AMCA); dipyrromethene boron difluorides sold under the trade names BODIPY® such as BODIPY® FR-Br ₂ , BODIPY® R6G, BODIPY® TMR, BODIPY® TR and BODIPY® 530/550 (excitation wavelength / emission wavelength, in nm), 558/567, 564/570, 576/589 , 581/591, 630/650 and 650/665 sold by Bio-Rad Inc. (USA); fluorophores derived from pyrene, such as, for example, Cascade Blue dyes (sold for example by Trilink BioTechnologies (USA) or Invitrogen), disazo derivatives such as DABCYL®, danzyl derivatives such as EDANS® (Eurogentec, BE), eosin, erythrosine and sulforhodamine derivatives such as sulforhodamine chloride / sulphonyl 101 also known as Texas Red The trifunctional reagents according to the present invention can also be used for the preparation of reagents luminescent, in particular fluorescent, also comprising an "acceptor compound" (Q) of the luminescence of the luminescent group In this case, such reagents constitute what is known as an energy transfer cassette (or FRET cassette ) usable especially for the sequencing of DNA.

Another object of the present invention is therefore the use of at least one trifunctional reagent as defined above for the preparation of a power transfer cassette.

In this case, the grafting of the luminescent group will be carried out on the deprotected oxyamine or aldehyde function unmasked from the unit B or via the thiol function or the maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide units of the unit C, and the grafting of the acceptor compound (Q) will be carried out respectively via the thiol function or the maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide units of the unit C or on the deprotected or aldehyde oxyamine function unmasked from unit B.

The invention therefore also relates to a transfer cassette of energy, characterized in that it consists of a trifunctional reagent as defined above, said reagent comprising a luminescent group (L) and an acceptor compound (Q ) of the luminescence of the luminescent group, L and Q being respectively and indifferently fixed on said trifunctional reagent via the deprotected oxyamine or aldehyde function unmasked from the unit B and the function thiol or the units maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide unit C.

In these two cases, it may be necessary to previously functionalize the acceptor compound (Q) to react by a complementary pattern of the function with which it wants to react.

The luminescent group (L) that can be used in these energy transfer cassettes may in particular be chosen from the luminescent groups (L) mentioned above and that can be used for the preparation of the luminescent trifunctional reagents in accordance with the present invention. According to the invention, the term "acceptor compound" (Q) is understood to mean any molecule making it possible to reduce or disappear the luminescence of the luminescent group (L) under certain conditions. This compound, of various natures, can in particular be a chemical compound (luminescent or not and such as for example fluorescent proteins), a heavy atom or a nanoparticle. Among such acceptor compounds (Q), mention may in particular be made of fluorescent compounds such as those mentioned above for L groups, in particular rhodamine and its derivatives such as tetramethyl rhodamine (TMR) and Rhodamine 6G (R6G). ) and QSY® 7, QSY® 9 and QSY® 21 dyes (Molecular Probes); but also non-fluorescent molecules of the family of azo dyes, such as the compounds sold under the trade names Black HoIe Quencher® (BHQ), for instance BHQ-O, BHQ-I, BHQ-2 and BHQ-3 (Biosearch Technologies); gold particles such as those having a diameter of 1.5 nm sold under the trade name Nanogold Particles ® (Nanoprobes); disazo dyes such as the products sold under the trade names Eclipse Dark Quencher® (Epoch Bioscience) or QSY® 35 (Molecular Probes); the ElleQuencher ® commercial product (Eurogentec); malachite green and the acceptor compounds ("Quenchers") of the cyanine family, such as the compounds sold under the trade names Cy5Q or Cy7Q by the company GE Healthcare. According to a particularly advantageous embodiment of these energy transfer cassettes, the luminescent group (L) and the acceptor compound (Q) are chosen from the following pairs (L / Q): Cy3 / Cy5, Cy5 / Cy7, Cy5 / Alexa Fluor ^® 750, Cy3 / Cy5Q, Cy3 / QSY ^® 7, Cy3 / QSY ^® 9, Cy5 / Cy7Q, Cy5 / QSY ^® 21, Cy5 / Cy5, Cy5.5 / Cy5.5, Cy7 / Cy7, R6 ) / Cy5, R6G / Alexa Fluor ^® 647, R6G / QSY ^® 21, Alexa Fluor ^® 532 / Cy5, Alexa Fluor ^® 532 / Alexa Fluor ^® 647, Alexa Fluor ^® 532 / QSY ^® 21, Alexa Fluor ^® 555 / Cy5, Alexa Fluor ^® 555 / Alexa Fluor ^® 647, Alexa Fluor ^® 555 / QSY ^® 21.

According to another particular embodiment of the invention, the trifunctional reagents can be used for the preparation of mixed bioconjugates comprising at least one biological molecule and at least one luminescent group. The present invention therefore also relates to a mixed bioconjugate, characterized in that it consists of a trifunctional reagent as defined above on which are fixed at least one biological molecule and at least one luminescent group. In addition, these mixed bioconjugates may also comprise an acceptor compound (Q) for the luminescence of the luminescent group (L).

These mixed bioconjugates are therefore chosen from the trifunctional reagents on which are fixed: i) one or two biological molecules of interest and a luminescent group; or ii) a biological molecule of interest, and two luminescent groups; iii) a biological molecule of interest, a luminescent group, and an acceptor compound (Q) of the luminescence of the luminescent group; said biological molecules and said luminescent moieties and acceptor compound (Q) being attached to the functional reagent at the terminal ends of units A, B and C as described above.

Trifunctional reagents and bioconjugates according to the present invention, modified or not by a luminescent group at unit C, and in which the oxyamine or aldehyde function of unit B is free (ie non-functionalized by a biological molecule of interest, a fluorescent group or an acceptor compound) can be used for the functionalization of solid supports comprising at least one surface having one or more carbonyl groups, in particular one or more aldehyde or ketone functions or one or more oxyamine functions, respectively. Consequently, the subject of the present invention is also a solid support, characterized in that it comprises at least one surface functionalized, covalently, with one or more trifunctional reagents, and / or with one or more bioconjugates, said reagents trifunctional and bioconjugated being optionally modified by a luminescent group at the level of the unit C and as defined above.

According to the invention, the nature of the solid support is not critical from the moment when it comprises at least one surface having, naturally or after chemical modification, one or more carbonyl groups, in particular one or more aldehyde or ketone functions or respectively one or more primary amine functions, said groups or functions being able to react with the deprotected oxyamine function or respectively with the unmasked aldehyde function of the unit B of the tri functional reagent or the bioconjugate.

Among such supports, there may be mentioned glass, plastic, and metals.

According to a particular embodiment of the invention, the solid support comprises at least one surface functionalized with at least one bioconjugate and then constitutes a biochip such as for example a nucleic acid chip, a protein chip, a polysaccharide chip or a peptide chip, or a biosensor such as for example an immunosensor.

Such supports can be prepared according to a process comprising the following steps: deprotection of the protected oxyamine function, or respectively the unmasking of the masked aldehyde function, present on unit B of at least one trifunctional reagent or from less a bioconjugate according to the invention to obtain a trifunctional reagent or a bioconjugate bearing a deprotected oxyamine function, respectively undefined aldehyde, - the formation of an oxyme bond by contacting said reagent or said bioconjugate carrying the oxyamine function deprotected or respectively aldehyde unmasked with a solid support of which at least one surface has a or more carbonyl groups, respectively primary amine, said oxyme bond ensuring the covalent attachment of said reagent or said bioconjugate to the surface of the support.

Such supports may in particular be used for the detection of molecules of interest, in particular for the detection of analytes in a liquid medium.

According to another embodiment, the invention also relates to the use of at least one trifunctional reagent as described above for the preparation of a probe for functional proteomics.

Another object of the present invention is therefore a probe for functional proteomics, characterized in that it consists of a trifunctional reagent comprising:

a group allowing detection (or visualization) and / or purification of a target protein (reporter tag or "reporter tag"),

a pattern recognized by said target protein (recognition unit or "recognition unit"), and

a reactive group making it possible to establish a covalent link with the active site of the targeted protein (reactive group).

According to the invention, the term "pattern recognized by said target protein", any ligand of the targeted protein or any substrate if it is an enzyme (peptide sequence for example).

According to a preferred embodiment of the invention the target protein is an enzyme.

According to a first embodiment of these probes for functional proteomics and when the target protein is an enzyme, the pattern recognized by the enzyme and the reactive group allowing a covalent link with the active site of this enzyme belong to the same entity. (This is the case for example irreversible inhibitors and / or suicidal substrates used in medicinal chemistry) and are therefore carried by the same unit of trifunctional pseudopeptide reagent according to the invention. In this case the other two units of said trifunctional reagent can be used to fix a group allowing detection (fluorophore for example) and a group facilitating purification (biotin, poly tag (histidine), ...). According to this embodiment, the three entities can be fixed indifferently on any unit of the trifunctional pseudopeptide reagent according to the invention.

According to a second embodiment of these probes for functional proteomics and when the target protein is an enzyme, the pattern recognized by the enzyme and the reactive group do not belong to the same entity and are therefore carried by two distinct units of the trifunctional pseudopeptide reagent according to the invention. In this case, it is preferable to fix them on the two units closest to each other (B and C) so that the reaction of the reactive group takes place in the targeted (active) site. Thus the last unit of the trifunctional pseudopeptide reagent according to the invention will be used to fix the group allowing detection and / or purification.

In addition to the foregoing, the invention also comprises other provisions which will emerge from the description which follows, which refers to examples of synthesis of trifunctional reagents according to the invention. EXAMPLE 1 PREPARATION OF A TRIFUNCTIONAL REAGENT CONFORMING TO THE INVENTION

In this example is described the synthesis of a trifunctional reagent (1-1) according to the present invention and corresponding to the following formula (1-1):

(M) wherein:

the unit A is a pseudo-PEG binding arm having an activated carbamate unit reactive with respect to the compounds having a primary amine function,

unit B is a lysine bearing on its side chain an oxyamine function protected by a Fmoc group, reactive with respect to surfaces having aldehyde functions, and - Unit C is a lysine having on its side chain a maleimide unit, reactive vis-à-vis the compounds having a thiol function.

