AU2012200508A1 - Labeling reagents, methods for the synthesis of such reagents and methods for the detection of biological molecules - Google Patents

Abstract

"Labeling reagents, methods for the synthesis of such reagents and methods for the detection of biological molecules" - BIOMERIEUX S.A. The present invention relates to a temperature-stable labeling reagent of formula (0): R -(Z-(CH,)m-CL)n--Y--X4 in which: " R1 represents H or an alkyl, aryl or substituted aryl group, * R2 represents a detectable marker or at least two detectable markers interlinked by at least one multimeric structure, * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, * R3 and R4 represent, independently of one another: H, NO 2, Cl, Br, F, I, R2 -(L),-Y-X-, OR, SR, NR2, R, NHCOR, CONHR, COOR, -CO-NH1-(CH 2)3-(O-CH 2-CH 2)3-CH2-NH-R 2, -CO-NH-(CH2) 3 (O-CH2-CH 2)4-CH 2-NH-R2 with R = alkyl or aryl, " A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or1, " -Y-X- represents -CONH-, -NHCO-, -CH 20-, -CH2S-, " -Z- represents -NH-, -NHCO-, -CONH- or -0-, " m is an integer between 1 and 10, preferably between I and 3, and " p is an integer between I and 10, preferably between 1 and 3. The present invention also describes a method for the synthesis of said labels and also applications for the labeling of biological molecules, in particular of nucleic acids, with a labeling reagent bearing the diazomethyl function. The invention is particularly suitable for use in the field of diagnostics.

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: bioMerieux Actual Inventor(s): Eloy Bernal-Mendez, Ali Laayoun Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: LABELING REAGENTS, METHODS FOR THE SYNTHESIS OF SUCH REAGENTS AND METHODS FOR THE DETECTION OF BIOLOGICAL MOLECULES Our Ref: 934233 POF Code: 472077/472077 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1 sooQg la Labeling reagents, methods for the synthesis of such reagents and methods for the detection of biological molecules The present application is a divisional application from Australian patent application number 5 2005225589, the entire disclosure of which is incorporated herein by reference. The present invention relates to novel reagents for labeling biological molecules, to a method for the synthesis of said labels and to applications for the labeling of biological molecules, in particular in the field of diagnosis using nucleic acid analysis. 10 The prior art shows that numerous methods exist for labeling nucleotides, oligonucleotides or nucleic acids. A first method consists in attaching the label to the base, whether the latter is natural or 15 modified. A second method proposes attaching the label to the sugar, here again whether it is natural or modified. A third method aims to attach the label to the phosphate. Labeling on the base has in particular been used in the approach consisting in labeling the nucleic acids by incorporation of directly labeled nucleotides. 20 Labeling on the sugar is often used in the case of nucleic acid probes prepared by chemical synthesis. Labeling on the phosphate has also been used to introduce functionalized arms and markers 25 during the chemical synthesis of oligonucleotides. In fact, those skilled in the art, who must perform labeling of a nucleotide, or of a nucleotide analog or of a nucleic acid, are inclined to carry out this attachment to the base or to the-sugar, which offer them greater convenience and more alternatives. This is, moreover, what emerges 30 from the study of numerous documents, such as EP-A-0.329.198, EP-A-0.302.175, EP-A 0.097.373, EP-A-0.063.879, US-A-5,449,767, US-A-5,328,824, WO-A-93/16094, DE-A 3.910.151, EP-A-0.567.841 for the base or EP-A-0.286.898 for the sugar. 30.01.2012 2101318.1 2 The attachment of the label to the phosphate is a technique which is more complex than the technique consisting in functionalizing the base or the sugar and has been used to a much lesser extent, in particular due to the weak reactivity of the phosphate (see, for example, Jencks W.P. et al. J. Amer. Chem Soc., 82, 1778-1785, 1960). Similarly, in the review by 5 O'Donnel and Mc Laughlin (< Reporter groups for the analysis of nucleic acid structure n, p 216-243, in a Bioorganic Chemistry: Nucleic Acids >, Ed Hecht S.M., Oxford University Press, 1996) relating to methods for introducing probes into oligonucleotide fragments, effective alkylation of the internucleotide phosphodiester is considered to be impossible. 10 Patent application WO-A-99/65926 describes a method for labeling a synthetic or natural ribonucleic acid (RNA) which consists in fragmenting the RNA and in labeling at the end phosphate. This document describes a certain number of functions which can be used for the labeling in connection with the fragmentation, such as hydroxyl, amine, hydrazine, alkoxyamine, alkyl halide, benzyl-type alkyl halide, in particular the 5 15 (bromomethyl)fluorescein derivative. These functions make it possible to label nucleic acids, but it is necessary to combine a fragmentation step in order to have efficient labeling because this labeling takes place on the phosphate freed during fragmentation. Furthermore, a large excess of labeling reagent compared with the RNA must be added in order to obtain efficient labeling, which brings about problems of background noise generated by the excess label. 20 Finally, this method does not function efficiently on double-stranded DNA. There exists therefore a need for novel reagents which are efficient from the point of view of labeling yield, which are specific at the level of the labeling position and in particular which do not affect the properties of hybridization of the bases involved in the formation of the 25 double helix, by means of hydrogen bonds, which can be used both for DNA and RNA and, finally, which allow labeling, without distinction, of nucleotides, oligonucleotides or nucleic acids which may be natural or prepared by enzymatic amplification. The Applicant has already proposed such novel labels which satisfy the abovementioned 30 conditions and which use the diazomethyl function as reactive function for the labeling. This is, for example, the case in patent applications WO-A-02/090319 and WO-A-02/090584 or in 3 the article by Laayoun et al., published in Bioconjugate Chem. 2003, 14, 1298-1306 and entitled: "Aryldiazomethanes for Universal Labelling of Nucleic Acids and Analysis on DNA Chips", to which the reader may refer in order to understand the methods of synthesis and of use of such constituents more clearly. 5 Thus, the diazomethyl function (of formula -C(N 2 )-) has already been used for the aikylation of phosphate groups, but a certain number of problems arise. Firstly, reagents which incorporate at least one diazo function are generally unstable by themselves, which poses problems for the use of these reagents in a labeling kit, which is totally unacceptable if the 10 function of the labeled product is to demonstrate the presence of a target biological molecule in any sample. Finally, reagents bearing the diazomethyl function and associated with certain labels, such as biotin, are relatively water-insoluble, which leads to the use of organic solvents which are 15 water-miscible for the coupling with biological molecules, which are soluble only in water or aqueous buffers, but these solvents, present in high concentrations in the labeling reaction, slow down the reaction rate and therefore adversely affect the coupling efficiency. The discussion of documents, acts, materials, devices, articles and the like is included in this 20 specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 25 The labeling reagents recommended by documents WO-A-02/090319 and WO-A-02/090584 mentioned above also solve these technical problems. The content of these applications is incorporated herein for reference. However, even though these molecules and labeling methods are particularly efficient, the 30 Applicant has succeeded in finding novel molecules and novel methods which further improve the labeling efficiency. The invention consists of the use of polyaminated arms 4 which, like ethylene glycol arms, make it possible to distance the biotin from the reactive center (diazo function). Thus, a better solubility in aqueous medium is obtained through the introduction of the hydrophilic arm, with the possibility of protonation of the amines in an aqueous medium at neutral pH, which produces an attraction between the nucleic acids, 5 which are negatively charged, and the label, with two main consequences: * more rapid labeling, which may be particularly advantageous for samples at low concentration, and e stabilization of the double helix by neutralization of the negative charges of the 10 phosphates. Furthermore, these novel molecules make it possible to carry out methods which can function in an acidic medium, which is particularly advantageous for molecules which incorporate diazo functions. Thus, the selectivity of the reagents bearing a diazo function is therefore 15 greater in an acid medium. The solubility introduced into the polyamide chains therefore facilitates washing while at the same time decreasing the background noise during the subsequent detection, or even purely and simply elimination of the purification step. According to a first embodiment of the invention, the latter proposes a temperature-stable 20 labeling reagent of formula (0): R3 RR R 2 (Z -(CH 2 )p)m (L)& Y - X R A " R

N

2 in which: * Ri represents H or an alkyl, aryl or substituted aryl group, * R 2 represents a detectable marker or at least two detectable markers interlinked by at 25 least one multimeric structure, 5 * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, * R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L)n-Y X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3 5 CH 2

