EP3191501A1

EP3191501A1 - Fluorescent carbohydrate conjugates, the preparation method and uses thereof

Info

Publication number: EP3191501A1
Application number: EP15766098.6A
Authority: EP
Inventors: Sylvie FOURNEL-GIGLEUX; Sandrine GULBERTI; Jean-Claude Jacquinet; Chrystel LOPIN-BON; Mohamed OUZZINE; Nick RAMALANJAONA; Isabelle BERTIN JUNG
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Lorraine
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Lorraine
Priority date: 2014-09-11
Filing date: 2015-09-11
Publication date: 2017-07-19
Also published as: FR3025796A1; WO2016038205A1

Abstract

The invention relates to fluorescent conjugates of formula (I) (I) where CG is a carbohydrate compound selected from the monosaccharide residues, the oligosaccharides and the polyoligosaccharides comprising at least one monosaccharide residue in cyclic form, substituted or not, L is a group grafted onto a marker position of said monosaccharide and selected from -O-NH-, -O-(CH₂)_nNH-, -O-(CH₂)_nS-, where n is an integer equal to 1 or 2, a group derived from a glycosylamine, a group derivesd from a thioglycoside, the nitrogen or sulphur atom being linked to the carboxylic carbon of the anthraniloyl group, R is H, CH₃, R' occupies the 4 free positions of the benzene ring of the anthraniloyl group and is independently selected from H, CH₃, -CH-(CH₃)₂, F, Cl and Br. Such conjugates can be used, in particular, to study protein-sugar interactions and to search for effectors on a high flow sieve.

Description

FLUORESCENT GLUCIDIC CONJUGATES, PROCESS FOR THEIR PREPARATION AND USES THEREOF

The present invention relates to fluorescent carbohydrate conjugates, processes for their preparation, and their use in particular for high throughput microplate screening of carbohydrate-protein interactions by fluorescence and for studying sugar-receptor protein interactions.

Compared to other biomolecules, glycotherapies constitute a field of investigation and research that deserves to be exploited much more widely. There is therefore an undeniable interest in the development of carbohydrate molecules for therapeutic purposes which are currently underused and have important fields of application.

Specifically targeted are especially carbohydrate compounds which find a wide field of application in biomedical research (substrates, enzyme inhibitors, receptor ligands) in the fields of diabetology, regenerative medicine (in particular rheumatic pathologies of degenerative origin), infectiology (antibiotic, antibacterial, antifungal), diabetes or that of cosmetology. Carbohydrate molecules have a wide variety of biological and pharmacological properties. For example, heparin (sulfated glycosaminoglycan) with anticoagulant property represents a global market estimated at nearly 4 billion euros in 2010. However, the therapeutic exploitation of carbohydrate structural molecules is currently limited due to the lack of simple and rapid tests of evaluation of their biological activity.

The study of glycosidic substrate enzymes has led to the development of fluorescence-based techniques that can be adapted to the development of high-throughput microplate screening due to sensitivity to detection. Thus, for the study of certain glycosidases, many chromogenic substrates are today developed and marketed by biotechnology companies (Invitrogen®). In the kinetic study of glycosyltransferases, a kit for fluorescence detection of nucleoside biphosphates (co-produced during the enzymatic reaction) is marketed by the company R & DSystem®. In another context, two fluorescent glucose analogues, 2-NBDG and 6-NBDG, recognized by some glucose-specific protein transporters, have been widely exploited in the study of glucose uptake by cells, but have seen their misuse as part of the development of a screening protocol (Jung D.-W.E et al., (201) Mol BioSyst 7, 346-358). Together, these examples demonstrate the difficulty of developing a screening method that can be applied to a broader panel of glycosidic ligand / receptor pairs. To overcome this lack, users must systematically set up screening systems specifically adapted to the proteins / enzymes they are working on.

The application WO 94/2842 describes compounds obtained by reaction of modified saccharides of the form oligo-Ch-NHR 'with isatoic anhydride where the nitrogen may carry a methyl group. These compounds are used in electrophoretic separation processes. However, the process described in this document consists in deriving the reducing end of the sugar to be labeled so as to obtain an intermediate of hydrazone then hydrazine type so that it can subsequently react with isatoic acid anhydride. (or one of its derivatives). This strategy involves the opening of the sugar-labeling cycle from the hemi-acetal form to the aldehyde form in which it reacts, making it unsuitable for experiments requiring its interaction with a possible target protein. Adapted for the fluorescence monitoring of the separation of oligosaccharides (on electrophoresis gel for example), this technique remains too invasive vis-à-vis the sugar structure marked by these methods and is therefore not suitable for studies involving their interaction with target proteins.

In the article proposed by Niranjan et al. "Analysis of steady-state Fôrster resonance energy transfer data by avoiding pitfalls: interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides". Anal Biochem. Nov 15

2013; 442 (2): 213-222, the fluorescence of an adenosine-5'-triphosphate (ATP) molecule labeled with the N-methyl-anthraniloyl group. (MANT) is used to monitor its interaction with a protein named JAK2 belonging to the Janus kinase family using the FRET technique. The authors show that it is possible to accurately estimate the affinity of this conjugate for JAK2. However, it should be noted that the conjugate used for these experiments consists in fact in a mixture of ATP labeled with MANT at the 2 'and 3' position of the ribose. However, the article by Suryanarayana et al. Distinct interactions of 2'- and 3'-O- (N-methyl) anthraniloyl-isomers of ATP and GTP with adenylyl cyclase toxin of Bacillus anthracis, edema factor Biochem Pharmacol Aug. 1, 2009; 78 (3): 224 -230 "shows that the position of the ribose on which the MANT is grafted influences the affinity of the target protein (here the oedematogenic factor of the anthrax toxin) with respect to ΓΑΤΡ or GTP labeled by the MANT, but also the spectroscopic behavior of the fluorescent conjugate. Thus, the affinity constant (Kd) observed by Niranjan et al., Probably reflects an average affinity of JAK2 with respect to the 2 'and 3' labeled ATP mixture of ribose by MANT. Indeed, it is not possible to distinguish which of the 2 'or 3' labeled conjugates is the main contributor in the monitoring of this interaction of ATP-MANT with JAK2, insofar as: i) the relative percentage of ATP-MANT labeled 2 'of ribose with respect to that labeled at 3' is not specified ii) the affinity of JAK2 with respect to ΓΑΤΡ-ΜΑΝΤ labeled at 2 'of ribose and that labeled at 3' is not known iii) the spectroscopic behavior of ΜΑΝΤ-ΜΑΝΤ labeled 2 'ribose and that labeled 3' in the presence of the protein JAK2 is also not known. These problems arise from the reactivity of the -OH groups at the 2 'and 3' position of the ribose with respect to the MAN.