The strategy for synthesizing the compound (1-1) consists in separately preparing three correctly protected precursors (AI), (BI) and (CI) and then assembling them via coupling reactions to obtain the trifunctional reagent of formula (I-1 ) above according to the present invention. 1) Synthesis of the precursor (A-I): Boc-PEG link arm

Precursor (AI) The precursor (AI) is a hydrophilic linker having four ethylene glycol units as well as a carboxylic acid function which will allow the subsequent coupling with the units (BI) and (CI) and a primary amino function that it It is essential to keep protected until the last conversion stage to "activated carbamate". It results from the combination of two molecules of 8-amino-3,6-dioxaoctanoic acid, said molecules having been prepared according to the methods described by Rensen, PC N et al, J. Med. Chem., 2004, 47, 5798-5808; Dondoni, A. et al, J. Org. Chem., 2005, 70, 5508-5518 or Dhawan, R. et al Bioconjugate Chem., 2000, 11, 14-21. This amino acid was then converted into two protected derivatives (N-Boc derivative (6) and methyl ester (7)) according to the methods described for example by Nakatani, K. et al, Bioorg. Med. Chem. Lett, 2004, 14, 105-1108 or Rachele, J., J. Org. Chem., 1963, 28, 2898, which could be coupled together according to the method described for example by Han, S. -Y. et al, Tetrahedron, 2004, 60, 2447-2467 to conduct, after saponification, the precursor (AI) sought. a) First step: Synthesis of 2- (2- (2-azidoethoxy) ethoxy) ethanol (3)

1.29 ml (8.9 mmol) of 2- (2- (2-chloroethoxy) ethoxy) ethanol were added to a suspension of sodium azide (0.7 g, 10.7 mmol) and iodide sodium (0.14 g, 0.93 mmol) in anhydrous ethanol. The resulting yellow mixture was refluxed for 5 days under an argon atmosphere. The reaction was checked for completeness by thin layer chromatography (TLC) sold under the reference DC Kieselgel 60 F254 by Merck using a dichloromethane / methanol solvent mixture (9: 1, v / v). The mixture was then filtered through Celite 545 to remove the sodium salts and then evaporated. The resulting oily residue was dissolved in about 10 ml of dichloromethane and stored at a temperature of 4 ° C for 1 hour. After filtration on cotton and concentration, the expected compound (3) was obtained in the form of a colorless oil (quantitative yield).

¹ H NMR analysis (300 MHz, CDCl ₃ ): δ = 2.63 (t, J = 6.0 Hz, 1H, OH), 3.43 (t, J = 4.9 Hz, 2H), 3, 61-3.77 (m, 10H).

VV) Second step: Synthesis of 2- (2- (2-azidoethoxy) ethoxy) acetic acid (4)

1.56 g (8.9 mmol) of the compound (3) obtained above in the previous step were dissolved in 90 ml of acetone and the resulting solution was cooled to a temperature of 4 ° C. 8.9 ml of freshly prepared Jones 3M reagent (for example by dissolving 26.72 g of CrO ₃ in 23 ml of concentrated sulfuric acid and then adding water to a volume of 100 ml) were then added dropwise (a green precipitate was immediately formed), and the resulting reaction mixture was stirred at room temperature for 1 hour. The reaction was checked for completeness by TLC as in the previous step and the reaction was stopped by adding about 4 ml of propan-2-ol. After 15 min, 100 ml of acetone was added and the green precipitate of Cr (III) salts was removed by filtration through Celite ^® 545. The filtrate was then evaporated to dryness. The resulting oily residue was purified by column chromatography on silica gel (50 g) using a gradient of methanol (0-5%) in dichloromethane as the mobile phase. Compound (4) (1.53 g, 8.1 mmol) was obtained as a yellow oil (91% yield). ¹ H NMR analysis (300 MHz, CDCl ₃ ): δ = 3.43 (t, J = 4.9 Hz, 2H),

3.66-3.80 (m, 6H), 4.19 (s, 2H).

¹³ C NMR analysis (75.5 MHz, CDCl ₃ ): δ = 50.7; 68.6; 70.2, 70.6; 71.4; 174.2. c) Third step: Synthesis of 2- (2-aminoethoxy) ethoxy) acetic acid (5)

A mixture of 1.53 g (8.2 mmol) of the compound (4) obtained above in the previous step and 2.5 ml (65.5 mmol) of formic acid was dissolved in 150 ml of ethanol, and the resulting solution was cooled to a temperature of 4 ° C.

0.32 g of Pd / C (Pd / C) 10% Pd was added to this solution and the resulting reaction mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere. It was then checked that the reaction was complete by

TLC as in the previous step but using an 8: 2 (v / v) mixture of dichloromethane (CH ₂ Cl ₂ ) / methanol (MeOH), then the mixture was filtered through Celite ^®

545 to remove Pd / C. The filtrate was then evaporated to dryness to an oily residue which was dried in vacuo to yield the expected compound (5) as a yellow oil (quantitative yield). (d) Fourth step: Synthesis of 2- (2-aminoethoxy) ethoxy) acetic acid methyl ester (6)

0.48 g (2.92 mmol) of the compound (5) obtained above in the preceding step were suspended in 20 ml of 2,2-dimethoxypropane, then 2.92 ml of acid were added. concentrated hydrochloric acid (37% HCl)). The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction was then checked for total TLC using CH ₂ Cl ₂ / MeOH / triethylamine (TEA) 80: 20: 2 (v / v / v) and the mixture was evaporated to dryness. The resulting oily residue was then subjected to 4 successive lyophilizations with the addition of 10 ml of osmosis water each time to finally obtain the expected product in the form of a yellow oil (quantitative yield).

¹ H NMR analysis (300 MHz, CD ₃ CN + 5% D ₂ O): δ = 3.08 (t, J = 5.3 Hz, 2H), 3.56 to 3.76 (m, 1 H) , 4.13 (s, 2H). e) Fifth step: Synthesis of 2- (2-tert-butyloxycarbonyl) aminoethoxy) ethoxy) acetic acid (7)

0.78 g (3.75 mmol) of the compound (5) obtained above in stage c) were dissolved in 12 ml of a mixture of tetrahydrofuran (THF) / H ₂ O (2: 1, v / v). 5 ml of a freshly prepared aqueous solution of 2 M sodium hydroxide was then added and the resulting solution was cooled to 4 ° C. 0.89 g (4.12 mmol) of di-t-butyl dicarbonate was then added and the reaction mixture was stirred at room temperature for 1 hour. It was then checked that the reaction was complete by TLC using CH ₂ Cl ₂ / MeOH (7: 3, v / v). The reaction mixture was then acidified by adding about 25 ml of a 1 M aqueous potassium hydrogen sulfate solution (KHSO ₄ ). This solution was then extracted with 3 times 50 ml of ethyl acetate. The organic phase was dried over sodium sulphate (Na ₂ SO ₄ ) and then evaporated to dryness. The resulting oily residue was purified by column chromatography on silica gel (40 g) using a gradient of methanol (0 to 6%) in CH ₂ Cl ₂ as the mobile phase. Compound (7) (0.50 g, 1.87 mmol) was obtained as a colorless oil (50% yield).

¹ H NMR analysis (300 MHz, CDCl _3): δ - 1, 44 (s, 9H), 3.34 (bm, 2H), 3.50 to 3.77 (m, 6H), 4.17 (s , 2H), 4.97 (bs, 1H, NH). f) Fifth step: Synthesis of the precursor (AI)

0.15 g (0.83 mmol) of the compound (7) obtained above in the preceding step and 0.23 g (0.87 mmol) of the compound (6) obtained above in step d) were dissolved in 8 ml of anhydrous acetonitrile. 0.44 ml (2.5 mmol) of 4,4'-diisopropylethylamine (DIEA) and 0.37 g (0.83 mmol) of benzotriazol-1-yl-Λf-oxytris hexafluorophosphate (dimethyl) were then added. amino) -phosphonium (BOP) then the reaction mixture was stirred at room temperature overnight under an argon atmosphere. It was then checked that the reaction was complete by TLC using a CH ₂ Cl ₂ / MeOH mixture (8: 2, v / v), and then the reaction mixture was evaporated to dryness. The resulting residue was then taken up in 75 ml of ethyl acetate, washed successively with 50 ml of a 10% aqueous solution of citric acid, 50 ml of a saturated solution of sodium bicarbonate (NaHCO ₃ ) and 50 ml of a saturated aqueous solution of NaCl and then dried over Na ₂ SO ₄ and evaporated to dryness. The orange oily residue was then dissolved in 5 ml of MeOH and the solution was cooled to 4 ° C. 0.83 ml of a 1M aqueous sodium hydroxide solution was then added and the reaction mixture was stirred at room temperature for 30 minutes. It was then checked that the reaction was complete by TLC using CH ₂ Cl ₂ / MeOH (7: 3, v / v). The reaction mixture was then acidified by addition of about 1 ml of an aqueous solution of 1 M KHSO _4. The solution was then evaporated to dryness without heating and the resulting residue was purified by chromatography on a gel column. silica (30 g) using as mobile phase a gradient of methanol (0-50%) in CH ₂ Cl ₂ . 0.17 g (0.42 mmol) of the linker precursor (AI) was thus obtained in the form of a colorless oil with a yield of 80%. ¹ H NMR analysis (300 MHz, CDCl _3): δ = 1.43 (s, 9H), 3.30 (bm,

2H), 3.47-3.66 (m, 14H), 4.01 (s, 2H), 4.04 (s, 2H), 5.16 (bs, 1H, NH), 7.34 (bs); , 1H, NH).

¹³ C NMR analysis (75.5 MHz, CDCl ₃ ): δ = 28.8 (3C); 38.9; 70.3 (3C); 70.5 (3C); 71.1 (3C); 79.8; 156.5; 171.6; 175.0. MS (ESI, positive mode) m / z: 431.27 (M + Na) ⁺ , 453.27 (M-H + 2Na) ⁺ .