-NH-R

2

,-CO-NH-(CH

2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl, , e A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or 1, * -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, 10 e -Z- represents -NH-, -NHCO-, -CONH- or -0-, * m is an integer between I and 10, preferably between I and 3, and * p is an integer between 1 and 10, preferably between I and 3. According to a second embodiment of the invention, the latter relates to a labeling reagent, as 15 described in the first embodiment, of formula (1): R3

R-(NH-(CH

2 )p)m-(L)n-Y -xR (

N

2 in which: * R' represents H or an alkyl, aryl or substituted aryl group, * R 2 represents a detectable label or at least two detectable labels interlinked by at least 20 one multimeric structure, * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, * R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L)n-Y X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3 25 CH 2

-NH-R

2 , -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2 -NH- R 2 with R = alkyl or aryl, and 6 e -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, * m is an integer between I and 10, preferably between 1 and 3, and e p is an integer between I and 10, preferably between 1 and 3. 5 Advantageously, according to a variant of the first two embodiments, the value p is less than or equal to the value m in formula (0) or (1) of the reagent. According to a third embodiment, the present invention proposes a reagent, as described in the first and second embodiments, of formula (2): R' R'-(NH-CH2-CH 2 )q-(L)f--Y -X R

N

2 10 in which: * R' represents H or an alkyl, aryl or substituted aryl group, e R 2 represents a detectable label or at least two detectable labels interlinked by means of at least one multimeric structure, 15 0 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, * R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2

-(L),-Y-X

, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2 NH-R 2 , -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl, and 20 e q is an integer between 1 and 10, preferably between 1 and 3. According to a fourth embodiment, the present invention describes a reagent, as described in the first and second embodiments, of formula (3): 6a R 3

R-(NH--CH

2 -CH2) 3 -NH-CO-CH2-CH2-CO-NHR4 5 R

N

2 in which: * R 1 represents H or an alkyl, aryl or substituted aryl group, 5 * R 2 represents a detectable label or at least two detectable labels interlinked by means of at least one multimeric structure, e L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, and * R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2

-(L),-Y-X

10 , OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2 NH-R 2 , -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl.

7 According to a variant associated with the fourth embodiment of the invention, R 2 consists of a D-biotin residue of formula (4): 0 HN NH 0 S

(CH

2 4

-C

Advantageously, and whatever the embodiment previously mentioned for the reagent, R1 5 consists of : CH 3 , and R 3 and R 4 each represent: H. Advantageously, and whatever the embodiment or variant previously mentioned for the reagent is, the structure -(L),- consists of: * spermine or N,N'-bis(3-aminopropyl)-1,4-diaminobutane:

NH

2

-(CH

2

)

3

-NH-(CH

2

)

4

-NH

10 (CH 2

)

3

-NH

2 , or " spermidine or N-(3-aminopropyl)-1,4-butanediamine: H 2

N-(CH

2

)

4

-NH-(CH

2

)

3

-NH

2 , or " a derivative containing an alanine motif: NH 2

-CH

2

-CH

2 -COOH. According to a fifth embodiment of the invention, the latter also relates to a temperature 15 stable labeling reagent of formula (6):

N

2 II C R2-(Z-(CH 2 )p)m-(L)-YX (A)u '(A X - Y -(Ln-((CH 2 )p-Z)m-R2 R R in which: e R' represents H or an alkyl, aryl or substituted aryl group, * R 2 represents a detectable label or at least two detectable labels interlinked by at least one 20 multimeric structure, * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 8 " R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L)n-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2

-NH-R

2 ,

-CO-NH-(CH

2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl, " A is a linker arm comprising at least one covalent double bond enabling the conjugation of 5 the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or 1, " -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, " -Z- represents -NiH-, -NHCO-, -CONH- or -0-, e m is an integer between 1 and 10, preferably between I and 3, and 10 e p is an integer between 1 and 10, preferably between I and 3. According to a sixth embodiment, the invention proposes a temperature-stable labeling reagent of formula (7):

N

2 II C R2(Z-(CH 2 )p)m-(L)-Y-X X - Y-(L-((CH 2 )p-Z)m-R 2 15 in which: * R' represents H or an alkyl, aryl or substituted aryl group, SR2 represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an 20 integer equal to 0 or 1, * R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L).-Y-X-, OR, 2 SR, NR 2 , R; NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2 -NI-R, _

CO-NH-(CH

2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl, * -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, 25 * -Z- represents -NH-, -NHCO-, -CONH- or -0-, * m is an integer between 1 and 10, preferably between I and 3, and " p is an integer between I and 10, preferably between I and 3.

9 Advantageously, and whatever the embodiment or variant previously mentioned for the reagent, L comprises a motif -(O-CH 2

-CH

2 )-, repeated from I to 20 times, preferably from 1 to 10 times, and even more preferably from 2 to 5 times, -Z- then being represented by -NH-, NHCO- or -CONH-. 5 The invention also relates to a method for the synthesis of a labeling reagent, according to the above embodiments, comprising the following steps: a) a label or a label precursor having a reactive function R 6 is provided, b) a linker arm of formula (8): 10

R-(Z-(CH

2 )p)m-R is provided, in which formula: " -Z- represents -NH-, -NHCO-, -CONH- or -0-, " m is an integer between I and 10, preferably between I and 3, 15 e p is an integer between 1 and 10, preferably between 1 and 3, " R 7 and R 8 represent two reactive functions which may be identical or different, c) the reactive function R 6 of said label or label precursor and the function R 7 of the linker arm of formula (8) are reacted together in the presence of at least one coupling agent so as to form a covalent bond, R 6 and R 7 being complementary, 20 d) a derivative of formula (9): O RI (A)u R-(L)ny -X is provided, in which formula: 25 e R' represents H or an alkyl, aryl or substituted aryl group, 10 " L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, " R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L).-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2 -NH-R2, 5 -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl, " -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, * A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazomethyl function with the aromatic ring and u is an integer equal to 0 or 1, and * R9 represents a reactive function complementary to R, 10 e) the reactive function R9 of the derivative of formula (9) and the function R 8 of the linker arm of formula (8) are reacted together in the presence of at least one coupling agent so as to form a covalent bond, f) the hydrazine or one of its derivatives is reacted with the ketone or aldehyde function so as to form a hydrazone, and 15 g) the hydrazone is converted to a diazomethyl function by means of an appropriate treatment. Advantageously, the method of synthesis can also comprise: * an additional step consisting of protection of the ketone or aldehyde function of compound (9), and 20 * a subsequent additional step consisting of deprotection of said ketone or aldehyde function. The invention also relates to a method for the labeling of a biological molecule, in particular a nucleic acid, comprising bringing into contact, in homogeneous solution, in a substantially aqueous buffer, a biological molecule and a reagent, according to the embodiments mentioned 25 above. The invention also relates to a labeled biological molecule which can be obtained by means of the method, according to the labeling claim above. 30 The invention also relates to a method for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid, comprising the following steps: 11 * fragmenting the nucleic acid, " attaching a label to at least one of the fragments by means of a labeling reagent chosen from the reagents according to the embodiments mentioned above, said reagent coupling covalently and predominantly on at least one phosphate of said 5 fragment. Advantageously, the labeling and fragmentation method is carried out using a labeling reagent which is chosen from the compounds of formula (3): R3

R-(NH-CH

2

-CH

2

)

3

-NH-CO-CH

2

-CH

2 -CO-NH R

N

2 10 in which: " R represents H or an alkyl, aryl or substituted aryl group, " R2 represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, " L is a linker arm comprising a linear chain of at least two covalent bonds and n is an 15 integer equal to 0 or 1, and " R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L)n-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2

)

3

-(O-CH

2

-CH

2

)

3

-CH

2 -NH-R2,

CO-NIH-(CH

2

)

3

-(O-CH

2

-CH

2

)

4

-CH

2

-NH-R

2 with R = alkyl or aryl. 20 According to a first variant of implementation of the labeling and fragmentation method, the fragmentation and the labeling are carried out in two steps. According to a second variant of implementation of the labeling and fragmentation method, the fragmentation and the labeling are carried out in one step. 25 Whatever the labeling and fragmentation method, the labeling is carried out in a substantially aqueous homogeneous solution.