This shows that there are no simplified methods for studying the properties of proteins (enzymes, receptors, lectins, etc.) interacting with sugars.

The object of the present invention is to propose a simple, fast and inexpensive methodology for fluorescence-based screening applicable to saccharide molecules of biomedical interest. For this purpose, and in accordance with the present invention, it is proposed new fluorescent conjugates of formula (I)

or

CG is a carbohydrate compound chosen from the residues of oste, the oligosides (comprising from 2 to 10 atoms) and the polysaccharides (comprising more than 10 monosaccharides) comprising at least one residue of osé in cyclic form, substituted or unsubstituted,

L is a single group grafted on a single labeling position of said ose and is selected from -O-NH-, -O- (CH2) _n NH-, -O- (CH2) _n S-, where n is an integer equal at 1 or 2, a group derived from a glycosylamine, a group derived from a thioglycoside, the nitrogen or sulfur atom being bonded to the carboxylic carbon of the anthraniloyl group,

R is H, CH ₃ ,

R 'occupies the 4 free positions of the benzene ring of the anthraniloyl group and is independently selected from H, CH3, -CH- (CH3) 2, F, Cl, Br.

CG is therefore a carbohydrate compound comprising at least one osse residue derived from an ose in hemiacetal form and which has retained its cyclic form, preserving its closed base structure.

CG does not include nitrogen base or phosphate chain. Thus, CG is not derived from a nucleotide.

Preferably, CG is chosen from an oste residue, a diholoside, a triholoside comprising at least one cyclic osse residue. More preferentially, CG is a residue of osé or a diholoside comprising at least one residue of osé in cyclic form. Thus, CG is chosen for example from a residue of xylose, maltose, lactose comprising at least one residue of osé in cyclic form.

The free positions of the diene residue may be independently unsubstituted bearing H or OH or substituted with a group selected from -O-PO ₃ Na ₂ , -O-SO ₃ Na, -O-CO-CH 3, -N-CO-CH 3 , -NH ₂ , -CH ₃ , -CH ₂ -CH ₃ , -F, -

Cl, -Br, -SH.

Preferably, L is chosen from -O-NH-, -O- (CH2) 2NH-, -O- (CH2) 2S-, a group derived from a glycosylamine, a group derived from a thioglycoside, the atom nitrogen or sulfur being bonded to the carboxylic carbon of the anthraniloyl group.

In a particularly advantageous manner, L is -O- (CH 2) 2 NH- or -O- (CH ₂ ) ₂ S-.

L can be grafted onto any free position of the oste residue according to the grafting techniques known to those skilled in the art, but preferably L is grafted in the 1-anomeric position of the diene residue.

For the anthraniloyl group, R is preferably H and the 4 positions R 'are occupied by an H atom or R is preferably CH3 and the 4 R' positions are occupied by an H atom.

A preferred conjugate of the invention is that for which CG is an oste residue or a diholoside comprising at least one ring-shaped oste residue, L is -O-NH- or -O-CH2-CH2-NH- grafted in the 1-anomeric position of the diene residue and R 'is H and R is CH3.

Thus, a compound (A) of the invention, derived from xylose and an ethanolamine L group precursor, is

(AT) Another compound (Α ') of the invention, derived from xylose and a precursor of the hydroxylamine L group, is

Another compound (B) of the invention, derived from maltose and an ethanolamine L group precursor, is

Another compound (C) of the invention, derived from lactose and an ethanolamine L group precursor, is

The present invention also relates to a process for preparing a fluorescent conjugate of formula (I) as defined above, said process comprising the steps of: preparing a CG-LH activated carbohydrate compound from a complete CG carbohydrate compound and an L group precursor,

labeling the activated carbohydrate compound CG-LH by reaction with a compound of formula (II)

(II)

to obtain the fluorescent conjugate of formula (I),

CG, L, R, R 'being as defined above.

More specifically, the step of preparing the CG-LH activated carbohydrate compound comprises the steps of:

activating the labeling position of a complete carbohydrate CG compound, said labeling position preferably being the anomeric position 1 of the complete monosaccharide in cyclic or hemiacetal form,

chemical grafting on the activated marking position of the group L,

- optionally substitution of one or more free positions of the ose,

optionally lyophilization of the activated carbohydrate compound CG-LH obtained.

Advantageously, the reducing end of the complete ose is free.

CG will be "activated" most often in position 1, at the reducing end of the molecule. "Activate" means to increase the reactivity of the position 1 of the carbohydrate compound with N-methyl isatoic acid anhydride or isatoic acid anhydride. This activation step requires the chemical grafting of L on the carbohydrate compound, preferably at position 1. This step of preparation of the activated carbohydrate compound CG-LH is summarized in Scheme I below applied to a residue xylose activated in position 1, the precursor of L being ethanolamine:

D-xylose

R2 = OH, H, OPO ₃ Na ₂ , etc. R3, R4 = OH, H, etc.

Diagram 1

The grafting of L starts with a series of steps for the protection of oxygen in positions 2, 3 and 4 of xylose with acetyl groups and then for activating the oxygen atom in position 1 of the xylose with trichloroacetonitrile (a). . The grafting of L is then carried out by reacting the compound formed with the protected ethanolamine on the primary amine with a carbobenzoxy (Z) (b) group.

The nature of the group L (length and reactive group) can be adapted according to the needs of the user.

Preferably, the precursor of the L group is chosen from the compounds HO-NH ₂ , HO- (CH ₂ ) n _-NH ₂ n HO- (CH ₂ ) n -SH, a glycosylamine, a thioglycoside.

The choice of a short (HO-NH ₂ ) or long (HO- (CH ₂ ) ₂ -NH ₂ , HO- (CH ₂ ) ₂ -SH) precursor will depend on the fluorescence screening technology employed by the user. (FRET, fluorescence intensity or fluorescence anisotropy).

Depending on the needs of the user, the carbohydrate portion of the conjugate may be modified in step (c). Carbohydrate molecules "activated" and substituted by different groups at certain positions will thus be able to to be proposed. For example, a molecule of D-xylose "activated" in position 1 by an ethanolamine L precursor can be substituted in the 2-position by a phosphate group (Compound D) or in the 4-position by a fluorine (Compound E).

The "activation" of xylose ends with the removal of the protective group (d). These steps will be done in the laboratory. The labeling on other positions of the carbohydrate compound may be considered "custom" depending on the user's requests, so as not to interfere with the biological activity studied.

The activated carbohydrate compound CG-LH is then used for the labeling reaction with the compound of formula (II).

The compound of formula (II) is commercially available.

However, in a particularly advantageous manner, the compound of formula (II) is first purified to a degree of purity greater than or equal to 90%, preferably 95%.