MS (ESI, negative mode) m / z: 407.47 (MH) ^- calcd for C ₁₇ H ₃₂ N ₂ O ₉ 408.45 2) Synthesis of the precursor (BI)

Precursor (BI)

The precursor (BI) was synthesized in two steps from the protected N-Fmoc derivative of the aminooxyacetic acid (8) previously prepared by reaction between the commercial aminooxyacetic acid and the chloroformate of 9-fluorenylmethanol under Schotten-Baumann conditions (Cipolla L. et al, Bioorg Med Chem, 2002, 10, 1639-1646). a) First preliminary step: Synthesis of 9-fluorenylmethoxycarbonylaminooxyacetic acid (8)

0.5 g (4.6 mmol) of carboxymethoxylamine hemihydrochloride was dissolved in 20 ml of an aqueous solution containing 1.2 g of Na ₂ CO ₃ and then the resulting solution was cooled to 4 ° C. 1.31 g (5.0 mmol) of 9-fluorenylmethyl chloroformate in 10 ml of anhydrous dioxane were then added dropwise to the reaction medium, which was then stirred at room temperature overnight. The reaction mixture was concentrated, acidified to pH 4-5 by addition of about 5 ml of 5% aqueous hydrochloric acid solution and the product precipitated in the solution. It was recovered by filtration and washed once with 20 ml of distilled water and then once with 20 ml of pentane. The residual water was removed by lyophilization to afford 1.05 g (3.4 mmol) of the compound (8) in crude form. After purification by chromatography on a column of silica gel (30 g) using a mobile gradient gradient of methanol (0-50%) in dichloromethane, 0.64 g (2.0 mmol) of the compound ( 8) as a white foam (yield 43%). ¹ H NMR analysis (300 MHz, CD ₃ OD): δ = 4.22-4.27 (m, 3H), 4.44

(d, J = 6.8 Hz, 2H), 7.29-7.42 (m, 4H), 7.64 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H). b) Second Preliminary Step: Synthesis of N - (tert-butyloxycarbonyl) -L-lysine (10)

1.0 g (2.63 mmol) iy _{^: Ctert-butyJoxycarbony.} l) _: N! -:

_{(benzyloxycarbonyl)):} The ly_sine _τ (commercial product) was dissolved in 50 ml of ethyl acetate and then the solution was cooled to 4 ° C. 0.1 g Pd / C at 10% Pd was then added and the resulting reaction mixture was stirred at room temperature for 21 hours under a hydrogen atmosphere. The reaction was checked for completeness by TLC (CH ₂ Cl ₂ / MeOH 85:15, v / v) and then the reaction mixture filtered through Celite 545 to remove Pd / C. Due to the low solubility of the compound (10) in ethyl acetate was conducted washes Celite ^® 545 with 2 times 50 ml of methanol to recover the compound (10). The filtrate was evaporated to dryness and the resulting oily residue was dried in vacuo to afford 0.41 g (1.67 mmol) of the compound (10) as a white foam in 64% yield. (c) First step: Synthesis of 9-fluorenylmethoxycarbonylaminooxyacetic acid succinimidyl ester (9)

0.560 g (1.80 mmol of the compound (8) obtained above in step a) were dissolved in 15 ml of a mixture of ethyl acetate / dioxane (2: 1, v / v), then the solution was cooled to a temperature of 4 ° C. 0.225 g (1.95 mmol) of N-hydroxysuccinimide and 0.404 g of Λζ N'-dicyclohexylcarbodiimide (DCC) were added successively to this solution and the resulting reaction medium was stirred at room temperature overnight under an atmosphere of argon. Converting the compound (8) N-hydroxysuccinimidyl ester corresponding (9) was monitored by high performance reverse phase liquid chromatography (RP-HPLC) on a Thermo Hypersil GOLD column "Ci ₈ 5 .mu.m and dimensions 4.6 x 150 mm, using acetonitrile and a 0.1% (v / v, pH 2) aqueous solution of trifluoroacetic acid (TFA) as eluents (80% 0.1% aqueous TFA solution with a linear gradient of 20 to 90% acetonitrile in a minute, at a flow rate of 1.0 ml / min Double UV detection was performed at 210 and 254 nm The compound (9) ( _R = 20, 72 min) was then immediately used in the subsequent coupling reaction without purification. d) Second step: Synthesis of N ^a - (tert -butyloxycarbonyl) - N ^β - ^ - fluorenylmethoxycarbonyl-aminooxyacétvQ-L-lysine (B- 1)

To a suspension of β1-Boc-L-lysine (10) (0.41 g, 1.67 mmol) in 6 ml of anhydrous DMF / NMP (5: 1, v / v) mixture was added. DIEA (0.29 ml, 1.67 mmol) and the crude solution of N-hydroxysuccinimide ester (9) obtained above in the previous step were successively added. The resulting reaction medium was stirred at room temperature for 30 minutes. An aqueous solution of DIEA (0.15 ml in 5 ml) was then added and the resulting solution was stirred at room temperature for 90 minutes. The reaction was followed by HPLC-PI (system identical to that used above in the previous step for the analysis of the compound (9)). The reaction mixture was acidified to pH 5-6 by addition of a 5% aqueous solution of HCl (~ 3 ml) and then evaporated to dryness. The residue thus obtained was taken up in 50 ml of ethyl acetate and the insoluble solid residue (precipitated DCU) removed by filtration on Celite ^® 545. The filtrate was washed with 30 ml of an aqueous acid solution 10% citric, dried over Na ₂ SO ₄ and then evaporated to dryness. The oily yellow residue thus obtained was purified by chromatography on a column of silica gel (45 g) using a gradient of MeOH (0-6%) in dichloromethane as the mobile phase. 310 mg (0.57 mmol) of compound (BI) was obtained in the form of a white foam with a yield of 34%. ¹ H NMR analysis (300 MHz, CD ₃ CN): δ = 1.37-1.83 (m, 15H),

3.18 (q, J = 6.4 Hz, J = 12.6 Hz, 2H), 4.00 (m, 1H, α-CH), 4.04 (s, 2H), 4.26 (t. , J = 6.8 Hz, 1H), 4.49 (d, J = 6.8 Hz, 2H), 5.61 (bd, J = 7.5 Hz, 1H, NH), 7.31-7 , 44 (m, 4H), 7.51 (bs, 1H, NH), 7.63 (d, J = 7.5 Hz, 2H), 7.83 (d, J = 7.5 Hz, 2H) , 8.9 (bs, 1H, NH). ¹³ C NMR analysis (75.5 MHz, CD ₃ CN): δ = 23.5; 28.5 (3C); 29.5

; 31.6; 38.9; 47.7; 54.2; 68.0; 76.3; 79.8; 120.9 (2C); 126.0 (2C); 128.1 (2C); 128.7 (2C), 142.1 (2C); 144.6 (2C); 156.7; 158.9; 169.3; 174.5.

MS (Maldi-TOF, positive mode) m / z: 564.5709 (M + Na) ⁺ , 580.5535 (M + K) ⁺ , calculated for C ₂₈ H ₃₅ N ₃ O ₈ 541.61. 3) Synthesis of the precursor (CI)

This synthesis is represented in Diagram 1 below:

As shown in Scheme 1, the precursor (CI) was synthesized in four steps from β-Boc-β-ZL-lysine (11); in this compound Z represents the benzyloxycarbonyl group. In a first step, the carboxylic acid function is converted to carboxamide (12) by reaction of ammonia with the mixed anhydride intermediately formed according to the method described, for example, by Hofmann, K. et al., J. Am. Chem. Soc., 1978, 100, 3585-3590. Then, the ε-NH _{2 function} protected by a Z group was released (13) by catalytic hydrogenation in order to be able to couple the maleimide derivative of glycine (14) previously prepared by reaction between glycine and N- (methyloxycarbonyl) maleimide according to the method described by Keller O. et al, HeIv. Chim. Acta, 1975, 58, 531-541. The maleimide derivative (15) was then isolated by chromatography on silica gel with a yield of 51%. Finally, the N-terminus was deprotected by treatment with trifluoroacetic acid to yield the precursor (CI). a) First step: Synthesis of N - (tert-butyloxycarbonyl) -2- (benzyloxycarbonyl) ₇ L _: lysine-carboxamide 2 1.0 g (2.63 mmol) of n - (tert-butyloxycarbonyl) -

(Benzyloxycarbonyl) -L-lysine (11) was dissolved in 18 ml of anhydrous ethyl acetate and the solution was cooled to -15 ° C in a bath consisting of dry ice (solid CO ₂ ) and ethylene glycol. 0.29 ml (2.63 mmol) of N-methylmorpholine (NMM) and 0.34 ml (2.63 mmol) of isobutyl chloroformate was then added and the resulting reaction medium was stirred at -15 ° C for 10 minutes under argon atmosphere. Then 1.3 ml of aqueous ammonia (50% v / v) was added to this mixture and the reaction mixture was stirred at 0 ° C for 1 hour. A voluminous white precipitate immediately formed; this was recovered by filtration and washed with 25 ml of distilled water and then with 25 ml of pentane. The residual water was removed by lyophilization to yield 0.705 g (1.86 mmol) of the compound (12). The filtrate was, moreover, taken up in 20 ml of ethyl acetate, washed with 20 ml of distilled water, dried over Na ₂ SO ₄ and evaporated to dryness. The white solid obtained was washed with twice 25 ml of pentane and dried under vacuum. An additional amount of compound (12) (0.25 g, 0.66 mmol) was thus obtained. The total yield was 95%.

¹ H NMR analysis (300 MHz, CD ₃ CN): δ = 1.31-7.78 (m, 15H), 3.10 (q, J = 6.4 Hz, J = 12.9 Hz, 1H) , 3.92 (m, 1H, α-CH), 5.06 (s, 2H, CH ₂ -Bn), 5.53 (bs, 1Η, NH), 5.66 (bs, 1H, NH), 5.71 (bs, 1H, NH), 6.31 (bs, 1H, NH), 7.30-7.42 (m, 5H). b) Second step: Synthesis of N - (tert-butyloxycarbonyl-L-lysine-carboxamide (13)

0.95 g (2.5 mmol) of the compound (12) obtained above in the previous step were dissolved in 50 ml of a mixture of ethyl acetate / ethanol (4: 1, v / v) and the solution was cooled to 4 ° C. 0.1 g Pd / C at 10% Pd was added and the resulting reaction mixture was stirred at room temperature for 3 hours under an argon atmosphere. Was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / Me0H 9: 1, v / v) and the mixture was filtered through Celite ^® 545 to remove Pd / C. The filtrate was then evaporated to dryness to give an oily residue which was then evaporated in vacuo to yield the expected compound (13) as a white solid (quantitative yield).