12 Whatever the labeling and fragmentation method, the fragmentation is carried out via an enzymatic, physical or chemical process. The present invention also relates to any labeled nucleic acid which can be obtained by means 5 of the labeling and fragmentation method above. The present invention also relates to a kit for the detection of a target nucleic acid, comprising a labeled nucleic acid as defined above. 10 The present invention always relates to a solid support to which is attached at least one reagent as defined above. Finally, the present invention relates to a method for the capture of nucleic acids, comprising the following steps: 15 e providing a solid support to which is directly or indirectly attached at least one biological molecule, defined above, or a nucleic acid, also defined above, the biological molecule or the nucleic acid comprising a diazomethyl function, * bringing into contact a biological sample which may contain free nucleic acids, and * washing the solid support where the molecule(s) is(are) covalently attached at least to one 20 nucleic acid. The term "multimeric structure" is intended to mean a polymer formed of repeat units of chemical or biological synthons. An example is mentioned in example 34.2 of the description of patent application WO-A-02/090319. Those skilled in the art are requested to refer to this 25 document should they find the information developed hereinafter insufficient for their complete understanding of this subject. Many variants of such structures which can be used in the present invention are known, such as, for example: " linear polymers (EP-A-0.561.722, EP-A-0.669.991), " branched polymers (WO-A-01/92361), 30 * particles (EP-A-0 827 552), " dendrimers (US-A-4,507,466; US-A-4,568,737; US-A-6,083,708), 13 " polynucleotides, and " polypeptides. Should it prove necessary, those skilled in the art can also refer to these documents for a complete understanding of the subject. 5 The term "detectable label" is intended to mean at least one label capable of directly or indirectly generating a detectable signal. A nonlimiting list of these labels follows: " enzymes which produce a detectable signal, for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, p-galactosidase or 10 glucose-6-phosphate dehydrogenase, " chromophores, such as fluorescent, luminescent or dye compounds, " groups with an electron density detectable by electron microscopy or by virtue of their electrical property, such as conductivity, amperometry, voltametry or impedance, " detectable group, for example the molecules of which are sufficiently large to induce 15 detectable modifications of their physical and/or chemical characteristics; this detection can be carried out by optical methods such as diffraction, surface plasmon resonance, surface variation or contact angle variation, or physical methods such as atomic force spectroscopy or the tunnel effect, e radioactive molecules such as 32 P, 35 S or 1251. 20 Preferably, the label is not a radioactive label, in order to avoid the safety problems associated with these labels. In one specific embodiment of the present invention, the label is electrochemically detectable, and in particular the label is a derivative of an iron complex, such as ferrocene. 25 Indirect systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand/anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/sequence complementary to 30 the polynucleotide. In this case, it is the ligand which bears the diazomethyl reactive function. The anti-ligand can be directly detectable by means of the labels described in the above 14 paragraph or can itself be detectable by means of another ligand/anti-ligand pair. This stacking system is illustrated in the examples. Another example of indirect systems uses a specific covalent bond between the ligand and the 5 anti-ligand, for example methyl ketone and alkoxyamine. Examples of this system are described in patent applications WO-A-00/40590 and WO-A-98/05766. These indirect detection systems can produce, under certain conditions, an amplification of the signal and reference may be made to the prior patent applications WO-A-00/07982, WO-A-01/92361 and WO-A-95/08000 for examples of chemical amplification using polymers, or to application 10 WO-A-01/44506 for systems of chemical amplification by stacking. In one specific embodiment of the signal amplification, at least two labels are present on the labeling reagent. 15 In one preferred embodiment of the invention, the tracer is a fluorescent compound with low steric hindrance, such as fluoresceine, hexachlorofluoresceine (HEX), dansyl (edans), rhodamine, tetramethylrhodamine (5- or 6-TAMRA), carboxy-X-rhodamine (ROX), chromophores of the NIR type (LI-COR Inc, Lincoln NE, USA), cyanine derivatives such as Cy5 and Cy3 (Randolph J.B. el al., Nucleic Acids Res., 25(14), p2923-2929, 1997), and in 20 particular Cy5 derivatives, or else the tracer is a hapten with low steric hindrance, such as biotin dinitrophenyl, or an abietane derivative (see application WO-A-00/07982). The term "low steric hindrance" is intended to mean a molecular weight of less than 1000 g/mol. In the case of a fluorophore, it is preferable to work with fluorophores for which the excitation 25 wavelength is greater than 450 nm, preferably greater than 600 nm. When the tracer is a hapten which does not produce any signal by itself, for instance biotin, the detection is carried out by means of the recognition of a labeled anti-ligand as described above. In the case of biotin, use is preferably made of streptavidin or an anti-biotin antibody 30 coupled to a fluorescent compound such as fluoresceine, Cy5 or phycoerythrin. in the case of abietane, a monoclonal antibody as described in patent application WO-A-00/07982 is used.

15 In particular, the labeling reagents of the invention are soluble in polar solvents such as DMF, DMSO, CH 3 CN, THF, DMA (dimethylacetamide), NMP (N-methylpyrrolidone) or DME (dimethoxyethane). 5 The labeling reagents are preferably soluble in DMSO or water. The term "water-miscible solvent" is intended to mean a solvent which is miscible in a proportion of at least 5% by volume with water or an aqueous buffer containing salts. 10 Advantageously, in the above formulae, the arm L comprises an ethylene glycol or polyethylene glycol motif in order to increase the water-solubility of the reagent. A is a linker arm comprising at least one double bond of ethylenic type enabling the 15 conjugation of the diazomethyl function with the aromatic ring. The function of the linker arm A is to distance the diazomethyl function from the ring in order to decrease the steric hindrance while at the same time conserving the stability of the diazomethyl function. The term "conjugation" is intended to mean electron delocalization of the aromatic ring along the carbon chain of the linker arm. By way of example, the arm A can have the following 20 structure:

-

r-RN - Rio -- CH=CH or -C=CH-_ o -C=CH CH=CH -- v L - v1 in which: * v is an integer of between 1 and 10, preferably v is I or 2, and a R1 0 is H or an alkyl group, preferably R1 0 is H, methyl or ethyl. 25 These reagents can thus attach in the homogeneous phase to the biological molecules, the homogeneous phase consisting of a substantially aqueous solution, i.e. a solution containing at least 50% of water.

16 The term "biological molecule" is intended to mean a compound which has at least one recognition site allowing it to rcact with a target molecule of biological interest. By way of example of biological molecules, mention may be made of nucleic acids, antigens, antibodies, polypeptides, proteins, haptens. 5 The term "nucleic acid" means a chain of at least two deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example at least one nucleotide comprising a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5 deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine, or any other 10 modified base allowing hybridization. This polynucleotide can also be modified at the level of the internucleotide bond, such as, for example, phosphorothioates, H-phosphonates or alkyl phosphonates, or at the level of the backbone, such as, for example, alpha-oligonucleotides (FR 2 607 507) or PNA (M. Egholm et al., J. Am. Chem. Soc., 114, 1895-1897, 1992) or 2' 0-alkyl riboses. The nucleic acid can be natural or synthetic, an oligonucleotide, a 15 polynucleotide, a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, or a nucleic acid obtained by means of an enzymatic amplification technique, such as: " PCR (Polymerase Chain Reaction), described in patents US-A-4,683,195, US-A-4,683,202 and US-A-4,800,159, and its derivative RT-PCR (Reverse Transcription PCR), in particular in a one-step format, as described in patent EP-B-0.569.272, 20 * LCR (Ligase Chain Reaction), disclosed, for example, in patent application EP-A 0.201.184, * RCR (Repair Chain Reaction), described in patent application WO-A-90/01069, " 3SR (Self Sustained Sequence Replication) with patent application WO-A-90/06995, " NASBA (Nucleic Acid Sequence-Based Amplification) with patent application WO-A 25 91/02818, " TMA (Transcription Mediated Amplification) with patent US-A-5,399,491, and " RCA (Rolling Circle Amplification (US-6,576,448). The term "amplicons" is then used to denote the nucleic acids generated by an enzymatic 30 amplification technique.