For example, when the compound of formula (II) is N-methyl-isatoic acid anhydride, it can be repurified to ensure a control on the degree of purity of the reagent and thus to improve the conditions of the reaction. marking. The repurification process consists for example of a double recrystallization of the precursor in acetone. This process makes it possible to obtain an N-methyl-isatoic acid anhydride with a degree of purity greater than 95% characterized by a cream-like texture and a melting point of between 166 ° C. and 167 ° C. According to the invention, the step of labeling the activated carbohydrate compound CG-LH by reaction with the compound of formula (II) comprises the steps of:

mixing the activated glucidic compound CG-LH in aqueous solution, preferably buffered, with the compound of formula (II) in an organic solvent suitable for the labeling step,

- agitation,

concentration and drying under vacuum of the fluorescent conjugate of formula (I).

In possession of the activated carbohydrate compound CG-LH, the user carries out the labeling reaction of said carbohydrate compound with the compound of formula (II), for example N-methyl-isatoic acid anhydride. The labeling reaction is carried out on the bench of the laboratory at room temperature, by mixing an aqueous solution of the activated carbohydrate compound CG-LH with a solution of the compound of formula (II), for example N-methyl acid anhydride. isatoic, in slight molar excess, concentrated in an organic solvent in the presence of a basic buffer such as NaHCO 3. The main operations of the marking can be schematized as shown below in Figure 2:

R2 = -OH, -H, -OP0 ₃ Na ₂ , te

R3, R4 = -OH, -H, etc.

Figure 2

The process according to the invention makes it possible to use the activated carbohydrate compound CG-LH directly, the labeling reaction being carried out in a single step, without protection / deprotection step of the carbohydrate compound. The reaction is done with a very good yield (about 82%).

The present invention also relates to a kit for screening carbohydrate compounds by fluorescence from a fluorescent conjugate of formula (I) as described above, said kit comprising:

at least one activated carbohydrate compound CG-LH as defined above, preferably in freeze-dried form,

a compound of formula (II) as defined above, preferably in freeze-dried form,

an organic solvent suitable for the marking step.

The possibility of separately storing a compound of formula (II) constituting the probe and the activated carbohydrate compound (s) allows ease of use, prolonged storage and reserve the probe for others. markings.

The present invention also relates to a method for screening carbohydrate compounds by fluorescence using a fluorescent conjugate of formula (I) as defined above. This screening method can use one of the techniques selected from FRET, fluorescence intensity and fluorescence anisotropy.

An advantage of the present invention resides first and foremost in the possibility for the user to separately dispose of at least one CG-LH activated carbohydrate compound of his choice and the compound of formula (II) derived from N-acid anhydride. methyl isatoic acid or isatoic acid anhydride. These products can advantageously be provided in freeze-dried form and can be stored at -20 ° C. for several months without major risk of degradation by hydrolysis. This presentation allows the user to invest only in the CG-LH activated carbohydrate compound (s) which he or she would find useful.

The present invention also has an advantage because of the simple nature of its application. Indeed, once it has the CG-LH activated carbohydrate compound synthesized, the user can, simply and quickly, dispose of its fluorescent conjugate following the simplified protocol preset. Thus, the user can have several series of molecules simple or complex carbohydrates labeled and usable for screening interactions with fluorescent receptor proteins.

In addition, the probe at the origin of the fluorescent character of the conjugate, derived from N-methyl anthranilate or anthranilate, is an inexpensive, small probe whose steric hindrance within the fixation pocket. a receptor protein can be minimized by modulating the size of the L group, allowing its use on a very large number of carbohydrate ligand-protein receptor systems. In addition, the N-methyl anthranilate derivatives have an absorption spectrum that allows high throughput screening by three fluorescence techniques:

1) FRET ("Forest Resonance Energy Transfer").

2) Fluorescence intensity. The N-methyl-anthranilate derivatives have the spectroscopic particularity of seeing their quantum yield and thus their fluorescence intensity increase in a hydrophobic environment. Thus, it is conceivable to exploit this property in the case of a protein receptor having a hydrophobic fixation pocket supporting the whole conjugate or only the carbohydrate moiety provided that the group derived from N-methyl anthranilate is in close proximity. of the surface of the receptor protein. Consequently, the conjugate according to the invention is little fluorescent in aqueous solvent when it is separated from its protein receptor. When the receptor binds the labeled ligand, the probe is near the protein surface or at best within the ligand binding pocket. If this surface is hydrophobic or if the interior of the ligand binding pocket is hydrophobic, the conjugate is increased in fluorescence intensity. Under these conditions, the conjugate according to the invention may be excited in wavelengths between 320 nm and 360 nm, while the increase in fluorescence emission after addition of the target protein will be followed between 400 and 500 nm. The exploitation of the method in competition is possible. Adding a competitor displacing the conjugate according to the invention is accompanied by a fall in the fluorescence intensity between 400 and 500 nm, making exploitation possible in the context of a high-throughput screening.

3) Fluorescence anisotropy. Under the conditions in which it is not possible to exploit the spectroscopic peculiarity of the probe derived from N-methyl anthranilate as presented in 2), the interaction of the fluorescent conjugate according to the invention with its protein target can be followed by increasing the level of fluorescence anisotropy. In this case, experience by competition is also possible. Thus, in the presence of an effective competitor, the displacement of the fluorescent conjugate of the receptor protein binding site will be followed by a decrease in the level of anisotropy.

Thus, the fluorescent conjugates according to the invention are particularly well suited to high throughput screening. In fact, whatever the envisaged strategy (FRET, fluorescence intensity (IF), fluorescence anisotropy (A)), the fixation of the conjugate on the target protein will be accompanied by an increase of a fluorescence signal (FRET , IF or A) between 400 nm and 500 nm while the effective displacement of the conjugate operated by a competitor can be followed by a decrease of the signal (FRET, IF or A) in this same range of wavelength. In the context of the screening of an effector specifically targeting a protein of interest, it will be easily possible to detect a "hit" by selecting the competitor corresponding to the well for which the fluorescence signal (FRET, IF or A) has been lowered. . In the presence of competitors, the fluorescent wells correspond to molecules that have not been able to displace the fluorescent conjugate of the protein receptor. On the contrary, non-fluorescent or weakly fluorescent wells correspond to "hits".

The present invention can be exploited in particular in the field of biomedical applications: the carbohydrate compounds of the invention can be used in the search for enzyme effectors (substrate, inhibitors, activators) by high throughput screening (in direct interaction or by competition) and in the qualitative study of the interactions between a ligand carbohydrate and a target (receptor). The targets of the glycans are multiple and the field of exploitation of the technology of the invention is therefore very broad. Thus, in addition to high throughput microplate screening of glycosidic effectors for industrial purposes, the present invention can also be used in basic research in qualitative and quantitative studies for the study of sugar ligand-protein receptor interactions.