¹ H NMR (300 MHz, CD ₃ OD): δ = 1.40-1.80 (m, 15H), 2.70 (t, J =

6.8 Hz, 1H), 4.03 (q, J = 4.9 Hz, J = 12, 1 Hz, 1H, α-CH). c) Step Schedule: Synthesis of N-maleoyl-L-Glycine (14) i) preliminary step: Preparation of N- (m4thyApΛycarpony _{ljmaléimide.}

1 g (10.3 mmol) of maleimide was dissolved in 20 ml of anhydrous ethyl acetate and then the solution was cooled to 4 ° C. 1.13 ml of NMM (10.3 mmol) and 0.80 ml (10.3 mmol) of methyl chloroformate were then added and the resulting reaction mixture was stirred at room temperature for 1 hour. A large white precipitate of NMM.HC1 immediately formed; it was removed by filtration and washed with about 20 ml of ethyl acetate. The filtrate was washed with 30 ml of saturated aqueous NaCl solution, dried over Na ₂ SO ₄ and evaporated to dryness. The resulting oily residue was dried under vacuum to give N- (methyloxycarbonyl) maleimide as a purplish brown solid (1.16 g, 7.5 mmol, 73% yield). ii) Preparation of the _. N-Maleoyl-L-Glycine (14) 0.5 g of glycine (6.7 mmol) was dissolved in aqueous NaHCO ₃ (2.8 g in 32 ml) and the solution was then cooled to room temperature. 0 ° C (NaCl / ice bath). 1.04 g (6.7 mmol) of the W- (methyloxycarbonyl) maleimide obtained in step i) above was added and the resulting reaction mixture was stirred vigorously at 0 ° C for 20 minutes. . Then, 60 ml of distilled water was added and the resulting aqueous solution was left at room temperature overnight. The reaction mixture was then acidified to pH 6 by adding H ₂ SO ₄ and then partially evaporated. Additional H ₂ SO ₄ was then added to pH 1-2, and the aqueous solution was extracted with ethyl acetate (2 x 50 mL). The organic phase was washed with saturated aqueous NaCl solution (50 ml), dried over Na ₂ SO ₄ and evaporated to dryness. The resulting oily residue was purified by column chromatography on silica gel (25 g) using as the mobile phase a mixture of CHCl ₃ / AcOH (95: 5, v / v). Finally, traces of AcOH were removed by lyophilization. N-Maleoyl-L-glycine (14) was then obtained as a colorless solid (0.61 g, 3.6 mmol, 54% yield). This compound (14) was then immediately used in the third step which is described below. ¹ H NMR analysis (300 MHz, acetone-d _6): δ = 4.29 (s, 2H, CH ₂ - Gly), 7.02 (s, 2H, CH-May).

¹³ C NMR analysis (75.5 MHz, ₆ -acetone): δ = 39.6, 136.3 (2C), 169.8 (2C), 171.7 (1C). d) Third step: Synthesis of 7V ^α - (tert-butyloxycarbonyl) - / V ^£ - (N-maleoyl-L-glycyl) -L-lysine-carboxamide (15).

0.29 g (1.73 mmol) of N-maleoyl-L-glycine (14) obtained above was dissolved in 5 ml of anhydrous CH ₃ CN. A solution of β-Boc-L-lysine-NH ₂ (11) (0.55 g, 2.24 mmol) in 5 ml of anhydrous DMF was added and the resulting solution was cooled to 4 ° C. 0.23 g (1.73 mmol) of hydroxybenzotriazole monohydrate (HOBt) and 0.39 g (1.90 mmol) of DCC were added. The resulting reaction mixture was stirred at room temperature overnight under an argon atmosphere. Was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / Me0H 8: 2, v / v) and the mixture was filtered through Celite ^® 545 to remove the white precipitate of 7V, N'-dicyclohexylurea (DCU) . The filtrate was then evaporated to dryness and dried under vacuum to remove DMF. The resulting residue was then taken up in 50 ml of ethyl acetate, washed with 30 ml of saturated NaHCO ₃ , 30 ml of a saturated aqueous solution of NaCl, then dried over Na ₂ SO ₄ and evaporated to dryness. The resulting residue was purified by column chromatography on silica gel (30 g) using a gradient of methanol (0-15%) in dichloromethane as mobile phase, to yield 0.35 g (0.88 mmol). ) of the expected compound (15) as a white solid with a yield of 51%.

IR analysis (KBr) v _max 835, 1063, 1166, 1256, 1432, 1525, 1555, 1665 (broad), 1710 (broad), 2926, 3218, 3342 (broad), 3416 cm ^-1 . ¹ H NMR analysis (300 MHz, CD ₃ CN): δ = 1, 29-1, 78 (m, 15H),

3.15 (q, J = 6.4 Hz, J = 13.0 Hz, 1H), 3.88-3.95 (m, 1H, α-CH), 4.05 (s, 2H), , 52 (bs, 1H, NH), 5.72 (bs, 1H, NH), 6.34 (bs, 1H, NH), 6.62 (bs, 1H, NH), 6.85 (s, 2H). ).

¹³ C NMR analysis (75.5 MHz, CD ₃ CN): δ = 24.1, 29.0 (3C), 30.1, 33.0, 40.1, 41.4, 55.7, 80, 2, 136.1 (2C), 157.1, 167.8, 172.1, 176.0. e) Fourth step: Synthesis of N ^£ - (7V-maleoyl-L-glycyl) -L-lvsine-carboxamide

(Precursor C-I)

0.33 g (0.83 mmol) of N - (tert-butyloxycarbonyl) -N, -OM-maleoyl-L-glycyl) -L-lysine-carboxamide (15) was dissolved in 7 ml of a TFA / H ₂ O (95: 5, v / v) then the solution was left at room temperature for 90 minutes.

The TFA was then evaporated and the product was precipitated with ether, washed with ether and lyophilized. The crude TFA salt of precursor (CI) was purified by reversed-phase flash chromatography on a grafted silica column Ci ₈ (20 g, elution with an aqueous solution of 0.1% TFA). Fractions containing the product were lyophilized to give 91 mg (0.22 mmol, 27% yield) of the expected precursor (Cl) as a white solid.

¹ H NMR analysis (300 MHz, D ₂ O): δ = 1.33 to 1.42 (m, 2H) ₅ 1,49-

1.59 (m, 2H), 1.83-1.90 (m, 2H), 3.21 (t, J = 6.8 Hz, 2H), 3.98 (t, J = 6.8 Hz). , IH, α-

CH), 4.22 (s, 2H), 6.92 (s, 2H). 4) Synthesis of the functional reagent (I-1) according to the invention and not fluorescent:

Assembly of precursors (A-I), (B-I) and (C-I)

The trifunctional compound (1-1) was synthesized in 5 steps from the precursors (AI), (BI) and (CI) prepared above in the previous steps. The synthesis protocol of the compound (1-1) is summarized (the three main steps only) in the following scheme 2:

Precursor (B-1-C-1)

FIGURE 2

The coupling between the precursors (BI) and (CI) was carried out after pre-activation of the precursor (BI) in the form of hydroxysuccinimide ester according to the method described for example in the article by Knorr, R. et al. Tetrahedron Lett., 1989, 30, 1927-1930. The activated ester was then reacted with the precursor (CI) to yield a coupling product (16) which was isolated by silica gel chromatography in 32% yield. Then, treatment with TFA made it possible to eliminate the Boc group and thus obtain the precursor (BlC-1). The final coupling between the precursor (AI) and the precursor (BlC-1) was carried out using the BOP as a coupling reagent. The expected product, in still protected form, was purified by chromatography on silica gel. After deprotection, by treatment with trifluoroacetic acid, the N-terminus was converted to activated carbamate by treatment with TV, 4'-disuccinimidyl carbonate (DSC) in anhydrous DMF in the presence of triethylamine. Total conversion of the pseudo-peptide to the compound of formula (I-1) was observed after 30 minutes. The compound of formula (1-1) expected was then purified by flash reverse phase chromatography on a grafted silica column Ci ₈ . a) First step: Synthesis of Λ ^ - (tert-butyloxycarbonylV7V ^g - (9- fluorenylmethyloxycarbonyl-aminooxy-acetyl) -L-lvsine-N ^g -maléoyl-L-lysine-carboxamide (16)

0.2 g (0.36 mmol) of the precursor (BI) obtained above in step 2) d) were dissolved in 2 ml of anhydrous acetonitrile. 67.4 μl (0.38 mmol) of DIEA and 1.16 mg (0.38 mmol) of O- (N-succinimidyl) -N, N, N ', N'-tetramethyl-uronium tetrafluoroborate were then added. (TSTU) and the reaction mixture was stirred at room temperature for 45 minutes under an argon atmosphere. The conversion of the precursor (BI) to its N-hydroxysuccinimide ester was monitored by TLC (CH ₂ Cl ₂ / MeOH, 85:15, v / v). A solution of 91 mg (0.23 mmol) of N - (N-maleoyl-L-glycyl) -L-lysine-carboxamide (precursor CI) obtained above in step 3 e) and 40 μl ( 0.23 mmol) of DIEA in 3 ml of anhydrous CH ₃ CN / DMF (2: 1, v / v) was then added dropwise and the resulting reaction mixture was stirred at room temperature for 1 hour. 1 hour under an argon atmosphere. It was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / MeOH), 85:15, v / v /) and then the mixture was evaporated in vacuo to remove the DMF. The oily residue thus obtained was purified by chromatography on a column of silica gel (25 g) using a gradient of MeOH (0-10%) in dichloromethane as the mobile phase. There was thus obtained 60 mg (0.075 mmol) of the compound (16) as a white foam with a yield of 32%.

¹ H NMR analysis (300 MHz, CDCl ₃ ): δ ≈ 1.25-1.91 (m, 21H), 3.07-3.29 (m, 4H), 4.08-4.42 (m, 7H, 2 x α-CH, CH-Fmoc, 2 x CH ₂ ), 4.48 (d, J = 6.8 Hz, 2H ₅ CH ₂ -Fm ₀ C), 5.61 (bd, J = 6 , 4 Hz, 1H, NH), 6.04 (bs, 1H, NH), 6.74 (s, 2H), 6.81 (bs, 1H, NH), 6.91 (bs, 1H, NH). , 7.27-7.42 (m, 4H), 7.57 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 9.2 ( bs, 1H, NH).

MS (MALDI-TOF, positive mode) m / z 706.6896 ((M-Boc) + H) ⁺ 808.7330 (M + H) ⁺ , 828.7064 (M + Na) ⁺ , calculated for C ₄₀ H _5I N ₇ On 805.89. b) Second step: Synthesis of N - (9-fluorenylmethyloxycarbonyl-aminooxy- acetyl) -L-lysine - / V ^ε -maléoyl-L-glycyl) -L-lysine-carboxamide (BLC-1) 60 mg (0.075. mmol) of the compound (16) obtained above in the previous step were dissolved in 2 ml of dichloromethane and the solution was cooled to 4 ° C. 306 μl (4.12 mmol) of TFA was added dropwise and the mixture The resulting reaction mixture was stirred at room temperature for 90 minutes. It was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / MeOH, 85:15, v / v) and then the mixture was evaporated to dryness. The oily residue thus obtained was dissolved in 5 ml of CH ₃ CN / H ₂ O (1: 1, v / v) and then lyophilized to give 58 mg (0.071 mmol) of the compound (Bcl-1) under the form of a white powder (94% yield).