17 Each of these modifications can be taken in combination as long as at least one phosphate is present in the nucleic acid. The term "polypeptide" is intended to mean a chain of at least two amino acids. 5 The term "amino acids" is intended to mean: * the primary amino acids which encode proteins, e the amino acids derived after enzymatic action, such as trans-4-hydroxyproline, * amino acids which are natural but are not present in proteins, such as norvaline, N-methyl L leucine, staline (see Hunt S. in Chemistry and Biochemistry of the amino acids, Barett 10 G.C., ed., Chapman and Hall, London, 1985), and * amino acids protected by chemical functions which can be used in solid-support or liquid phase synthesis, and unnatural amino acids. The term "hapten" denotes nonimmunogenic compounds, i.e. compounds incapable by 15 themselves of promoting an immune reaction through the production of antibodies, but capable of being recognized by antibodies obtained by immunization of animals under known conditions, in particular by immunization with a hapten-protein conjugate. These compounds generally have a molecular mass of less than 3000 Da, and most commonly less than 2000 Da and may for example be glycosylated peptides, metabolites, vitamins, hormones, 20 prostaglandins, toxins or various medicaments, nucleosides and nucleotides. The term "antibody" includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments such as Fab or F(ab') 2 . 25 The term "antigen" denotes a compound capable of generating antibodies. The term "protein" includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and glycoproteins, both fibrous and globular in their characteristic conformational form. 30 Advantageously, the biological molecule has a phosphate group, i.e. a group having at least one motif: 18 0- 0 -O-P-0- or -0-P-O' II I o 0 which is either naturally present in the biological molecule, or can be introduced, for example, by chemical or enzymatic modification. Examples of chemical modification for proteins are 5 given in "Chemistry of protein conjugation and cross linking", S.S. Wong, CRC Press, 1991. Preferably, the biological molelcule is a nucleic acid. Certain advantageous reagents of the invention are: 10 a) of formula (10): 0 R2-(L)n-C-NH Nj

N

2 b) of formula (11): 0 R2-(L),-C-NH R

N

2 c) of formula (12): 15 R2--(L)a---NH K 15 N 2 in which: " R represents H or an alkyl, aryl or substituted aryl group, " R2 represents a detectable label or at least two detectable labels interconnected by at least one multimeric structure, 20 * L is a linker arm comprising a linear chain of at least two covalent bonds, and 19 n is an integer equal to 0 or 1. Preferably, the labeling reagent has the: a) formula (13): 0 0 11 NH N2-N b) formula (14): 0 00 -- NH H H R1 N2 c) formula (15): 0 0 H HN~/.yCN 0 0i 10 in which R' represents a methyl or phenyl group. Whatever the variant and the embodiment of the reagent, L can comprise a motif -(NH-CH 2 CH 2 )-, repeated from I to 20 times, preferably from 1 to 10 times, and even more preferably from 2 to 5 times. 15 The term "hydrazine derivative" is intended to mean a molecule having the NH 2

-NH

function. Tosylhydrazine is an example of such a derivative.

20 The conversion of hydrazone to diazomethyl is carried out by the usual methods, in particular oxidation with MnO 2 . Other methods can be used, as described in X. Creary, Organic Syntheses, Wiley: New York, 5 Coll. Vol. VII, p438-443, 1990; H. Zollinger, Diazo Chemistry II, VCH, Weinheim, p34 47, 1995; T. L. Holton and H. Shechter, J. Org. Chem., 60, 4725-4729, 1995. In the case of the use of a tosylhydrazine derivative, the method is described in X. Creary, Organic Syntheses; Wiley: New York, Coll. Vol. VII, p438-443, 1990. 10 In one specific embodiment of any one of the methods of synthesis, said method comprises: " an additional step consisting of protection of the ketone or aldehyde function (where R' is H) of compound (9), and " a subsequent additional step consisting of deprotection of said ketone or aldehyde function. 15 This protection is carried out by means of an acetal group, for example. The deprotection is carried out by an appropriate means, such as in an acidic medium for the acetal group. Those skilled in the art will determine, according to the compounds, at which step of synthesis these two protection and deprotection steps occur. 20 In the case of signal amplification, the method of synthesis is similar to those mentioned above. The label precursor can have formula (17) below

GPI-NH-(CH

2 )p COOH

GP

2 -NH in which GPI and GP 2 represent two amine function-protecting groups, which may be 25 identical or different, and p is an integer between I and 10, advantageously 2 and 6, preferably 4. Advantageously, GPI and GP 2 are different in order to be able to add several motifs, as explained below. Examples of protective groups GP1 or GP2 which can be used in the present invention are 30 given in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, 21 John Wiley and Sons, New York, 1991, preferably those commonly used in peptide synthesis, such as Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenylmethyleneoxycarbonyl), Cbz (carboxybenzyl) or Alloc (allyloxycarbonyl). 5 In particular, GPI and GP2 are, respectively, the protective groups Boc and Fmoc. The reaction between this precursor which has a carboxyl function and the derivative of formula (18), hereinafter, is carried out in the presence of a coupling agent so as to form the amide bond. O Ri 10 A NH 2 After deprotection, under usual conditions, of one of the two protective groups, for example Fmoc, with a base such as piperidine, the freed amine function is used to couple another molecule of formula (17). This process is repeated as many times as necessary to obtain a multitide of NH 2 functions protected with a protective group, for example a Boc function. 15 The motif is added between one (I) and a hundred (100) times, preferably between one (I) and twenty (20) times. Hydrazine is reacted with the ketone function originating from the phenyl ketone derivative so as to form a hydrazone, and then oxidation is carried out in the presence of MnO 2 so as to 20 form a diazomethyl residue. Then, after deprotection of the amine function bearing the Boc group, a tracer, for example a biotin activated with an N-hydroxysuccinimide group, is coupled to the amine functions so as to produce a reagent in which the motif R 2 -(L),- is that of formula (5). 25 The invention is also directed to a method, and also the products obtained by means of this method, for the labeling of a biological molecule, in particular a nucleic acid, 22 comprising bringing into contact in solution, in a substantially aqueous homogeneous solution, a biological molecule and a labeling reagent according to the invention. The term "substantially aqueous solution", is intended to mean a solution containing at least 5 50% of water. This solution preferably contains salts, such as a buffer solution. The term "homogeneous solution" is intended to mean a single-phase solution such as a water/DMSO solution, as opposed to a two-phase solution such as a water/chloroform solution. 10 The specific conditions for the labeling reactions vary according to the biological molecules and to the label. As regards nucleic acids, a pH of between 5 and 8 allows efficient labeling. In particular, a pH of between 5.5 and 7.0 is preferred for all the reagents of the invention. With the reagent of formula (11), the pH range is broader for the labeling. Good labeling efficiency 15 is obtained for a pH of between 3 and 8 for this reagent. This labeling and fragmentation method is particularly useful when the labeled nucleic acid must hybridize with a multitude of nucleic acids, in particular oligonucleotides, attached to the solid support at a predetermined position so as to form a DNA chip. The term "DNA chip" is 20 intended to mean a solid support of small size, to which is attached a multitude of capture probes at predetermined positions. Specifically, the density of the nucleic acids attached to the solid support imposes considerable steric constraints during hybridization and fragmentation makes it possible to improve this hybridization step. Examples of these DNA chips are given, for example, in the publications by G. Ramsay, Nature Biotechnology, 16, p40-44, 1998; 25 F. Ginot, Human Mutation, 10, pl-10, 1997; J. Cheng et al., Molecular diagnosis, 1(3), p183 200, 1996; T. Livache et al., Nucleic Acids Research, 22(15), p2915-2921, 1994; J. Cheng et al., Nature Biotechnology, 16, p541-546, 1998. The fragmentation and the labeling are carried out in one step or in two steps and the labeling 30 can be carried out, without distinction, before, after or simultaneously with the fragmentation.

23 Preferably, the labeling and fragmentation are carried out simultaneously, i.e. the reagents required for these two steps are placed together in a substantially aqueous homogeneous solution with the nucleic acid, for example. This is in particular the case for chemical or enzymatic fragmentation. In the case of mechanical fragmentation by a physical means, 5 "labeling and fragmentation being carried out simultaneously" signifies that the physical means is applied to a substantially aqueous homogeneous solution containing at least the nucleic acids and the labeling reagent. The fragmentation of the nucleic acid is carried out by an enzymatic, chemical or physical 10 process. The fragmentation of the nucleic acid by an enzymatic process is carried out, for example, with nucleases. 15 The fragmentation of the nucleic acid by a physical process is carried out, for example, by sonication or by radiation. The fragmentation by a chemical process, if the nucleic acid is an RNA, is carried out by the usual methods (see, for example, Chem. Rev, 98, 961-990, 1998 by Oivanen M. et al.). 20 Metal complexes, as described in the review by G. Pratviel et al., Adv. Org. Chem., 45, p 2 51 312, 1998 or the review by G. Pratviel et al., Angew. Chem. Int. Ed. Engl., 34, p746-769, 1995, can be used for the fragmentation of DNA or RNA. 25 In a first embodiment, the chemical fragmentation of RNA is carried out with metal cations possibly associated with a chemical catalyst. In this case, the metal cations are Mg2+, Sr 2 +, Ba2+, Pb2+, Zn2+, Cd2+, Mn 2+ Fe2+, C02