The following examples illustrate the present invention without, however, limiting its scope.

1) Study of Xyl-MANT Compound A (precursor of L: Ethanolamine) a) Preparation D-xylose is activated with an ethanolamine-type L group precursor at position 1. Referring to scheme 1 above, for which R2 , R 3 and R ₄ are OH groups, the grafting of the group L starts by a step of protection and activation of the oxygen atom in position 1: a) (1) Ac ₂ O-NaOAc, 100 ° C, 30 min. ; (2): Hydrazine acetate, DMF, TA, 30 min. ; (3) CI3C-CN, DBU, CH2Cl2, TA, 20 min. Activation of oxygen in position 1 allows the grafting of the ethanolamine precursor previously protected by a carbobenzoxy (Z) group: b) (1): N, Z-Ethanolamine, TMSOTf, 0 ° C, 30 min. Then the protecting group Z is removed: d) H 2, Pd / C 10%, MeOH, TA, 16 h. These steps are all done in the laboratory. The activated carbohydrate compound is obtained, which is then labeled with N-methyl-isatoic acid anhydride. The marking is simple and can be done by the user himself according to a pre-established protocol.

For this, an aqueous solution of xylose (39 mg, 20 μηηοΙ in 1.5 ml) previously activated in position 1 by means of a precursor of the L group of ethanolamine type is mixed with a solution of NaHCO3 (1 M, 30 μηηοΙ in 30 μl) and is stirred at room temperature. A solution of N-methyl isatoic anhydride (recrystallized, 59 mg, 30 μηηοΙ) in 1, 4- dioxane (0.8 ml) is added to the above mixture. The new reaction mixture is then stirred for 2 hours at room temperature. The progress of the reaction is monitored by thin layer chromatography (TLC) of silica in the solvent mixture mentioned above. The complete disappearance of activated xylose using the ethanolamine precursor

(Rf = 0, ninhydrin positive, revealing spray of the amino compounds, Rf designates the frontal ratio) could be visualized in favor of the product (Rf = 0.4, positive in UV). The excess reagent, N-methylisatoic acid anhydride, is on a TLC in the form of a task characterized by a Rf of 0.9. TLC verification of the progress of the reaction after 2 hours of reaction shows an almost total disappearance of the activated carbohydrate compound associated with the appearance of the labeled reagent while there is a clear resorption of the stain associated with the excess of N-methyl isatoic acid anhydride (data not shown).

Confirmation has been given that the reaction product is indeed xylose labeled in position 1 by N-methyl anthranilate via an ethanolamine L group precursor (compound A). To do this, the reaction mixture was concentrated and dried in vacuo so that the resulting sample could be quantified and analyzed by mass spectroscopy and NMR. Thus, the residue is extracted with CH 2 Cl 2: MeOH (9: 1) (2 × 5 mL) and then chromatographed on a column of silica gel (10 g). The excess of N-methyl-isatoic acid is eluted before the compound A (data not shown). The mobile phase used consists of the same solvent mixture as mentioned above. These conditions made it possible to collect approximately 54 mg of product, ie a reaction yield of 82%. The product is in the form of a cream mousse.

Compound A obtained was then analyzed by high resolution mass spectroscopy. The ratio m / z calculated for C15H23N2O6 [M + H] ⁺ is 327.15506 while that found experimentally is 327.15512. Compound A was also analyzed by ¹ H NMR and ¹³ C NMR. The two NMR spectra are consistent with the expected structure. ¹ H NMR (400 MHz, CD3OD-CDCl3): δ 7.42 (m, 1H, Ar-H), 7.28 (m, 1H, Ar-H), 6.61 (m, 1H, Ar-H), 6.55. (m, 1H, Ar-H), 4.22 (d, 1 H, Ji, ₂ 7.4 Hz, H-1), 3.93 (m, 1H, O-CH), 3.95 (dd, 1H, J ₄ , _5eq 5.2, J ₅ ax, 5e _q 1 1.5 Hz, H-5eq), 3.71 (m, 1H, O-CH), 3.60 (m, 1H, N-CH), 3.52 3.42 (m, 2H) , N-CH, H-4), 3.31 (t, 1H, J ₂ , s = Js, 4 = 8.9 Hz, H-3), 3.21 (dd, 1H, H-2), 3.18 (dd, 1H, J ₄ , _5ax 8.0 Hz, H-5ax), 2.80 (s, 3H, N-CH 3).

¹³ C NMR (100 MHz, CD3OD-CDCl3): δ 171.40 (1 C, NH-CO), 150.58, 133.1 1, 126.43, 1 16.25, 1 15.28, 1 1 1.36 (6c, Ar-C), 104.37 ( 1 C, C-1), 76.74 (1 C, C-3), 73.88 (1 C, C-2), 70.19 (1 C, C-4), 69 .37 (1 C, O-CH ₂ ) , 66.17 (1 C, C-5), 39.93 (1 C, N-CH ₂ ), 29.64 (1 C, N-CH 3). b) Application to FRET

Figure 1 shows the normal fluorescence absorption (A, B respectively) and emission (D, C respectively) spectra of compound A and a tryptophan residue belonging to a target protein in an aqueous solvent. The normalized absorption spectrum of compound A is spectrum A. At around 274nm, the excitation wavelength characteristic of aromatic amino acid residues (spectrum B), the absorption of light by the conjugate is negligible. while it is significant between 290 and 375nm, range of emission wavelength of aromatic amino acid residues (spectrum C). Thus, the conjugates prepared by the user can therefore be used for any quantitative study of the ligand-FRET protein receptor interactions by excitation of the tyrosines and tryptophans (at 274 nm) present in the binding site following the evolution of the emission of the fluorescent conjugate between 400 and 500 nm after adding the competitors in a high-throughput screening (spectrum D). c) Quantitative Study of the Interaction of Compound A with 4GalT7 by FRET Methods and Fluorescence Anisotropy

1 - Determination of the affinity (Kd) of compound A for β4ΘθΙΤ7 by FRET on a 96-well microplate In the context of the FRET evaluation of the affinity (Kd) of the bond between the 4GalT7 and the compound A, it is considered that the FRET donor is constituted by the β4ΘθΙΤ7 whereas the FRET recipient is the compound A given the spectral properties of each of the partners (Fig. 1). The experiment is carried out in a 96-well plate (BD Falcon Microtest 96 well, Flat plate optilux, Black / Clear bottom surface standard REF: 353293). 160 μl of 4GalT7 are placed in the presence of increasing concentrations of compound A (0 to 2 mM), in a 50mM BisTris buffer pH6, 0.05mM MnC, 2mM UDP. After stirring for 1 min, the FRET is read at ambient temperature by exciting the mixture at the wavelength of 274 nm and following the emission spectrum obtained between 290 and 500 nm. The apparatus used is a Xenius (SAFAS) equipped with a rack adapted for plate readings.