MS (MALDI-TOF, positive mode) m / z 706.6488 (M + H) ⁺ , calcd for C ₃₅ H ₄₃ N ₇ O ₉ 707.77.

This compound was then used in the next step without purification. c) Third step: Synthesis of the trifunctional reagent (1-1) in totally protected form

58 mg (0.071 mmol) of the compound (BlC-1) obtained above in the preceding step and 32 mg (0.077 mmol) of the precursor (AI) obtained previously in step 1) f) were dissolved in 2 ml anhydrous dichloromethane. 25.9 μl (0.15 mmol) of DIEA and 34.4 mg (0.077 mmol) of BOP were then added and the reaction mixture was stirred at room temperature for 30 minutes under an argon atmosphere. Was checked that the reaction was complete by RP-HPLC (System B, that is to say using a Thermo Hypersil GOLD ^® Ci ₈ column of 5 microns and of dimensions 4.6 x 150 mm with an eluent of acetonitrile and 0.1% aqueous TFA solution (v / v, pH 2) by passing the mixture to 80% TFA for 5 minutes and then a linear gradient ranging from 20 to 90% CH ₃ CN minutes with a flow rate of 1.0 ml / min, a double UV detection was made at 210 and 254 nm), then an additional amount of precursor (AI) (14.4 mg, 0.035 mmol), DIEA (18, 5 μl, 0.10 mmol) and BOP (15.6 mg, 0.077 mmol) was added. After mixing at room temperature for 90 minutes, the reaction mixture was dissolved in 25 ml of dichloromethane. The resulting organic phase was washed successively with 25 ml of 10% citric acid and 25 ml of brine, then dried over Na ₂ SO ₄ and evaporated to dryness. The oily residue thus obtained was purified by chromatography on a column of silica gel (10 g) using as mobile phase a methanol gradient (0-15%) in dichloromethane to yield 24 mg (0.022 mmol) of the compound of formula (1-1) completely protected in the form of a yellow foam with a yield of 31%. d) Fourth step: Synthesis of the TFA salt of the compound of formula CI-I) completely protected 24 mg (0.022 mmol) of the compound of formula (1-1) in fully protected form and as obtained above at previous step were dissolved in 1 ml of dichloromethane. 81 μl (1.08 mmol) of TFA was then added dropwise and the resulting reaction mixture was stirred at room temperature for 1 hour. It was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / MeOH, 85:15, v / v) and then the mixture was evaporated to dryness. The crude TFA salt of the compound of formula (1-1) as fully protected was purified by flash reverse-phase chromatography on a grafted silica column Ci ₈ (5 g) using as mobile phase a gradient of CH ₃ CN (0-23%) in 0.1% aqueous TFA solution. The fractions containing the product were lyophilized to give 12 mg (0.01 mmol of the TFA salt of the trifunctional reagent of formula (1-1) completely protected as a white foam.) Step 5: Synthesis trifunctional reagent of formula (1-1)

12 mg (0.01 mmol) of the TFA salt of the trifunctional reagent of formula (1-1) in fully protected form as obtained above in the previous step were dissolved in 200 .mu.l of anhydrous DMF. 1.5 μl (0.011 mmol) of TEA and a solution of the DSC reagent (4.15 mg, 0.016 mmol) were then added to 100 μl of anhydrous DMF. The reaction mixture was then stirred at room temperature for 1 hour. The reaction was checked for completeness by HPLC-PI using System B as described above in step 4) b). The mixture was then taken up in 5 ml of an aqueous solution of 0.1% TFA and purified by reversed-phase flash chromatography on a grafted silica column Ci ₈ (5 g) using as mobile phase a gradient of CH ₃ CN (0-50%) in 0.1% aqueous TFA solution. Fractions containing the product were lyophilized to give 6.70 mg (0.0059 mmol) of the expected trifunctional reagent of formula (1-1) as a white powder. MS (MALDI-TOF, positive mode) m / z: 996.6590 ((M-NHS carbamate) + H) ⁺ , 1159.5921 (M + Na) ⁺ , 1175.5654 (M + K) ⁺ , calculated for C ₅₂ H ₆₈ N ₁₀ O ₁₉

1,137.18.

EXAMPLE 2 Synthesis of a Trifunctional Reagent

PSEUDOPEPIECQUE COMPLIES WITH THE INVENTION (1-2)

In this example is described the synthesis of a trifunctional pseudopeptide reagent (1-2) according to the present invention and having the following formula:

(1-2)

In this example, the synthesis of a trifunctional reagent different from the reagent of formula (1-1) previously synthesized above in Example 1) is illustrated by the nature of the amino acid to support the fixing of a fluorophore. (precursor C). This example shows that it is also possible to use a cysteine whose thiol function of the side chain has been protected in the form of disulphide with the aid of the S-ethyl group (SEt), in order to make it react on a fluorophore previously functionalized with a maleimide or iodoacetyl unit.

To do this, it was used a convergent synthesis strategy, as in Example 1) of separately preparing the precursors (A), (B) and (C) and to assemble them. As precursor (A), the precursor (AI) synthesized in Example 1) above was used. As a precursor (B), a precursor of formula (BI) was synthesized in a substantially different route and improved with respect to that used for the synthesis of the precursor (BI) of Example 1). Finally the synthesis of the precursor (C-2) was carried out from the commercial Boc-Cys (SEt) -OH. 1) Synthesis of the Precursor (BI): Λ -Frn-butyloxyearbonyl-1-fluorenylmethyloxycarboxyl-aminooxy-acetyl-L-lysine

The precursor (B-I) was synthesized from the protected N-Fmoc derivative of the aminooxyacetic acid (8) previously prepared as in Example 1), step 2) a).

0.230 g (0.73 mmol) of compound (8) was dissolved in 9 ml of CH ₃ CN / DMF (1: 1, v / v). 1,19 mg (0.88 mmol) of hydroxybenzotriazole monohydrate and 182 mg (0.88 mmol) of DCC were then added, and the reaction mixture was stirred at room temperature for 2 hours under an argon atmosphere. A solution of 180 mg (0.731 mol) of β-Boc-L-lysine

(10) as obtained above in step 2), b) of Example 1 in 2 ml of anhydrous DMF was then added and the reaction mixture was stirred at room temperature under an atmosphere of argon. The reaction was monitored by TLC

(CH ₂ Cl ₂ / MeOH, 80:20, v / v). After stirring for 2 hours, the mixture was evaporated to dryness. The residue thus obtained was taken up in 50 ml of ethyl acetate, washed with 50 ml of a 10% aqueous solution of citric acid, 25 ml of distilled water, dried over Na ₂ SO ₄ and finally purified. by chromatography on a column of silica gel (20 g) using a gradient of ethyl acetate (0-80%) in dichloromethane as the mobile phase.

296 mg (0.36 mmol) of compound (B-1) was obtained as a white foam in 50% yield.

¹ H NMR analysis (300 MHz, CD ₃ CN): δ = 1.37-1.83 (m, 15H),

3.18 (q, J = 6.4 Hz, J = 12.6 Hz, 2H), 4.00 (m, 1H, α-CH), 4.04 (s, 2H), 4.26 (t. , J =

6.8 Hz, 1H), 4.49 (d, J = 6.8 Hz, 2H), 5.61 (bd, J = 7.5 Hz, 1H, NH), 7.31-7.44 (b.p. m,

4H), 7.51 (bs, 1H, NH), 7.63 (d, J = 7.5 Hz, 2H), 7.83 (d, J = 7.5 Hz, 2H), 8.9 ( bs, 1H, NH).

¹³ C NMR analysis (75.5 MHz, CD ₃ CN): δ = 23.5; 28.5 (3C); 29.5; 31, 6; 38.9; 47.7; 54.2; 68.0; 76.3; 79.8; 120.9 (2C); 126.0 (2C); 128.1 (2C); 128.7 (2C), 142.1 (2C); 144.6 (2C); 156.7; 158.9; 169.3; 174.5.

MS (MALDI-TOF, positive mode) m / z: 564.5709 (M + Na) ⁺ , 580.5535 (M + K) ⁺ , calculated for C ₂₈ H ₃₅ N ₃ O ₈ 541.61. 2) Synthesis of the precursor (C-2)

The precursor (C-2) was synthesized in two steps from 7V- (tert-butyloxycarbonyl) -S- (S-ethyl) -cysteine (19) according to scheme 3 below:

SCHEME 3

According to this scheme and in a first step, the carboxylic acid function of the compound (19) is converted into carboxamide (20) by reaction of the ammonia with the mixed anhydride intermediately formed according to the method described for example by Hofmann, K. and al, J. Am. Chem. Soc., 1978, 100, 3585-3590. Next, the N-terminus of the compound (20) is deprotected by treatment with trifluoroacetic acid to yield the precursor (C-2). a) First step: Synthesis of N- (tert-butyloxycarbonyl) -5 '- (S-ethyl-3-carboxyamide (6) 500 mg (1.08 mmol) of N- (tert-butyloxycarbonyl) -S- (S-ethyl) Cysteine (19) was dissolved in 15 ml of ethyl acetate and the solution was cooled to -15 ^° C. in a bath of dry ice and ethylene glycol 0.119 ml (1.08 mmol) NMM and 0.140 mg (1.08 mmol) of isobutyl chloroformate were then added and the reaction mixture was stirred at -15 ° C for 10 minutes under an argon atmosphere and added to this anhydrous solution. a 15% aqueous solution of ammonia (0.38 ml, 3.24 mmol) and then the mixture The reaction mixture was stirred for 30 minutes at a temperature of 4 ° C. The mixture was then evaporated to dryness and the residue was taken up in 30 ml of ethyl acetate and then washed with 20 ml of water. The organic phases were dried over Na ₂ SO ₄ and then evaporated to dryness. Compound (20) was obtained in quantitative yield as a white solid.

¹ H NMR analysis (300 MHz, CDCl ₃ ): δ = 1, 24-1.29 (t, J = 6.8 Hz, 3H, CH ₃ (SEt)), 1.39 (s, 9H, tBu), 2 , 63-2.70 (q, J = 7.1 Hz, 2H, CH ₂ (SEt)), 2.99-3.01 (d, J = 6.0 Hz, 2H, CH ₂ β), 4 , 36-4.39 (m, 1H, CH α), 5.25-5.28 (d, J = 9.0 Hz, 1H, NH), 5.63 (bs, 1H, NH), 6, 31 (bs, 1H, NH). ¹³ C NMR analysis (75.5 MHz, CDCl ₃ ): δ = 13.8 (CH ₃ (SEt)), 27.8

(tBu), 32.0 (CH ₂ (SEt)), 39.9 (CH ₂ β), 53.0 (CH α), 80.1 (Cq tBu), 155.1 (Cq), 172.6 (Cq).