+

, Ni 2 +, Ru'*, Ce'*, Eu'*, Tb*, Tm *, Yb 3 3or Lu3* ions, the chemical catalyst consists of imidazole, a substituted analog, for example, N-methyl imidazole, or any chemical molecule having an affinity for RNA and bearing an imidazole 30 ring or a substituted analog. The conditions for fragmentation using metals are well described 24 in patent application WO-A-99/65926. Advantageously, the metals are Mg 2 +, Mn2, Zn 2 + Tb 3 or Ce 3 *, preferably Mg 2+, Mn2+, Zn2+. Efficient fragmentation conditions are obtained with a concentration of metal cation such as 5 Mn** of between 2 and 100 mM, and a concentration of imidazole of between 2 and 100 mM. Especially efficient conditions are obtained with a concentration of cation such as Mn* of between 3 and 15 mM, and a concentration of imidazole of between 20 and 50 mM, in particular 30 mM. 10 The pH of the reaction must be slightly basic. Advantageously, the pH is between 8.5 and 9, which represents a very advantageous compromise for carrying out the labeling and fragmentation combination with RNA. 15 In a second embodiment, the chemical fragmentation of RNA is carried out by the action of a polyamine, such as spermine, putrescine or cadaverine. Concentrations of 5 to 100 mM allow fragmentation. The latter is complete starting from 10 mM of polyamine. In a third embodiment, the chemical fragmentation of RNA is carried out by the action of an 20 artificial nuclease (see G. Pratviel et al., Adv. Inorg. Chem.,45, p 2 5 1-312, 1998; D. S. Sigman et al. Chem. Rev., 93, p 2 2 95

-

2 3 16, 1993), such as 1,10-phenanthroline associated with a metal cation such as iron, copper or zinc. These cations originate, respectively, from FeSO 4 , CuCl 2 or ZnCl 2 in solution. Concentrations of between 2 and 50 mM of 1,10-phenanthroline are used for RNA fragmentation, in particular of between 4 and 10 mM. 25 The fragmentation of DNA by a chemical process is carried out by bringing the nucleic acid together with a chemical means of creating an abasic site. The formation of an abasic site results from cleavage of the N-glycoside bond which links the 2-deoxyribose sugar to the nucleic base. This involves a depurination via the loss of a negatively charged purine 30 (guanine, adenine), or a depyrimidination in the case of the loss of a pyrimidine (cytosine, thymine), which is positively charged.

25 This depurination is spontaneous under physiological conditions (pH 7.4 at 37*C), but the reaction rate is very low, of the order of 3x10." depurination per second, i.e. unusable for efficient fragmentation. In order to increase the reaction rate, alkylating agents which make 5 the N-glycoside bond fragile are used or uracil-DNA glycosylases are used on DNAs which incorporate uracils. The abasic site obtained by depurination or depyrimidation is very unstable. The fragmentation at this site is obtained at ambient temperature in a basic medium. In acidic 10 medium, high temperature also accelerates this fragmentation. The use of molecules capable of initiating the p-elimination phenomenon also accelerates fragmentation. A preferred embodiment of the fragmentation is obtained by using an acidic pH, i.e. a pH of less than 5. Advantageously, the pH is 3. 15 A sodium formate buffer at pH 3 makes it possible to efficiently fragment according to the invention. This buffer is compatible with the conditions for one-step labeling as will be demonstrated in the examples. Even more advantageously, an acidic medium (HCI, carbonate,

H

2

SO

4 ) is used. 20 In one specific embodiment of the present invention, and with the aim of further increasing the fragmentation, the deoxyribonucleic acid contains at least one modified base capable of generating an abasic site more readily. 25 Various modified bases can be used, such as N7-alkyl purines, N3-alkyl purines, 06-alkyl purines, 8-bromopurines, 8-thiopurines, 8-alkylthiopurines, 8-azidopurines or 8 alkylsulfonylpurines. When the nucleic acid to be labeled is generated by an enzymatic amplification technique 30 such as PCR, the use of an 8-bromopurine makes it possible to have efficient incorporation during the amplification, which facilitates accordingly the fragmentation and labeling method 26 according to the invention, while at the same time conserving excellent sensitivity for the enzymatic amplification step. The present invention describes a labeled biological molecule, and in particular a labeled 5 nucleic acid, which can be obtained by means of any one of the methods according to the invention. The present invention also relates to a kit for the detection of a biological molecule, in particular a target nucleic acid, comprising a labeling reagent according to the invention. 10 Depending on the uses of the kit, other elements, such as, for example lysis means (microorganisms and/or cells) and/or concentration means (such as silica or magnetic particles) and/or enzymatic amplification means are incorporated into the kit. The invention relates to the use of a labeled biological molecule, in particular a labeled 15 nucleic acid as defined above, as a probe for the detection of a target biological molecule, and in particular of a target nucleic acid. The invention also relates to the use of a nucleic acid as defined above, as a labeled target which can attach to a capture probe. 20 In order to allow the detection and/or the quantification and/or the purification of the target biological molecule, the labeled biological molecule is capable of forming a complex with.the target biological molecule. By way of example, for demonstrating a target molecule of nucleic acid type, the labeled nucleic acid is sufficiently complementary to the target to hybridize 25 specifically according to the reaction conditions, and in particular the conditions of temperature or of salinity of the reaction medium. The detection method is applicable for sequencing, the expression profile of messenger RNAs or the screening of mutations for research purposes and also the screening of drugs in the 30 pharmaceutical industry, the diagnosis of infectious or genetic diseases, or food product or industrial controls.

27 The tendency in terms of diagnosis, and in particular for infectious diseases (AIDS or tuberculosis, for example), is to reduce the level of sensitivity, until a single molecule is detected in a sample, which can represent several milliliters in the case of a liquid sample such 5 as blood, urine or cerebralspinal fluid. This level of sensitivity can also be obtained if all the steps from the taking of the sample to the delivery of the result, are optimized. The various means of the invention allow this optimization without difficulty because the reagents, methods and methods of the invention are very broadly applicable to various biological molecules. In particular, when an enzymatic amplification step is necessary in order to obtain 10 the required sensitivity (viral or bacterial infection such as HIV, HCV or tuberculosis), a labeling and/or fragmentation method, as described in the present invention, makes it possible not to affect the sensitivity of the amplification technique, either because it is not necessary to replace the deoxyribonucleotides or the ribonucleotides used in the enzymatic amplification technique, or because the ribonucleotides or deoxyribonucleotides incorporated do not impair 15 the sensitivity. The grafting chemistry described in the present invention has characteristics such, from the reactivity and specificity point of view, that other applications are described hereinafter: e In a first embodiment, this grafting chemistry is applied to the covalent attachment of 20 nucleic acids to a solid support. * In a first variant of the method, a precursor of the diazomethyl function, such as a ketone or a hydrazine as described above, is introduced during the chemical synthesis and the diazomethyl function is introduced onto the nucleic acids in a second step. * In a second preferred variant of the method, the diazomethyl functions are introduced onto 25 the solid support and the nucleic acids are attached to the solid support by means of the phosphates of the nucleic acids, and in particular of the end (5' or 3') phosphates. The introduction of phosphate at the 3' or 5' end of nucleic acids is known (see "Protocols for Oligonucleotides and Analogs, Synthesis and Properties" edited by S. Agrawal, Humana 30 Press, Totowa, New Jersey).