Figure 2A shows the emission spectra of Compound A between 290 and 500nm from excitation at 274nm in the absence of 4GalT7 for different concentrations.

FIG. 2B shows the emission spectra of compound A obtained between 290 and 500 nm from excitation at 274 nm in the presence of 4GalT7. FIG. 2C represents the saturation curve obtained by transferring the values of the fluorescence signal at 430 nm as a function of the concentration of compound A.

The emission spectra reveal FRET between the aromatic amino acid residues of 4GalT7 and compound A (Figure 2B). Measurements were also performed on protein-free controls for each concentration of compound A (Fig. 2A). The "raw" fluorescence emission values read at the wavelength of 430 nm for each concentration of compound A in the presence of the enzyme (FIG 2B) were thus reduced from those obtained for each concentration of compound A in the absence of the enzyme. The "net" fluorescence emission values at 430 nm were then plotted as a function of the concentration of compound A. The saturation curve is shown in FIG. 2C. This curve was then adjusted by non-linear regression using the GraphPad Prism5 software. A preliminary value of the affinity constant (Kd) of 4GalT7 for compound A could be determined around 202 ± 68 μΜ. 2 - Determination of the affinity (Kd) of 4GalT7 for compound A by fluorescence anisotropy on a 96-well microplate

In the context of the fluorescence anisotropy evaluation of the affinity (Kd) of 4GalT7 for compound A, we follow the formation of the complex between compound A and the enzyme through the evolution of the level of anisotropy fluorescence of compound A as a function of increasing concentrations of 4GalT7. For this purpose, in a 96-well plate (BD Falcon Microtest 96 well, Assay plate optilux, Black / Clear bottom surface standard REF: 353293), 10 μl of compound A are brought together with different concentrations of 4GalT7 (0 to 1 mM). in 50mM BisTris pH6 buffer, 0.05mM MnC, 2mM UDP. After stirring for 1 minute, the anisotropic reading is carried out at room temperature by exciting at a wavelength 354 nm and making an end-point measurement at an emission wavelength of 430 nm. The device used is the Infinity® 200 pro (TECAN), equipped with an optical filter (TECAN) centered at excitation at 360 nm (± 35 nm) and centered at emission at 430 nm (± 20 nm) ).

A measurement in anisotropy is performed on a control without proteins and the value is directly subtracted from the values obtained in the presence of 4GalT7 by the apparatus.

FIG. 3 represents the saturation curve obtained from the fluorescence anisotropy values at 430 nm as a function of the 4GalT7 concentration. The anisotropy values read at 430 nm for each concentration of 4GalT7 were plotted against them.

The saturation curve obtained was then adjusted by non-linear regression using the GraphPad Prism5 software. A preliminary value of the affinity constant (Kd) of 4GalT7 for compound A could be determined at around 455 ± 51 μΜ. 2) Study of compound A 'Xyl-MANT (precursor of L: hydroxylamine) a) Preparation

Compound A 'is prepared in the same way as compound A but using as precursor of group L a precursor of hydroxylamine type. This makes it possible to bring the osidic structure of the aromatic body closer to the probe so as to position the latter in the vicinity of the hydrophobic amino acid residues present in the attachment pocket to the accepting substrate. Compound A 'is recrystallized from EtOH.

Mp 206-208 ° C (decomposition)

¹ H NMR (400 MHz, CD ₃ OD): δ 7.68 (m, 1H, arom H), 7.60 (m, 1H, H arom), 7.18 (m, 1H, H arom), 7.12 (m , 1 H, H arom), 4.65 (d, 1 H, Ji, ₂ 9.0 Hz, H-1), 3.97 (dd, 1H, J _4, _5ax 9.0, J _4, _5EQ 3.0 Hz, H-5EQ) , 3.96 (dd, 1H, J ₂ , s 9.0 Hz, H-2), 3.82 (dd, 1H, J ₃ , ₄ 9.0 Hz, H-3), 3.54 (m, 1H, H-4). , 3.35 (dd, 1H, H-5ax), 3.08

¹³ C NMR (100 MHz, CD3OD): δ 172.01 (1 C, NH-C = O), 146.44, 134.66, 131.79, 125.82, 123.35, 120.94 (aromatic 6C, C), 93.33 (1 C, C-1) , 79.05 (1 C, C-2), 78.74 (1 C, C-3), 78.41 (1 C, C-4), 68.26 (1 C, C-5), 37.17 (1 C, N-CH ₃₎ ). b) Determination of the affinity (Kd) of 4GalT7 for the compound A 'by direct fluorescence on a 96-well microplate

During this experiment, we follow the formation of the complex formed between the compound A 'and the enzyme by increasing the fluorescence signal emitted by the compound A' interacting with 4GalT7. For this study, 25μΜ of compound A 'were brought into contact with increasing concentrations of 4GalT7 ranging from 100 to 1000μΜ. The experiment was carried out in a 96-well plate (BD Falcon Microtest 96 well, Assay plate optilux, Black / Clear bottom surface standard REF: 353293) using a 50mM BisTris buffer pH6, MnCh 0.05mM and containing 2mM UDP. After stirring for 1 minute, the fluorescence intensity reading is carried out at room temperature. ambient by exciting at a wavelength 354nm and following the emission spectrum obtained between 370 and 600nm. The device used is a Varioskan® Flash (Thermo Scientific).

FIG. 4 shows the emission spectra of compound A 'between 370 and 600 nm from excitation at 354 nm in the presence of increasing concentrations of 4GalT7.

The results presented in FIG. 4 show that the fluorescence signal of compound A 'increases as the concentration of 4GalT7 increases (FIG. 4). The evolution of the signal seems to saturate from 750μΜ (Fig.4), assuming a Kd of between 100 and 300μΜ which remains in agreement with the data of the literature. c) Competition Experiment for Xylose tert-Butylcyclohexane on Compound A 'Involved in the Complex with 4GalT7 by FRET on a 96-Well Microplate

Compound A 'competition is monitored in complex with 4GalT7 by xylose tert-butylcyclohexane, non-natural and non-fluorescent ligand of 4GalT7, by FRET.

The xylose tert-butylcyclohexane (compound F) has the formula:

For this purpose, 200 μl of 4GalT7 and 600 μl of compound A 'were placed in the presence of xylose tert-butylcyclohexane at 600 μΜ, in a BisTris 50mM pH6 buffer, MnCl ₂ 0.05mM, 2mM UDP. After stirring for 1 minute, the reading

FRET was performed as before. The apparatus used is a Xenius (SAFAS) equipped with a rack adapted for plate readings.