MS (MALDI-TOF, positive mode) m / z = 303.13 (M + Na) ⁺ , calculated for C ₀ H ₂₀ N ₂ O ₃ S ₂ -Na 303.41. (b) Second step: Synthesis of precursor (C-2): (S-ethyl-cysteine-carboxamide

The compound (20) obtained above in the preceding step was slowly dissolved in 14 ml of a mixture of TFA / H ₂ O (95: 5 v / v) with stirring at a temperature of between 0 ° C. and room temperature for 1 hour. The reaction was checked by TLC (CH ₂ Cl ₂ / MeOH 90:10 v / v) and the reaction mixture was evaporated to dryness. A minimum amount of osmosis water was then added and the resulting aqueous solution was lyophilized to yield the precursor (C-2) as a yellowish powder with a yield of 89%.

¹ H NMR analysis (300 MHz, D ₂ O): δ = 1.26-1.31 (t, J = 7.1 Hz, 3H, CH ₃ (SEt)), 1.39 (s, 9H), 2.72-2.80 (q, J = 7.1 Hz, 2H, CH ₂ (SEt)), 3.25-3.26 (d, J = 6.0 Hz, 2H, CH ₂ β), 4.30-4.35 (m, 1H, CH α).

¹³ C NMR analysis (75.5 MHz, D ₂ O): δ = 13.8 (CH ₃ (SEt)), 30.1 (CH ₂ (SEt)), 38.3 (CH ₂ β), 52, 2 (CH α), 170.8 (Cq).

MS (MALDI-TOF, positive mode) m / z = 180.00 (M + Na) ⁺ , calculated for C ₅ H ₁₂ N ₂ OS ₂ -Na 180.29. 3) Synthesis of the trifunctional reagent of formula (1-2)

The reagent (1-2) was synthesized in three steps from the precursors (A-I), (B-I) and (C-2) previously synthesized according to scheme 4 below:

Trifunctional reagent (1-2)

SCHEMA 4

The coupling between the precursors (BI) and (C-2) was carried out after pre-activation of the precursor (BI) in the form of hydroxybenzotriazole ester according to the method described for example in the article by Konig, W. and al, Chem. Ber., 1970, 103, 788-798. The activated ester was then reacted with the precursor (CI) to yield a coupling product (21). As the committed amines are in the form of TFA salt, it was necessary to add a base (DIEA). The intermediate (BlC-2) was purified by chromatography on silica gel with a yield of 61%. The N-terminus of the N-Boc trifunctional reagent (22) was deprotected with TFA and then converted to activated carbamate by DSC treatment in anhydrous DMF in the presence of TEA. (a) First step: Synthesis of CTC-tert-butyloxycarbonyl-N, N-fluorenylmethyloxycarbonylaminooxyacetyl-L-lysine-N-f-ethyl-L-cysteine carboxamide (21)

0.196 g (0.36 mmol) of the compound (BI) obtained above at the end of step 1) was dissolved in 3 ml of an anhydrous CH ₃ CN / DMF mixture (2: 1, v / v). 58.4 mg (0.43 mmol) of hydroxybenzotriazole monohydrate and 89.1 mg (0.43 mmol) of DCC were then added and the resultant reaction mixture was stirred at room temperature for 2 hours under an atmosphere of 'argon. Then, 1 ml of a DMF solution containing 106 mg (0.36 mmol) of the compound (C-2) was added and the reaction mixture was stirred at room temperature under argon atmosphere. After 2 hours, 31 μl (0.18 mmol) of DIEA were added. After another 2 hours, 31 μl (0.18 mmol) of DIEA was added again. Again after an additional 2 hours, 31 μl (0.18 mol) of DIEA and 44.9 mg (0.18 mmol) of DCC were further added. The vial containing the reaction medium was stored at a temperature of -20 ° C overnight. The next day, additional DIEA (31 μl, 0.18 mmol) and DCC (44.9 mg, 0.18 mmol) were added. After 2 hours, the reaction was judged complete and the reaction mixture was evaporated to dryness. The resulting residue was taken up with 25 ml of ethyl acetate, washed with 25 ml of a 10% aqueous citric acid solution, 25 ml of saturated NaHCO ₃ , 25 ml of distilled water, dried over Na ₂ SO ₄ and purified by column chromatography on silica gel (20 g) using a gradient of MeOH (0-10%) in dichloromethane as the mobile phase. 222 mg (0.31 mmol) of the compound (21) was obtained as a white foam in 88% yield). ¹ H NMR analysis (300 MHz, CDCl ₃ ): δ = 1.26-1.31 (t, 3H, J = 7.1

Hz, CH ₃ (SEt) Cys), 1.42 (s, 9H, tBu), 1.51-1.92 (m, 6H, CH ₂ β, δ, γ Lys), 2.65-2.72 (q, 2H, J = 7.5 Hz, CH ₂ (SEt) Cys), 3.08-3.10 (d, 2H, J = 6 Hz, CH ₂ β Cys), 3.26-3.33 (m, 2H, CH ₂ ε Lys), 4.04-4.06 (t, J = 6.0 Hz, 1H, CH α Lys), 4.20-4.25 (t, J = 7.2 Hz, 1H, CH Fmoc), 4.33 (s, 2H), 4.49-4.51 (d, 1H, J = 6.4 Hz, CH ₂ Fmoc), 4.71-4.78 (q, J = 6.4). Hz, 1H, CH? Cys), 7.26-7.78 (m, 8H, CH Fmoc).

¹³ C NMR analysis (75.5 MHz, CDCl ₃ ): δ = 14.2 (CH ₃ (SEt) Cys), 22.3 (CH ₂ γ, Lys), 28.6 (CH ₂ δ Lys), , 1 (CH ₂ (SEt) Cys), 32.4 (CH ₂ β Lys), 38.0 (CH ₂ β Cys), 39.3 (CH ₂ ε Lys), 46.7 (CH Fmoc), 52.3 (CH α Cys), 52.2 (CH α Lys), 68.0 (CH ₂ Fmoc) ), 76.1 (CH ₂ ), 80.8 (Cq tBu), 120.3 (Fmoc Cq), 125.1 (Fmoc Cq), 127.4 (Fmoc Cq), 128.1 (Fmoc Cq), 141.4 (Cq), 143.3 (Cq), 156.4 (Cq), 158.6 (Cq), 169.0 (Cq), 172.6 (Cq), 172.8 (Cq). HPLC-PI (system B): t _R ≈ 22.3 min, purity 84%

MS analysis (MALDI-TOF, positive mode) m / z = 726.77 (M + Na) ⁺ , 742.74 (M + K) ⁺ calcd for C ₃₃ H ₄₅ N ₅ O ₈ S ₂ -Na 726.88 . b) Second Step: Synthesis of Coupling Product (Bcl-2): 1- (Fluorylmethyloxycarbonylaminooxyacetyl) -L-lysine-S- (S-ethyl) -L-cysteine carboxamide

191 mg (0.27 mmol) of the compound (21) obtained above in the previous step were dissolved in 7 ml of dichloromethane and the solution was cooled to 4 ° C. 1.2 ml (16.6 mmol) of TFA was then added dropwise and the resulting reaction mixture was stirred at room temperature for 90 minutes. It was checked that the reaction was complete by TLC (CH ₂ Cl ₂ / MeOH, 80:20, v / v) and then the reaction mixture was evaporated to dryness. The oily residue thus obtained was dissolved in distilled water and lyophilized. 199 mg (0.27 mmol) of the coupling product (BlC-2) was obtained as a white powder in quantitative yield. This compound was then used in the next step without further purification.

¹ H NMR analysis (300 MHz, CD ₃ OD): δ = 1.26-1.28 (t, 3H, J = 3.8 Hz, CH ₃ (SEt) Cys), 1.40-1.56 ( m, 4H, CH ₂ β, γ Lys), 1.83-1.90 (m, 2H, CH ₂ δ Lys), 2.67-2.7 '4 (q, 2H, J = 7.2 Hz , CH ₂ (SEt) Cys), 2.93-2.96 (d, 2H, J = 9.4 Hz, CH ₂ β Cys), 3.20-3.28 (m, 2H, CH ₂ ε Lys ), 3.84-3.88 (t, J = 6.0 Hz, 1H, CH α Lys), 4.23 (s, 3H, CH Fmoc, CH ₂ ), 4.46-4.48 (d , 2H, J = 6.4 Hz, CH ₂ Fmoc), 4.63-4.68 (q, J = 4.9 Hz, 1H, CH α Cys), 7.26-7.78 (m, 8H). , CH Fmoc), 8.32 (bs, 1H, NH).

¹³ C NMR analysis (75.5 MHz, CD ₃ OD): δ = 14.7 (CH ₃ (SEt) Cys), 22.3 (CH ₂ γ, Lys), 30.7 (CH ₂ δ Lys), 32.2 (CH ₂ (SEt) Cys), 33.2 (CH ₂ β Lys), 39.5 (CH ₂ β Cys), 41.0 (CH ₂ ε Lys), 48.2 (CH Fmoc), 53.9 (CH α Cys), 54.2 (CH α Lys), 68.6 (CH ₂ Fmoc), 76.5 (CH ₂ ), 121.0 (CH Fmoc), 126.0 (CH Fmoc) , 128.2 (CH Fmoc), 129.0 (CH Fmoc), 142.7-174.3 (Cq). HPLC-PI (system B): / _R = 16.5 min, purity 74%. MS (MALDI-TOF, positive mode) m / z = 604.70 (M + H) ⁺ , 626.68 (M + Na) ⁺ , 642.66 (M + K) ⁺ , calcd for C ₂₈ H ₃₇ N ₅ O ₆ S ₂ 603.76. c) Third step: Synthesis of the Boc protected trifunctional reagent (22 ^" ) 78 mg (0.19 mmol) of the precursor (AI) as prepared previously in Example 1 were dissolved in 2 ml of anhydrous CH ₃ CN. mg (0.23 mmol) of hydroxybenzotriazole monohydrate and 47.3 mg (0.23 mmol) of DCC were then added and the resulting reaction mixture was stirred at room temperature for 2 hours under an argon atmosphere. 136.4 mg (0.19 mmol) of the compound (BlC-2) obtained above in the preceding step were then added, and then the reaction mixture was stirred at room temperature for 2 hours under an argon atmosphere. The flask containing the reaction mixture was stored at -20 ° C. overnight The next day 32 μl (0.19 mmol) of DIEA was added The reaction was monitored by HPLC-PI B) After 3 hours, an additional amount of DCC (12 mg, 58 μmol) and DIEA (16 μl, 95 μmol) was added After 90 minutes, 16 μl (95 μmol) of DIEA was added. 2 hours later, 16 μl (95 μmol) of DIEA was added once more. The reaction was stopped by adding 21 μl (360 μmol) of acetic acid and the flask containing the reaction medium was stored at a temperature of -20 ^° C. overnight. After dilution with 2 ml of CH ₃ CN / H ₂ O (2: 1, v: v), the expected compound (22) was purified by HPLC-PI (System C, i.e. using a Varian column Kromasil ^® Ci ₈ to 10 microns and of dimensions 21.2 x 250 mm eluting with a mixture of acetonitrile and water osmosis by passing 90% of RO water for 5 minutes, then a linear gradient ranging from 10 to 40% of CH ₃ CN in 15 minutes, then from 40% to 70% of CH ₃ CN, with a flow rate of 20.0 ml / min, a dual UV detection was made at 254 and 305 nm) . Two products were obtained and lyophilized to yield respectively 38 mg and 66 mg of two different trifunctional reagents (22-1) (22-2) in the form of a white powder with an overall yield of 61%. Compound (22-1) = expected compound (22): HPLC-PI (system B): t _R