28 In one specific embodiment of such a solid support, a labeling reagent bearing a ligand, in particular a hapten such as biotin or abietane, is attached to the solid support to which an anti ligand, such as streptavidin or an antibody, for example, is attached covalently or by adsorption. These solid supports are well known in the prior art and are even commercially 5 available (microtitration plate-streptavidin or latex-streptavidin, for example). The function of the label is no longer, in this case, to allow detection, but to allow attachment of the labeling reagent to the solid support. The diazomethyl function is then available to react with nucleic acids. The derivatives of formulae (13), (14) and (15) or the PDAM derivative are examples of reagents which can be used for the fabrication of such a solid support. The monoclonal 10 antibody technique makes it possible to prepare antibodies against a large number of labels such as fluorescein or a Cy5 derivative. Those skilled in the art can use a solid support with the labeling reagents of the present invention without excessive difficulty through this method of indirect preparation of the solid support in which a ligand/anti-ligand reaction is used to attach the diazomethyl function to the solid support. 15 A second embodiment of the solid support concerns particulate supports such as latexes. Various methods of polymerization can be used to prepare the particles bearing a diazomethyl function from a polymerizable functional monomer bearing either a diazomethyl function or preferably a precursor function of the diazomethyl function, such as an aldehyde or a ketone, 20 and in particular: e Closed-reactor polymerization termed "batch": the monomers are introduced into the reactor, before the beginning of the reaction, with the other ingredients and without subsequent addition. Because of the difference in reactivity of the monomers, this method often leads to the appearance of a drift in composition. This manifests itself through the 25 production of macromolecules having compositions which vary considerably as a function of the conversion. This method is relatively inefficient for surface incorporation, since there is a risk of a considerable portion of the functional monomer being lost either inside the particles, or in the form of water-soluble polymer. When the copolymerization is performed by "batch" with monomers of a polar nature, smaller particles are obtained, in 30 large number, but with a limited conversion. This behavior is linked to the considerable 29 water-solubility of these monomers, and it is attributed to the predominance of the homogeneous nucleation mechanism. . Semicontinuous polymerization: a portion at least of the monomers is introduced into the reactor over a period between the start of the reaction and the end of the latter. This 5 addition can be carried out at a fixed rate or else following a given profile. The aim is to control the addition of the monomer mixture in such a way as to obtain a copolymer of controlled composition (control of the composition of the interface); thus, addition conditions are often obtained such that the rate of polymerization is faster than the rate of addition. 10 * Polymerization by deferred addition termed "shot": once the polymerization reaction is in progress, the functional monomer alone, or in the presence of the basic monomer, is introduced into the system in a controlled manner. The success of the process therefore depends on the degree of prior knowledge of the kinetics of copolymerization. It is an efficient method for promoting surface incorporation. The selection of the experimental 15 conditions (degree of conversion at the time of addition, composition and concentration of the monomer mixture) makes it possible to optimize the surface yields. e Seed polymerization: it consists in introducing the functional monomer into the system containing a latex which is already constituted and completely characterized. The functional monomer can be added alone or as a mixture with the basic monomer of the 20 seed, in one step or semicontinuously. The seed polymerization, shot polymerization and semicontinuous polymerization techniques are preferred since they result in a maximum of incorporation of the derivative bearing the precursor of the diazomethyl function at the surface. Examples of particles bearing aldehyde 25 functions are given, for example, in B. Charleux et al., Die Makromolecular Chem., 193, p 187 and p. 205, 1992, or in patent EP-B-0.350.407. A third embodiment of the solid support consists in providing a solid support comprising a first nucleophilic or electrophilic reactive function, such as, for example, NH 2 , SH, OH, 0 30 NH 2 , alkyl ketone, aldehyde, isocyanate, isothiocyanate, maleimide, alkyl halide, N hydroxysuccinimide ester, or tosylate, and then in reacting an attachment intermediate, 30 comprising a reactive function complementary to the first reactive function of the solid support. This reaction between the solid support and the attachment intermediate is carried out in the presence, optionally, of a coupling agent so as to form a covalent bond. 5 Such a solid support comprising at least one diazomethyl function, according to the various embodiments described above, in particular a solid support to which is indirectly attached a labeling reagent of the invention, is also a subject of the present invention, as is the solid support comprising nucleic acids attached to the solid support by means of the diazomethyl functions. 10 A first application of such a solid support is the fabrication of DNA chips. Methods exist for distributing nucleic acids on the solid support in discrete and predetermined positions. Patent US-A-6,110,426 proposes a method for producing these DNA chips using a capillary, 15 which is brought into contact on a solid surface so as to deliver a controlled volume of liquid. An effective contact takes place between the end of the capillary and the solid support so that the drop is deposited by capillary action. Similarly, patent US-A-6,083,763 describes a set of capillaries which slide in a device so as to compensate for the differences in height of each of them. They are brought into contact with a planar surface for the deposition, by capillary 20 action, of specific oligonucleotides. Patent US-A-6,083,762 proposes a drop distribution system comprising a microdispenser coupled to a piezoelectric transducer for ejecting drop volumes of less than a nanoliter onto a solid surface. A similar result is obtained by applying a hot source to the wall of a capillary so 25 as to form a bubble which ejects a defined volume of solution (see T. Okamoto el al., Nature Biotechnology, 18, p438-441, 2000). The diazomethyl function thus makes it possible to covalently graft the nucleic acids onto the support. The grafting is simple, the bonding is stable, compared with adsorption in particular, 30 and the selectivity of the reaction with respect to the end phosphate makes it possible to 31 perform an oriented coupling of the nucleic acid on the solid support, which facilitates accordingly the subsequent hybridization steps by decreasing the steric hindrance. A second application of a solid support according to the invention is the purification of 5 nucleic acids. In the case of purification, this purification is either direct (the solid support bearing diazomethyl functions reacts with the nucleic acids to be purified) or indirect (capture nucleic acids are attached to the solid support). These capture nucleic acids are sufficiently 10 complementary to the target to be captured so that they hybridize with the desired degree of specificity, and it is the "capture nucleic acids/solid support" complex which enables the purification of the target nucleic acids. The solid support is preferably dispersed for the use in purification, such as latex particles, for 15 example magnetic particles. The term "purification step" is intended in particular to mean the separation of the nucleic acids from the microorganisms and the cellular constituents released in the lysis step which precedes the nucleic acid purification. These lysis steps are well known; as an example by way 20 of indication, use may be made of the lysis methods as described in patent applications: - WO-A-00/60049 regarding lysis by sonication, - WO-A-00/05338 regarding mixed magnetic and mecahnical lysis, - WO-A-99/53304 regarding electrical lysis, and - WO-A-99/15621 regarding mechanical lysis. 25 Those skilled in the art may use other well known lysis methods, such as heat shock or osmotic shock, or treatments with chaotropic agents, such as guanidium salts (patent US-A 5,234,809). This step generally makes it possible to concentrate the nucleic acids. By way of example, use 30 may be made of magnetic particles (in this respect see patents US-A-4,672,040 and US-A 5,750,338), and it is thus possible to purify the nucleic acids, which have attached to these 32 magnetic particles, by means of a washing step. This nucleic acid purification step is particularly advantageous if it is desired to subsequently amplify said nucleic acids. A particularly advantageous embodiment of these magnetic particles is described in patent applications WO-A-97/45202 and WO-A-99/35500. 5 The term "solid support" as used here includes all materials to which a nucleic acid can be attached. Synthetic materials or natural materials, optionally chemically modified, can be used as a solid support, in particular polysaccharides, such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate or nitrocellulose, or dextran; 10 polymers, copolymers, in particular based on styrene-type monomers, natural fibers such as cotton, and synthetic fibers such as nylon; mineral materials such as silica, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels, etc. The solid support can be in the form of a microtitration plate, of a membrane, of a particle or of a substantially planar glass or silicon plate, or derivatives. 15 Finally, the invention relates to a method for the capture of nucleic acids, comprising the following steps: " providing a solid support to which is directly or indirectly attached at least one molecule comprising a diazomethyl function, 20 * bringing into contact a biological sample which may contain free nucleic acids, and " washing the solid support where the molecule(s) is (are) covalently attached at least to a nucleic acid. Additional information can be found in another patent application by the Applicant, 25 W002/090584, filed under the priority of May 4, 2001. The attached examples and figures represent specific embodiments and cannot be considered to limit the scope of the present invention. 30 Figure 1 represents the structural formulae of various reagents used in the present invention and also the abbreviation denoting them (o- signifies ortho, m- meta and p- para).