FIG. 5A shows the emission spectra of compound A 'obtained between 300 and 500 nm from an excitation at 274 nm. The spectra were carried out on two references made without 4GalT7 in absence (curve E) and in the presence of compound A '(curve F). The competition experiment was carried out in the presence of 200 μΜ of enzyme and 600 μΜ of compound A 'in absence (curve G) then in the presence of Xylose tert-butylcyclohexane (curve H). The emission spectra allow the detection of FRET between the aromatic amino acid residues of 4GalT7 and the compound A '(FIG.5A).

Measurements were also performed on controls without protein on the one hand and without fluorescent conjugate or protein on the other hand. FIG. 5B represents the histogram obtained by transferring the values of the fluorescence signal of the compound A 'to 430 nm according to measurements made.

The "raw" fluorescence emission values read at the 430 nm wavelength for testing were thus plotted in the graph (Fig. 5B). The competition percentage at 600 μΜ of tert-butylcyclohexane xylose is calculated using as a reference the fluorescence value obtained in the absence of xylose tert-butylcyclohexane. A preliminary value of the competition percentage of xylose tert-butylcyclohexane 600μΜ on the compound A 'could be determined at around 20%.

3) Study of compound B Maltose-MANT (precursor of L: ethanolamine) a) Preparation

The synthesis protocol is identical to that described for compound A above. The reaction is carried out from the corresponding amine (200 mg, 0.5 mmol). The residue is chromatographed on a column of silica gel (20 g) in dichloromethane-methanol (5: 2, v / v) to give compound B as a colorless foam (205 mg, m.p.

79%).

[a] _D = +57 (c 1, MeOH)

¹ H NMR (400 MHz, CD ₃ OD, internal acetone): δ 7.44 (m, 1H, Ar-H), 7.32 (m, 1H, Ar-H), 6.64 (m, 1H, Ar-H) ), 6.56 (m, 1H, Ar-H), 5.12 (d, 1 H, Ji, ₂ 3.8 Hz, H-1 '), 4.31 (d, 1 H, Ji, ₂ 7.8 Hz, H-1) , 3.97 (m, 1H, O-CH), 3.80

(m, 3H, H-4, H-6'a, b), 3.73 (m, 1H, O-CH), 3.62 (m, 3H, H-6a, b, N-CH), 3.60 ( m, 2H, H-3, H-4 '), 3.50 (dd, 1H, J s = J ₃ = 4-9.0 Hz, H-3'), 3.48 (m, 1H, N-CH), 3.42 (dd, 1H, H-2 '), 3.36 (m, 1H, H-5'), 3.28 (m, 1H, H-5), 3.23 (dd, 1H, J ₂ , s 9.0 Hz, H-2), 2.78 (s, 3H, N-CH ₃ ).

¹³ C NMR (100 MHz, CD ₃ OD, internal acetone): δ 172.05 (1 C, NH-CO), 151.08, 133.47, 129.05, 1 16.96, 1 15.69, 1 1 1.66 (6 C, Ar-C), 104.16 (1 C, C-1) 102.60 (1 C, C-1 '), 80.90 (1 C, C-4), 77.34 (1 C, C-3), 76.28 (1 C, C-5), 74.71, 74.46, 74.33 (3 C, C-2 ', 3', 5 '), 73.78 (1 C, C-4'), 71.22 (1 C, C-2), 69.51 (1 C, OCH 2), 62.39, 61.76 (2 C, C-6, C-6 '), 40.38 (1 C, N-CH ₂ ),

HRMS: m / z calcd for C22H35N2O12 [M + H] ⁺ 519.71205. Find ; 519.71228. b) Quantitative study of the interaction of compound B with MBP by FRET and by fluorescence anisotropy

1 - Determination of the affinity (Kd) of compound B for MBP by FRET on a 96-well microplate

In the context of the FRET evaluation of the binding affinity (Kd) of the MBP (Maltose Binding Protein) and the compound B, the FRET donor consists of the MBP while the FRET recipient consists of the compound B in view of the spectral properties of each of the partners (Fig. 1). The experiment was carried out in a 96-well plate (BD Falcon Microtest 96 well, Flat plate optilux, Black / Clear bottom surface standard REF: 353293). To do this, 50 μl of MBP are placed in the presence of increasing concentrations of compound B ranging from 0 to 200 μΜ, in a Sodium phosphate 100 mM buffer, pH 7.2 in the presence of imidazole 18.75 μΜ. After stirring for 1 min, the FRET reading is carried out at ambient temperature by exciting at the wavelength of 274 nm and following the emission spectrum obtained between 290 and 500 nm. The apparatus used is a Xenius (SAFAS) equipped with a rack adapted for plate readings. Measurements can be made at the single emission wavelength of 430nm.

Figure 6A shows the emission spectra of compound B obtained between 290 and 500 nm from an excitation at 274 nm in the absence of MBP. FIG. 6B shows the emission spectra of compound B obtained between 290 and 500 nm from an excitation at 274 nm in the presence of MBP.

FIG. 6C represents the saturation curve obtained by transferring the values of the fluorescence signal at 430 nm as a function of the concentration of compound B.

The emission spectra allow the detection of FRET between the aromatic amino acid residues of MBP and the compound B (Figure 6B). Measurements were also performed on protein-free controls for each concentration of compound B (Fig. 6A). The "raw" fluorescence emission values read at the wavelength of 430 nm for each concentration of compound B in the presence of MBP (FIG 6B) were thus reduced from those obtained for each concentration of compound B in FIG. absence of MBP. The "net" fluorescence emission values at 430 nm were then plotted as a function of the concentration of compound B. The saturation curve is shown in FIG. 6C. This saturation curve was then adjusted by nonlinear regression using the GraphPad Prism5 software. The affinity constant (Kd) of MBP for compound B could be determined around 25 ± 9 μΜ. 2 - Determination of the affinity (Kd) of compound B for MBP by fluorescence anisotropy on a 96-well microplate

In the fluorescence anisotropy evaluation of the affinity (Kd) of MBP for compound B, the formation of the complex between compound B and the protein is monitored by the evolution of the level of anisotropy of fluorescence of compound B as a function of increasing concentrations of

MBP. For this purpose, in a 96-well plate (BD Falcon Microtest 96 well, Assay plate optilux, Black / Clear bottom surface standard REF: 353293), 10 μl of compound B are placed in the presence of different concentrations of MBP (0 to 400 μl). in 100mM Sodium phosphate buffer, pH 7.2. After stirring for 1 minute, the anisotropic reading is carried out at room temperature by exciting at a wavelength 354 nm and making an end-point measurement at an emission wavelength of 430 nm. The device used is Infinity® 200 pro (TECAN), equipped with an optical filter (TECAN) centered on excitation at 360 nm (± 35 nm) and centered at emission at 430 nm (± 20 nm). A measurement in anisotropy is performed on a control without proteins and the value is directly subtracted from the values obtained in the presence of MBP by the apparatus.