= 20.8 min, purity 86%, 66 mg. ¹ H NMR analysis (300 MHz, CDCl _3): δ = 1.25-1.30 (t, 3H, J = 7.1 Hz, CH ₃ (SEt) Cys), 1, 43 (s, 9H, tBu ), 1.52-1.90 (m, 4H, CH ₂ γ, δ Lys), 2.32 (bs, 2H, CH ₂ β Lys), 2.64-2.69 (q, 2H, J = 7.2 Hz, CH ₂ (SEt) Cys), 2.97-3.16 (m, 2H, CH ₂ β Cys), 3.30 (s, 2H, CH ₂ unit (Al)), 3.45 -3.65 (m, HH, CH ₂ ε Lys + 4 x CH ₂ unit (Al)), 4.0 (s, 3H, CH ₂ unit (Al) + CH Fmoc), 4.21-4.25 (q, J = 6.8 Hz, 1H, CH α Lys), 4.34 (s, 2H, CH ₂ ), 4.44-4.50 (d, 2H, J = 10.0 Hz, CH ₂ Fmoc), 4.70-4.72 (q, 1H, J = 2.3 Hz CH α Cys), 5.23 (bs, 1H, NH), 6.08 (bs, 1H, NH), 6, 9 (bs, 1H, NH), 7.26-7.77 (m, 8H, CH Fmoc).

¹³ C NMR analysis (75.5 MHz, CDCl ₃ ): δ = 14.4 (CH ₃ (SEt) Cys), 22.6 (CH ₂ γ, Lys), 27.6 (CH ₂ unit (Al) ), 28.5 (tBu), 28.7 (CH ₂ δ Lys), 31.7 (CH ₂ (SEt) Cys), 32.5 (CH ₂ β Lys), 38.6 (CH ₂ unit (Al) )), 38.8 (CH ₂ unit (AI)), 39.6 (CH ₂ ε Lys), 40.4 (CH ₂ β Cys), 47.0 (CH ₂ unit (Al)), 53.6 (CH α Lys), 68.0 (CH ₂ Fmoc), 70.0-71.2 (5 x CH ₂ unit (Al)), 77.5 (CH ₂ unit (Al)), 77.8 (CH 3) Fmoc), 79.5 (Cq tBu), 120.2 (CH Fmoc), 125.1 (CH Fmoc), 127.3 (CH Fmoc), 128.1 (CH Fmoc), 141, 4-173.0 (Cq).

MS (MALDI-TOF, positive mode) m / z = 1016.66 (M + Na) ^+, Calcd for C ₄₅ H ₆₇ N ₇ Oi ₄ S ₂ .NA 1017.20.

Compound (22-2) (not expected): HPLC-PI (System B): / _R = 21.6 min, purity 79%, 38 mg. MS (MALDI-TOF, positive mode) m / z = 871.47 (M + Na) ⁺ , calculated for C ₃₉ H ₅₆ N ₆ OnS ₂ -Na 871.04. d) Fourth step: Deprotection of the compound (22-1) and synthesis of the trifunctional reagent of formula (1-2)

62 mg (0.062 mmol) of the compound (22-1) obtained above in the previous step were dissolved in 3 ml of dichloromethane. The solution was cooled to 4 ° C and 368 μl (4.96 mmol) of TFA was then added dropwise. The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction was checked for completeness by HPLC-PI (System B) and then the mixture was evaporated to dryness. A minimal amount of osmosis water was then added and the resulting solution was lyophilized to yield 83 mg (0.082 mmol) of the compound (22-1) (TFA salt) in deprotected form (white powder). This compound was then used in the following reactions without further purification.

HPLC-PI (system B): t _R = 16.5 min, purity 88%. MS (MALDI-TOF, positive mode) m / z = 894.64 (M + H) ^+, calcd for C ₄₀ H ₅₉ N ₇ Oi ₂ S ₂ 894.08.

36 mg (36.7 μmol) of the TFA salt of the deprotected compound (22-1) obtained above were dissolved in 500 μl of anhydrous DMF, 5 μl (36.7 μmol) of TEA and a solution of DSC (24). mg, 91, 75 μmol) in 250 μl of anhydrous DMF was then added dropwise and the reaction mixture was stirred at room temperature for 90 minutes.The reaction was checked to be complete by HPLC-PI ( B) The mixture was then taken up with about 4 ml of 0.1% aqueous TFA solution and purified by HPLC-PI (System D, ie using a Thermo Hypersil GOLD ^® column. C] ₈ of 5 microns and dimensions 10 x 250 mm with as eluent a mixture of acetonitrile / 0.1% aqueous solution of TFA (v / v, pH 2) by passing the mixture at 90% TFA to 0, 1% for 5 minutes, then a linear gradient from 10 to 40% of CH ₃ CN in 15 minutes, then from 40% to 70% of CH ₃ CN at a flow rate of 5.0 ml / min; a double d UV reaction was made at 254 and 305 nm.) The fractions containing the product were lyophilized to yield 25 mg (24.2 μmol) of the trifunctional reagent of formula (1-2) as a white amorphous powder. , with a yield of 74%.

HPLC-PI (system B): t _R = 18.5 min, purity 97%. ¹ H NMR analysis (300 MHz, CDCl ₃ ): δ = 1.24-1.27 (t, 3H, J = 7.1 Hz, CH ₃ (SEt) Cys), 1.32-1.90 (m , 6H, CH ₂ β, γ, δ Lys), 2.63-2.69 (q, 2H, J = 7.1 Hz, CH ₂ (SEt) Cys), 2.81 (s, 4H, 2 x CH ₂ succinimidyl), 2.24-3.28 (m, 2H, CH ₂ β Cys), 3.24-3.69 (m, HH, 5 x CH ₂ unit (Al) + CH ₂ ε Lys), 4, 22-4.27 (t, 1H, CH Fmoc), 4.32 (s, 2H, CH ₂ ), 4.51-4.53 (d, 2H, J = 6.4 Hz, CH ₂ Fmoc), 4.58-4.66 (q, J = 7.5 Hz, 1H, CH α Lys), 4.70-4.76 (q, 1H, J = 6.0 Hz CH α Cys), 6.17 (bs, 1H, NH), 6.83 (s, 1H, NH), 7.27-7.83 (m, 8H, CH Fmoc), 9.2 (bs, 1H, NH).

MS (MALDI-TOF, positive mode) m / z 1035.17 (M + H) ⁺ , 1057.71 (M + Na) ⁺ , 1073.68 (M + K) ⁺ , calculated for C ₄₅ H ₆₂ N ₈ Oi ₆ S ₂ 1035.69 (M + H) ⁺ The trifunctional reagent of formula (1-2) may then be functionalized with any fluorescent ligand to yield the corresponding fluorescent trifunctional reagent.

Claims

A trifunctional pseudopeptide reagent, characterized in that it comprises at least the following three reactive structural units A, B and C: (a) a unit A consisting of a hydrophilic chain of pseudopoly (ethylene glycol) interrupted by at least one amide function and having two ends E1 and E2, said E1 end being free and having a terminal unit selected from amino group, activated carbamates and activated esters, and said E2 end having a terminal carbon atom carrying a carbonyl function, said carbon atom being engaged in an amide function (-C (O) -NH-) formed with the nitrogen atom of an α-amine function carried by the unit B;

(b) a unit B consisting of an amino acid chosen from α-amino acids of the L, D series and their racemic mixtures, said amino acid having on its side chain at least one oxyamine function protected by a protecting group or at least one minus a masked aldehyde function; and

(c) a unit C consisting of an amino acid selected from α-amino acids of the L, D series and their racemic mixtures, said amino acid having on its side chain at least one thiol, maleimide, iodoacetyl, azide unit, true alkyne, phosphane or cyclooctyne; said units B and C being linked to one another via an amide function formed between the carbon atom carrying the carbonyl function of the α-amino acid constituting the unit B and the nitrogen atom of the α-amine function of the α-amino acid constituting the unit C.

2. Trifunctional reagent according to claim 1, characterized in that the pseudo-poly (ethylene glycol) chain of unit A is chosen from the following chains of formula (A-I):

(HAVE) in which :

R 1 represents a primary amine, an activated carbamate unit or an activated ester unit,

- m and n, identical or different, are integers between 2 and 10 inclusive,

p is an integer from 1 to 10 inclusive,

the arrow represents the covalent bond connecting the amide function of the E2 end of the pseudo-poly (ethylene glycol) chain to the B or C unit.

3. Trifunctional reagent according to claim 2, characterized in that the pseudo-poly (ethylene glycol) chain is chosen from pseudopoly (ethylene glycol) chains of formula (AI) in which m = n = 2 and p = 1.

4. trifunctional reagent according to any one of the preceding claims, characterized in that the activated carbamate groups are chosen from N-hydroxysuccinimidyl carbamate, N-hydroxysuccinimidyl sulfo carbamate, N-hydroxyphthalimidyl carbamate, N-hydroxypiperidinyl, p-nitrophenyl carbamate and pentafluorophenyl carbamate.

5. trifunctional reagent according to claim 4, characterized in that the activated carbamate group is N-hydroxysuccinimidyl carbamate.

6. trifunctional reagent according to any one of claims 1 to 3, characterized in that the activated ester groups are chosen from the ester of TV-hydroxysuccinimidyl, the sulfo-N-hydroxysuccinimidyl ester, the cyanomethyl ester, the N-hydroxyphthalimidyl ester, the N-hydroxypiperidinyl ester, the p-nitrophenyl ester, the pentafluorophenyl ester, the benzotriazole esters, the hydroxybenzotriazole esters and the hydroxyazabenzotriazole esters.