33 Figure 2 represents the average value of the signal and the percentage similarity of m-bio TETA-PMDAM as a function of its concentration for rpoB. 5 Example 1: Synthesis of the reference reagent: meta-BioPMDAM: e Biotin meta-acetophenone compound la: 10 D-Biotin (1.0 gram (g), 4.1 millimol (mmol)) is solubilized in 45 milliliters (ml) of anhydrous DMF under hot conditions. The product is cooled to 0*C under argon, and then N methylmorpholine (590 microliters (pd), 5.33 mmol) and isobutyl chloroformate (840 1d, 6.60 mmol) are added successively. The mixture is left to stir for 30 minutes (min), and then 3-aminoacetophenone (824 mg, 6.10 mmol) and N-methylmorpholine (480 1, 4.35 mmol) in 15 10 ml of DMF are added. The solution is kept stirring at 0*C for 2 hours (h), and then evaporated to dryness. The residue is taken up in 3 ml of MeOH, and then 50 ml of water are added. The precipitate obtained is filtered off, and washed with water, CH 2 C1 2 and ether, to give 1.2 g (80%) of crude product la. Recrystallization from the couple MeOH-H 2 0 gives Ia (1.01 g, 70%) in the form of a white powder. 20 Mp 145*C. - IR (KBr): 3280, 2931, 2857, 1691, 1590, 1540, 1487, 1434, 1298, 1266 cm'. 1 H NMR (300 MHz, DMSO-d 6 ) 8 = 1.3-1.7 (m, 6 H); 2.33 (t, J = 8 Hz, 2 H); 2.55 (s, 3 H); 2.58; (d, J= 12 Hz, 1 H); 2.83 (dd, J= 12 and 5 Hz, 1 H); 3.13 (m, I H); 4.15 (m, I H); 4.31 (m, I H); 6.34 (s, 1 H); 6.41 (s, 1 H); 7.44 (t, J = 8 Hz, 1 H); 7.64 (d, J = 8 Hz, 1 H); 7.85 (d, J= 8 25 Hz, I H); 8.17 (s, I H); 10.05 (s, I H). - MS (FAB/glycerol), m/z: 362 [M+H) e meta-Hydrazone compound 2a: A solution of Ia (500 mg, 1.38 mmol) and of hydrazine monohydrate (200 pl, 4.15 mmol) in 30 absolute ethanol (8 ml) is refluxed for 2 h. After cooling to ambient temperature, the white 34 precipitate is filtered off, washed with water and then with ether, and dried. 385 mg (74%) of product 2a are thus obtained in the form of a white powder. Mp 185"C. - IR (KBr): 3298, 2931, 2857, 1698, 1665, 1626, 1541, 1494, 1470, 1446, 1330, 5 1265 cm 1 . 'H NMR (300 MHz, DMSO-d 6 ) 6 = 1.3-1.7 (m, 6 H); 1.98 (s, 3 H); 2.26 (t, J= 8 Hz, 2 H); 2.56; (d, J= 12 I-Iz, 1 H); 2.81 (dd, J= 12 and 5 Hz, 1 H); 3.11 (m, 1 H); 4.13 (m, 1 H); 4.29 (m, I H); 6.39 (s, 3 H); 6.42 (s, 1 H); 7.22 (m, 2 H); 7.50 (d, J = 8 Hz, 1 H) 7.84 (s, I H); 9.82 (s, I H). - MS (FAB/glycerol), m/z: 376 [M+H] 10 e meta-Diazomethane compound 3a: 2a (180 mg, 0.48 mmol) is solubilized in 2 ml of DMF. MnO 2 (340 mg, 3.9 mmol) is then added. After stirring for 30 minutes at normal temperature, the mixture is filtered through a sintered glass funnel containing celite (thickness: 0.5 cm) and powdered 3 A molecular sieves 15 (0.5 cm). The reaction mixture is concentrated to a volume of approximately 0.5 ml, and then 5 ml of ether are added. The resulting precipitate is filtered off, washed with ether, and then dried. Compound 3a (170 mg, 95%) is obtained in the form of a pink powder. Mp 160 0 C. - IR (KBr): 3278, 2935, 2859, 2038, 1704, 1666, 1605, 1577, 1536, 1458, 1430, 20 1263 cm 1 . - 'H NMR (300 MHz) 8 = 1.3-1.7 (m, 6 H); 2.11 (s, 3 H); 2.28 (t, J= 8 Hz, 2 H); 2.57; (d, J= 12 Hz, 1 H); 2.81 (dd, J= 12 and 5 Hz, 1 H); 3.11 (m, 1 H); 4.13 (m, 1 H); 4.29 (m, 1 H); 6.33 (s, 1 H); 6.41 (s, 1 H); 6.60 (m, 1 H); 7.25 (m, 3 H); 9.84 (s, 1 H)). Example 2: Synthesis of the reagent N-(3.6,9-triaminenonanyl)-biotinamide (Bio-TETA) 25 (1). D-Biotin (2.80 g, 11.40 mmol) is dissolved in 30 ml of anhydrous DMF. The addition of carbonyldiimidazole (1.5 eq.; 2.78 g) brings about, after a few minutes, the formation of a precipitate. After activation for 30 min, the resulting suspension is carefully added to 30 triethylenetetramine (2 eq.; 5.00 g) in suspension in 20 ml of DMF. The reaction is left for 2 h 35 on an oil bath at 60*C. The product is purified by flash chromatography on silica gel, with 20:80:3

CH

2 Cl 2 /MeOH/NH 4 0H as eluent. After evaporation of the fractions concerned, 1.94 g of product are obtained in the form of a white powder (46%). 5 'H NMR (200 MHz, DMSO- d) S = 7.75 (s, IH, -NH-CO-); 6.40 (d, 2H, -NH- biot); 4.30 (t, 1H, -CH- biot); 4.15 (d, 1H, -CH- biot); 3.30 (m, 12H, -CH2-NH-); 3.11 (in, 1H, -CH-S-); 2.8 (dd, 2H, -CH 2 -S-); 2.55 (m, 5H, -NH-CH 2 - & -NH 2 ); 2.04 (t, 2H, -CH 2 -CO-); 1.52 (in, 6H, CH 2 -). 10 N-(3'-Acetophenvi) succinamic acid (ACBA) (2) 3-Aminoacetophenone (5.0 g; 37 mmol) is dissolved in 50 ml of anhydrous acetonitrile under argon. Succinic anhydride (1.3 eq.; 4.62 g) is added and allowed to react for one hour under argon. Product 2 appears in the form of a precipitate. After filtration and washing of the precipitate with ether, 7.29 g of white powder are obtained (84%). 15 'H NMR (200 MHz, DMSO-d 6 ) S = 12.10 (s, lH, -OH); 10.17 (s, 1H, -NH-); 8.19 (s, lH); 7.82 (d, IH); 7.66 (d, I H); 7.50 (t, 1H); 2.56 (m, 7H, -(CH 2

)

2 - and -CH 3 ). N-(3'-Acetophenvl)-N'-(3.6-diamine-9-biotinoylaminononanvl)succinamide (Bio-(TETA)-AP) 20 The ACBA (2) (1.03 g; 4.39 mmol) is dissolved in 20 ml of anhydrous DMF under argon. The medium is cooled in ice, and N-methylmorpholine (1.25 eq.; 725 pl) and isobutyl chloroformate (1 eq.; 690 pl) are added successively; the medium becomes cloudy after 30 min. In parallel, the Bio-TETA (1) (0.8 eq.; 1.94 g) is solubilized under hot conditions in 50 ml of DMF and of triethylamine (0.8 eq.; 750 pl). It is added to the activated ACBA at 0"C, 25 for 30 min. The mixture is then left at ambient temperature overnight. The purification is carried out by flash chromatography on silica gel, with 85:30:3 CH 2

CI

2 /MeOH/NH 4 0H as elulent. The fractions containing product 3 are combined and the solvent is evaporated off. 1.01 g of white solid is obtained in the form of flakes (33%). 'H NMR (200 MHz, DMSO- d 6 ) S = 10.16 (s, I H, Ph-NH-CO-); 8.18 (s, lH); 7.97 (s, IH, 30 CO-NH-CH 2 -); 7.80 (d, 1H); 7.85 (s, 1H, -CH 2 -NH-CO-); 7.60 (d, 1H); 7.43 (t, IH); 6.38 (d, 2H, -NH- biot); 4.3 (t, 1H, -CH- biot); 4.10 (d, lH, -CH- biot); 3.35 (in, 12H, -_GH 2

-NH-);

36 3.10 (m, IH, -CH-S-); 2.80 (dd, 2H, -CH 2 -S-); 2.60 (s, 4H, -CH 2 -CO-); 2.55 (s, 3H, -CO