Figure 7 shows the saturation curve obtained from fluorescence anisotropy values at 430 nm as a function of MBP concentration. The anisotropy values read at 430 nm for each concentration of MBP were plotted against them.

The saturation curve obtained was then adjusted by non-linear regression using the GraphPad Prism5 software. The affinity constant (Kd) of MBP for compound B could be determined around 66 ± 7 μΜ. c) Competitive experiment of maltose on the compound B involved in the complex with MBP by 96 well microplate fluorescence anisotropy

The competition is monitored by maltose, the natural ligand of MBP, with compound B complexed with MBP by fluorescence anisotropy. For this purpose, 50 μl of MBP and 10 μl of compound B were placed in the presence of maltose concentrations ranging from 50 nm to 300 μm in a Sodium phosphate 100 mM buffer, pH 7.2. The dissociation of compound B is followed by the evolution of the fluorescence anisotropy value at 430 nm under an excitation length of 354 nm. The device used is the Infinity® 200 pro (TECAN), equipped with an optical filter (TECAN) centered at excitation at 360 nm

(± 35 nm) and centered at emission at 430 nm (± 20 nm). A measurement in anisotropy is performed on a control without proteins and the value obtained is directly subtracted from the values obtained in the presence of MBP by the apparatus.

Figure 8 shows the level of fluorescence anisotropy as a function of increasing concentration of maltose, natural substrate of MBP. Values of anisotropy read at 430 nm for each concentration of maltose were reported according to the latter.

The resulting competition curve was then adjusted according to a nonlinear regression using the GraphPad Prism5 software. The median inhibition constant (IC50) characterizing the efficiency with which maltose displaces compound B complexed with MBP could be determined around 15 ± 1 μΜ under the conditions of the experiment.

The fluorescence anisotropy value of compound B in its complexed form is about 5 times greater than that of its free form. This confirms that the level of sensitivity of the conjugates according to the invention allows their use in the context of a high-throughput screening by fluorescence anisotropy reading.

4) Study of compound C Lactose-MANT (precursor of L: ethanolamine) a) Preparation

The synthesis protocol is identical to that described for compound A above. The reaction is carried out from the corresponding amine (160 mg, 0.4 mmol). The residue is chromatographed on a column of silica gel (20 g) in dichloromethane-methanol (5: 2, v / v) to give compound C as a colorless foam (156 mg, 75%). [a] _D = +9 (c 1, MeOH)

¹ H NMR (400 MHz, CD ₃ OD, internal acetone): δ 7.45 (m, 1H, Ar-H), 7.36 (m, 1H, Ar-H), 6.66 (m, 1H, Ar-H) ), 6.54 (m, 1H, Ar-H), 4.35 (d, 1 H, Ji, ₂ 8.0 Hz, H-1 '), 4.34 (d, 1 H, Ji, ₂ 7.5 Hz, H-1) , 3.98 (m, 1H, O-CH), 3.82 (m, 3H,

H-4, H-6'a, b), 3.75 (m, 1H, O-CH), 3.62 (m, 2H, H-6a, b), 3.61 (m, 1H, N-CH), 3.60 (m, 3H, Η-2 ', 4', H-5), 3.48 (dd, 1H, Jz.s 1 1.0, J ₃ \ _^4! 3.6 Hz, H-3 '), 3.45 (m , 1H, N-CH), 3.42 (m, 1H, H-5), 3.36 (m, 1H, H-5 '), 3.26 (dd, 1H, H-1),

¹³ C NMR (100 MHz, CD3OD, internal acetone): δ 171.94 (1 C, NH-CO),

151.0, 133.37, 128.97, 1 16.91, 1 15.59, 1 1 1.56 (6 C, Ar-C), 104.73 (1 C, C-1) 103.96 (1 C, C-1 '), 80.29 (1 C, C -4), 76.66 (1C, C-3), 76.1 (1C, C-5), 75.87, 74.41, 74.36, 74.33, 72.14 (5C, C-2, C-2 ', 3', 4 ', 5'), 69.92 (1 C, OCH ₂ ), 62.14, 61.49 (2 C, C-6, C-6 '), 40.31 (1 C, N-CH ₂ ), 29.58 (1 C, N-CH ₃ ).

HRMS: m / z calcd for C22H35N2O12 [M + H] ⁺ 519.71205. Find ; 519.71232. b) Quantitative study of the interaction of compound C with Galectin-3 by FRET and by fluorescence anisotropy

1 - Determination of the affinity (Kd) of compound C for Galectin-3 by FRET on a 96-well microplate

In the context of the FRET evaluation of the dissociation constant (Kd) characterizing the affinity of Galectin3 for compound C, it is considered that the donor and the FRET recipient are respectively represented by Galectin3 and compound C given their spectral properties (Fig. 1). The experiment is carried out in a 96-well plate (BD Falcon

Microtest 96 well, Flat optilux assay, Black / Clear bottom surface standard REF: 353293). We put in the presence of 50μΜ of Galectin-3 increasing concentrations of compound C (0 to 200μΜ). The Galectin-3 used in these assays was expressed in E. coli bacteria from a complementary DNA sequence coding for the lactose binding domain, inserted into a pET-41a vector before bacterial transformation. The solvent used is a phosphate phosphate buffer, pH7. After stirring for 1 min, the FRET reading is carried out at ambient temperature by exciting at the wavelength of 274 nm and following the emission spectrum obtained between 290 and 500 nm. The apparatus used is a Xenius (SAFAS) equipped with a rack adapted for plate readings. Measurements can be made at the single emission wavelength of 430nm.

FIG. 9A represents the emission spectra of compound B obtained between 290 and 500 nm from excitation at 274 nm in the absence and then in the presence of increasing concentration of Galectin-3. FIG. 9B represents the saturation curve obtained by transferring the values of the fluorescence signal at 430 nm as a function of the concentration of compound B.

The emission spectra allow the detection of FRET between the aromatic amino acid residues of Galectin-3 and compound B (FIG.

9A). The fluorescence emission values read at the wavelength of 430 nm for each concentration of compound C in the presence of Galectin-3 were then plotted as a function of the concentration of compound C. The saturation curve is presented in FIG. Figure 9B. This saturation curve was then adjusted by nonlinear regression using the GraphPad Prism5 software. The affinity constant (Kd) of Galectin-3 for compound C could be determined around 35μΜ.