7. trifunctional reagent according to claim 6, characterized in that the activated ester group is the 7V-hydroxysuccinimidyl ester.

8. trifunctional reagent according to any one of the preceding claims, characterized in that the α-amino acids used for the unit B are chosen from lysine, homolysine, ornithine, 2,4- diaminobutanoic acid and 2,3-diaminopropanoic acid.

9. Trifunctional reagent according to claim 8, characterized in that the α-amino acid of unit B is lysine.

10. trifunctional reagent according to any one of the preceding claims, characterized in that the α-amino acids usable for the unit C, are chosen from lysine, cysteine, homolysine, ornithine, acid 2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid, aminomercapto-acetic acid, homocysteine, 5-mercapto-norvaline, 6-mercapto-norleucine, 2-amino-7-mercapto acid -heptanoic acid, and 2-amino-8-mercaptooctanoic acid.

11. Trifunctional reagent according to claim 10, characterized in that the α-amino acid of unit C is lysine or cysteine.

12. Trifunctional reagent according to any one of the preceding claims, characterized in that the protective groups of the oxyamine function of the side chain of the α-amino acid constituting the unit B, are chosen from 9-fluorenylmethoxycarbonyl groups. benzyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, pyridyldithioethoxycarbonyl, 2- (2-nitrophenyl) propyloxycarbonyl, azomethyloxycarbonyl, 2- (trimethylsilyl) ethanesulfonyl and phthalimide.

13. Trifunctional reagent according to any one of the preceding claims, characterized in that it is chosen from the following compounds of formula (I):

(I) in which:

R 1, m, n and p have the same meanings as defined in claim 2 or 3 for the unit of formula (A-I),

X ₁ and X ₂ , which are identical or different and, together with the carbon atom to which they are bonded, represent the hydrocarbon chain of an α-amine acid, Proc is a protecting group selected from 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, pyridyldithioethoxycarbonyl, 2- (2-nitrophenyl) propyloxycarbonyl, 1-azomethyloxycarbonyl, 2- (trimethylsilyl) ethanesulfonyl and phthalimide;

- Func is a thiol, maleimide, iodoacetyl, azide, true alkyne, phosphane or cyclooctyne unit.

14. trifunctional reagent according to claim 13, characterized in that Xi is an n-butyl chain.

15. trifunctional reagent according to claim 13 or 14, characterized in that X ₂ is an ethyl or n-butyl chain.

16. Reagent according to any one of claims 13 to 15, characterized in that the compounds of formula (I) are chosen from the following compounds of formula (I-1) and (1-6):

⁽ M ⁾

(1-2)

(1-5)

d-6).

17. Use of at least one trifunctional reagent as defined in any one of the preceding claims, for the preparation of a bioconjugate.

18. Bioconjugate, characterized in that it consists of a trifunctional reagent as defined in any one of claims 1 to 16, wherein the primary amine unit, activated carbamate or activated ester present at the free end of the pseudo-poly (ethylene glycol) chain constituting unit A and / or the oxyamine or aldehyde function carried by unit B after its deprotection or unmasking, is (are) functionalized by a biological molecule of interest .

19. bioconjugate according to claim 18, characterized in that the biological molecule of interest is chosen from antibodies, nucleic acid molecules and their analogues, polysaccharides, proteins, peptides, radionuclides, toxins, enzyme inhibitors and haptens.

20. Bioconjugate according to claim 18 or 19, characterized in that it is chosen from: i) a bioconjugate consisting of a trifunctional reagent in which only the primary amine function or only the activated carbamate unit or activated ester present at the free end of the pseudo-poly (ethylene glycol) chain constituting the unit A is functionalized by a biological molecule, ii) a bioconjugate consisting of a trifunctional reagent in which only the unmasked deprotected oxyamine or aldehyde function of the unit B is functionalized by a biological molecule, and iii) a bioconjugate comprising two biological molecules, consisting of a trifunctional reagent in which the primary amine function or activated carbamate or activated ester unit present at the free end of the pseudo-poly (ethylene glycol) chain. constituting unit A and the deprotected oxyamine or aldehyde function unmasked from unit B are each a functionalized by a biological molecule, said molecules being identical or different from each other.

21. Use of at least one trifunctional reagent as defined in any one of claims 1 to 16 for the preparation of a luminescent reagent.

22. Luminescent trifunctional reagent, characterized in that it consists of a trifunctional reagent as defined in any one of claims 1 to 16, and that it comprises a single luminescent group (L) attached to the unit B via the deprotected oxyamine function or the function aldehyde unmasked or on the unit C via the thiol function or the units maleimide, iodoacetyl, true alkyne, phosphane, cyclooctyne or even azide.

23. trifunctional reagent luminescent, characterized in that it consists of a trifunctional reagent as defined in any one of claims 1 to 16, and it comprises two luminescent groups, one being fixed on the unit B via the deprotected oxyamine function or the undefined aldehyde function and the other being attached to the unit C via the thiol function or the maleimide, iodoacetyl, true alkyne, phosphane or cyclooctyne units or else still azide.

24. trifunctional luminescent reagent according to claim 22 or 23, characterized in that the luminescent group or groups are chosen from polymethine chain fluorophores; fluorescent cyanines; fluorescein and its derivatives; rhodamine and its derivatives; water-soluble derivatives of rhodamine in the form of a TV-hydroxysuccinimide ester; rhodols and their derivatives; coumarin derivatives; fluorescent dyes with reactive amines; dipyrromethene boron difluorides; fluorophores derived from pyrene; disazo derivatives; danzyl derivatives; eosin; erythrosine and sulforhodamine derivatives.

25. Use of at least one trifunctional reagent as defined in any one of claims 1 to 16, for the preparation of a transfer cassette energy.

26. Energy transfer cassette, characterized in that it consists of a trifunctional reagent as defined in any one of claims 1 to 16, said reagent comprising a luminescent group (L) and an acceptor compound (Q) luminescence of the luminescent group, L and Q being respectively and indifferently attached to said trifunctional reagent via the deprotected oxyamine or aldehyde function unmasked from unit B and the thiol function or the units maleimide, iodoacetyl, alkyne true, phosphane , cyclooctyne or even azide of unit C.

27. Cassette according to claim 26, characterized in that the luminescent group (L) is selected from polymethine chain fluorophores; fluorescent cyanines; fluorescein and its derivatives; rhodamine and its derivatives; water-soluble derivatives of rhodamine in the form of N-hydroxysuccinimide ester; rhodols and their derivatives; coumarin derivatives; fluorescent dyes with reactive amines; dipyrromethene boron difluorides; fluorophores derived from pyrene; disazo derivatives; danzyl derivatives; eosin; erythrosine and sulforhodamine derivatives.

28. Cassette according to claim 25 or 26, characterized in that the acceptor compound (Q) is chosen from the luminescent groups (L) listed in claim 27, the non-fluorescent molecules of the family of azo dyes, the particles gold, disazo dyes, malachite green and acceptor compounds of the cyanine family.

29. Cassette according to any one of claims 25 to 28, characterized in that the luminescent group (L) and the acceptor compound (Q) are chosen from the following pairs (L / Q): Cy3 / Cy5, Cy5 / Cy7, Cy5 / Alexa Fluor ^® 750, Cy3 / Cy5Q, Cy3 / QSY ^® 7, Cy3 / QSY ^® 9, Cy5 / Cy7Q, Cy5 / QSY ^® 21, Cy5 / Cy5, Cy5.5 / Cy5.5, Cy7 / Cy7 , R6) / Cy5, R6G / Alexa Fluor ^® 647, R6G / QSY ^® 21, Alexa Fluor ^® 532 / Cy5, Alexa Fluor ^® 532 / Alexa Fluor ^® 647, Alexa Fluor ^® 532 / QSY ^® 21, Alexa Fluor ^® 555 / Cy5, Alexa Fluor ^® 555 / Alexa Fluor ^® 647, Alexa Fluor ^® 555 / QSY ^® 21.

30. Use of a trifunctional reagent as defined in any one of claims 1 to 16, for the preparation of a mixed bioconjugate comprising at least one biological molecule and at least one luminescent group.

31. Mixed bioconjugate, characterized in that it consists of a trifunctional reagent as defined in any one of claims 1 to 16 on which are fixed at least one biological molecule and at least one luminescent group.

32. Mixed bioconjugate according to claim 31, characterized in that it further comprises an acceptor compound (Q) of the luminescence of the luminescent group.

33. Mixed bioconjugate according to claim 31 or 32, characterized in that it is chosen from the trifunctional reactants on which are fixed: i) one or two biological molecules of interest and a luminescent group; or ii) a biological molecule of interest, and two luminescent groups; iii) a biological molecule of interest, a luminescent group, and an acceptor compound (Q) of the luminescence of the luminescent group; said biological molecules, said luminescent moieties and said acceptor compound (Q) being attached to the functional reagent at the terminal ends of the A, B and C units.

34. Use of a trifunctional reagent or a bioconjugate, whether or not modified with a luminescent group at unit C, as defined in any one of claims 1 to 16, 18 to 20, 22, 24 , 31 and 33, and wherein the oxyamine or respectively the aldehyde function of the unit B is free, for the functionalization of solid supports comprising at least one surface having one or more carbonyl groups or respectively one or more oxyamine functions.

35. Solid support, characterized in that it comprises at least one functionalized surface, covalently, with one or more trifunctional reagents, and / or with one or more bioconjugates as defined in any one of claims 1 to 16, 18 to 20, 22, 24, 31 and 33, said trifunctional and bioconjugated reagents being optionally modified by a luminescent group at the level of the unit C.

36. The solid support according to claim 35, characterized in that it comprises at least one surface functionalized with at least one bioconjugate and that it then constitutes a biochip or a biosensor.

37. Use of a support as defined in claim 35 or 36, for the detection of molecules of interest.

38. Use of at least one trifunctional reagent as defined in any one of claims 1 to 16 for the preparation of a probe for functional proteomics.

39. Probe for functional proteomics, characterized in that it consists of a trifunctional reagent as defined in any one of Claims 1 to 16, said reagent comprising:

a group allowing detection (or visualization) and / or purification of a target protein (reporter tag or "reporter tag"),

a pattern recognized by said target protein (recognition unit or "recognition unit"), and

a reactive group making it possible to establish a covalent link with the active site of the targeted protein (reactive group).

40. Probe according to claim 39, characterized in that the target protein is an enzyme.