2 - Quantitative Determination of Lactose-MANT Affinity (Kd) for Galectin-3 by Fluorescence Anisotropy on a 96-well Microplate

In the framework of the fluorescence anisotropy evaluation of the affinity (Kd) of Galectin-3 for compound C, the formation of the complex between compound C and the protein is monitored by the evolution of the level of Fluorescence anisotropy of compound C as a function of increasing concentration of Galectin-3. To do this, in a 96-well plate (BD

Falcon Microtest 96 well, Assay plate optilux, Black / Clear bottom surface standard REF: 353293), 10 μΜ of compound C are brought into increasing concentrations of Galectin-3 (0 to 400 μΜ) in a phosphate salt buffer, pH7. The Galectin-3 used in these assays was expressed in E. coli bacteria from a complementary DNA sequence coding for the lactose binding domain, inserted into a pET-41a vector before bacterial transformation. After stirring for 1 minute, the anisotropic reading is carried out at room temperature by exciting at a wavelength 354 nm and making an end-point measurement at an emission wavelength of 430 nm. The device used is Infinity®

200 pro (TECAN), equipped with an optical filter (TECAN) centered at 360 nm excitation (± 35 nm) and centered at emission at 430 nm (± 20 nm). A measure in anisotropy is performed on a control without protein and the value is directly subtracted from the values obtained in the presence of Galectin-3 by the apparatus.

FIG. 10 represents the saturation curve obtained from the fluorescence anisotropy values of compound C at 430 nm as a function of the Galectin-3 concentration. The anisotropy values read at 430 nm for each concentration of Galectin-3 were plotted against them.

The saturation curve obtained was then adjusted by non-linear regression using the GraphPad Prism5 software. The affinity constant (Kd) of Galectine3 for Lactose-MANT could be determined around 84 ± 25 μΜ (N = 1).

These results show that the conjugates of the present invention are effective in monitoring protein-sugar interactions.

They first have the advantage of being easily synthesized, without the use of a toxic reagent, by bringing into contact the carbohydrate compound chosen beforehand "activated" with a precursor of the group L with isatoic acid anhydride or a derivative thereof. in a practically equimolar mixture. The labeling yield under these conditions is around 82%. The reaction of the carbohydrate compound with isatoic acid anhydride or a derivative is substantially complete, which allows the user to avoid labeling conditions in which the isatoic acid anhydride must be in excess of to the carbohydrate molecule. This makes it possible to minimize the background noise associated with the unreacted isatoic acid or derivative acid anhydride probes and to ensure that the monitored fluorescence signal comes essentially from the carbohydrate conjugate. The labeling method is thus improved as well as the ability of the conjugates of the invention to sensitively shift their interaction with their target proteins.

Moreover, the process for preparing the carbohydrate conjugates according to the invention makes it possible to respect the closed cycle structure of the diene residue. labeled so that the resulting molecule remains suitable for studies involving its interaction with target proteins.

The conjugates of the invention also have the advantage of being able to be used as a fluorescent "reporter" for their interaction with their target protein, the Kd associated with the interactions of the protein receptor-saccharide ligand couples being able to be determined.

The conjugates of the invention also have the advantage that they can be used in competition experiments, the IC50 being determinable.

Thus, this results demonstrate that the conjugates of the invention are as well suited for the study of protein-sugar interactions as for the search for effectors for high-throughput screening.

Claims

claims

1. Fluorescent Conjugate of Formula (I)

or

CG is a carbohydrate compound chosen from the residues of oste, oligosides and polysaccharides comprising at least one residue of osé in cyclic form, substituted or unsubstituted,

L is a group grafted on a labeling position of said ose and is selected from -O-NH-, -O- (CH2) _n NH-, -O- (CH2) _n S-, where n is an integer equal to 1 or 2, a group derived from a glycosylamine, a group derived from a thioglycoside, the nitrogen or sulfur atom being bonded to the carboxylic carbon of the anthraniloyl group,

R is H, CH ₃ ,

The fluorescent conjugate according to claim 1, wherein L is grafted to the 1-anomeric position of the oste residue.

3. Fluorescent conjugate according to any one of the preceding claims, wherein CG is selected from a diene residue, a diholoside, a triholoside.

4. Fluorescent conjugate according to claim 3, wherein CG is selected from a xylose residue, maltose, lactose.

A fluorescent conjugate according to any one of the preceding claims, wherein the free positions of the oste residue are independently unsubstituted bearing H or OH or substituted with a group selected from -O-PO ₃ Na ₂ , -O-SO ₃ Na , -O-CO-CH 3, -N-CO-CH 3, -NH ₂ , -CH ₃ , -CH ₂ -CH ₃ , -F, -Cl, -Br, -SH. Fluorescent conjugate according to any one of the preceding claims, wherein L is -O-CH 2 -CH 2 -NH- grafted in the anomeric position 1 of the diene residue and R 'is H and R is CH ₃ .

Process for the preparation of a fluorescent conjugate of formula (I) according to claims 1 to 6 comprising the steps of:

preparation of a CG-LH activated carbohydrate compound from a complete carbohydrate CG compound and an L group precursor,

labeling of the activated carbohydrate compound CG-LH by reaction with a compound of formula (II)

(II)

to obtain the fluorescent conjugate of formula (I)

CG, L, R, R 'being defined according to the preceding claims.

The method of claim 7, wherein the step of preparing the CG-LH activated carbohydrate compound comprises the steps of:

activating the labeling position of a complete carbohydrate CG compound, said labeling position preferably being the anomeric position 1 of the complete monosaccharide in cyclic form,

chemical grafting on the activated marking position of the group L,

- optionally substitution of one or more free positions of the ose,

9. The method of claim 8, wherein the reducing end of the complete ose is free.

10. Process according to any one of claims 7 to 9, wherein the precursor of the group L is chosen from the compounds HO-NH2, HO- (CH2) _n -NH2, HO- (CH2) _n -SH, a glycosylamine a thioglycoside.

A process according to any one of claims 7 to 10, wherein the compound of formula (II) is previously purified to a degree of purity greater than or equal to 90%, preferably 95%.

The method according to any one of claims 7 to 11, in the step of labeling the activated carbohydrate compound CG-LH by reaction with the compound of formula (II) comprises the steps of:

mixing the activated glucidic compound CG-LH in aqueous solution with the compound of formula (II) in an organic solvent suitable for the labeling step,

- agitation,

13. A kit for screening carbohydrate compounds by fluorescence from a fluorescent conjugate of formula (I) according to any one of claims 1 to 6, said kit comprising:

at least one activated carbohydrate compound CG-LH, preferably in freeze-dried form,

a compound of formula (II), preferably in freeze-dried form,

an organic solvent suitable for the marking step.

14. A method of screening carbohydrate compounds by fluorescence using a fluorescent conjugate of formula (I) according to any one of claims 1 to 6.

15. Screening method according to claim 14, using one of the techniques selected from the FRET, the fluorescence intensity and the fluorescence anisotropy.