CH

3 ); 2.50 (m, 2H, -CH 2 -CH2-CO-); 2.15 (in, 2H, -NH-); 1.40 (m, 6H, -CH 2 -). N-3'-(1 -Hydrazonoethvl)phenll-N'-(3, 6 -diamine-9-biotinoylaminononanvl)succinamide 5 (Bio-(TETA)-HY) (4) The Bio-(TETA)-AP (3) (1.0 g; 1.71 mmol) is suspended in 25 ml of ethanol under hot conditions (60*C). At reflux, hydrazine monohydrate (9 eq.; 750 pl) is added. The reaction is left at reflux for 2 h, and then cooled on ice. A precipitate forms after a short time. The two phases are separated and the precipitate is placed under vacuum. 690 mg of a flocculant solid 10 are obtained (68%). 'H NMR (200 MHz, DMSO- d 6 ) 8 = 9.95 (s, 1H, Ph-NH-CO-); 8.0 (s, lH, -CO-NH-CH 2 -); 7.90 (s, IH); 7.80 (s, 1H, - CH 2 -NH-CO-) ; 7.5 (d, 1 H); 7.28 (in, 2H); 6.41 (s, I H, -NH- biot); 6.36 (d, 3H, -NH- biot & -NH 2 ); 4.64 (t, IH, -CH- biot); 4.30 (d, IH, -CH- biot); 3.43 (in, 12H, -_CH2-NH-); 3.12 (in, 1H, -CH-S-); 2.80 (dd, 2H, -CH 2 -S-); 2.60 (s, 4H, -CH-C 2 -CO-); 15 2.50 (s, 3H, -CO-CH 3 ); 2.10 (in, 2H, -CH 2

-NH-CH

2 -); 1.50 (in, 6H, -CH 2 -). N-(3'-(1-Diazoethyl)phenyll-N'-(3,6-diamine-9-biotinovlaminononanvl)succinamide (m-Bio (TETA)--PMDA M) (5) The Bio-(TETA)-Hy (4) (150 mg; 250.4 4mol) is solubilized in 1 ml of anhydrous DMSO 20 under argon. It is left to react for 30 minutes with MnO 2 (s) (15 eq.; 330 mg) and then the mixture is filtered through a sintered glass funnel No. 4 with celite (0.5 cm thickness) and 3 A molecular sieves (0.5 cm thickness). 100 pl are used for the NMR with the addition of 380 PI of DMSO-d 6 and 20 pl of methanol-d 4 . The final volume is adjusted to 4.5 ml with anhydrous DMSO and 4% of methanol. The mixture is aliquoted in 250 pl-aliquots in a glovebox under 25 argon. The compound is fuschia pink. The degree of purity (diazomethyl content) is verified by 'H NMR and UV-vis spectrophotometry (diazomethyl absorbance peak at 516 nm). 'H NMR (200 MHz, DMSO- d 6 ) 6 = 9.93 (s, 1H, Ph-NH-CO-); 7.3 (s, 3H, Haomacic); 6.6 (s, IH, Homatic); 6.4 (s, IH, -NH- biot); 6.3 (s, 1H, -NH- biot); 4.3 (t, 1H, -CH- biot); 3.3 (in, 12H, -CH 2 -NH-); 3.3 (in, lH, -CH-S-); 2.9 (dd, 2H, -CH 2 -S-); 2.5 (4H, -CH 2 -CO-); 2.1 (s, 3H, 30 -CH 3 ); 2.0 (in, 2H, -CH 2

-NH-CH

2 -); 1.50 (m, 6H, -CH 2

-).

37 Example 3: Preparation of DNA and RNA nucleic acids: Example 3.1: Preparation of DNA amplicons: 5 The DNA amplicons are generated by PCR from Mycobacterium tuberculosis 16S genomic DNA targets (10+4 copies as starting targets) using the Fast Start kit from Roche, 0.2 mM of each deoxyribonucleotide (d-ATP, d-CTP, d-GTP, d-TTP), 0.3 pM of primers and 0.4 pl of enzyme. 10 The PCR parameters are as follows: - 95"C: 4 min then 35 cycles (95'C: 30 sec; 55 0 C: 30 sec; 72*C: 30 sec) then 4*C. The amplicons are analyzed qualitatively by agarose gel electrophoresis (1.5%, 0.5X TBE). The volume loaded is 5 pl and the migration is carried out for 20 min at 100 volts (V). The PCR products are visualized under a UV lamp after staining with ethidium bromide. 15 The conditions for the culturing, the extraction of the mycobacteria and also the amplification primers are given in patent application WO-A-99/65926. Example 3.2: Preparation of transcribed RNAs: 20 The transcriptions are carried out from PCR target (Mycobacterium tuberculosis 16S RNA fragment) using the MEGAscript kit from Ambion: 7.5 mM of each nucleotide (ATP, CTP, GTP and UTP) and 2 pLl of enzyme (RNA polymerase). The incubation time is 3 hours (h) at 37*C. The PCR amplification primers carry a T3 or T7 polymerase promoter, as described in application WO-A-99/65926 or in the article J. Clin Microbiol. 37(1), p 49-55, 1999, which 25 makes it possible to carry out the transcription. The transcripts are analyzed by agarose gel electrophoresis (1.5%; 0.5X TBE). The volume loaded is 5 pl and the migration is carried out for 20 min at lOOV. The transcripts are visualized under a UV lamp after staining with ethidium bromide. 30 Identical results, from the point of view of the invention, can be obtained using other amplification techniques such as NASBA or TMA, which generate RNA amplicons directly.

37a Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. 5 A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims. 30.01.2012 2101318.1

Claims (31)

1. 2. A temperature-stable labeling reagent as claimed in claim 1, wherein u is 0 or 1.
2. 3. A temperature-stable labeling reagent as claimed in claim 1 or 2, wherein m is 25 an integer between I and 3. 39
3. 4. The reagent as claimed in any one of claims 1 to 3, wherein p is less than or equal to m.
4. 5. The reagent as claimed in one of claims I to 4, of formula (2): R3 R-(NH-CH 2 -CH 2 )q-(L)j--Y -X R 5 N 2 in which: R', R 2 , R 3 , R 4 , L, -Y-X, and n are as defined in any one of claims 1 to 4; and where q is an integer between I and 10.
5. 6. The reagent as claimed in claim 5, wherein q is between 1 and 3. 10
6. 7. The reagent, as claimed in any one of claims I to 6, of formula (3): R3 R-(NH-CH 2 -CH 2 ) 3 -NH-CO-CH 2 -CH 2 -CO-NHR R N 2 in which: R', R 2 , R 3 and R4 are as defined in any one of claims I to 6. 15 8. The reagent as claimed in claim 7, wherein R 2 consists of a D-biotin residue of formula (4) 0 HN NH 0 S (CH 2 ) 4 -C- 40
7. 9. The reagent as claimed in any one of claims I to 8, wherein R' consists of: CH 3 , and R3 and R 4 each represent: H.
8. 10. The reagent as claimed in any one of claims I to 6, in which the structure 5 -(L),- consists of: * spermine or N,N'-bis(3-aminopropyl)-1,4-diantinobutane: NH 2 -(CH 2 ) 3 -NH-(CH 2 ) 4 -NH (CH 2 ) 3 - NH 2 , or e sperm idine or N-(3-am inopropyl)- 1,4-butanediamine: H 2 N-(CH 2 ) 4 -NH-(CH 2 ) 3 - NH 2 , or e a derivative containing an alanine motif: NH 2 -CH 2 -CH 2 -COOH. 10
9. 11. A temperature-stable labeling reagent of formula (6): N 2 I I R2_(Z-(CH 2 )p)m-(L),-Y-X R X - Y-(L)-((CH 2 p-Z)m-R 2 QR7 R in which: " R 2 represents a detectable label or at least two detectable labels interlinked by at least one 15 multimeric structure, " L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, " R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L),-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 3 -CH 2 -NH 20 R 2 ,-CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 4 -CH 2 -NH-R 2 with R = alkyl or aryl, e A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, * -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, * -Z- represents -NH-, 25 e m is an integer between I and 10, and " p is an integer between I and 3. 41
10. 12. A temperature-stable labeling reagent as claimed in claim 11, wherein u is 0 or 1.
11. 13. A temperature-stable labeling reagent as claimed in claim I1 or 12, wherein m 5 is an integer between I and 3.
12. 14. The labeling reagent, as claimed in any one of claims I1 to 13, of formula (7): N 2 C R2-(Z-(CI2)p)m-(L)n-Y -X X - Y -(L)- ((CH)- Z)m- R2 R 3 R in which: R 2 , R 3 ,R 4 , L, -Y-X-, Z, m, n and p are as defined in any one of claims I1 to 13. 10
13. 15. The reagent as claimed in any one of claims 1 to 6, 11 to 14, wherein L comprises a motif -(O-CH 2 -CH 2 )-, repeated from I to 20 times.
14. 16. The reagent as claimed in claim 15, wherein the motif -(O-CH 2 -CH 2 )- is 15 repeated I to 10 times.
15. 17. The reagent as claimed in claim 16, wherein the motif -(O-CH 2 -CH 2 )- is repeated 2 to 5 times. 20 18. A method for the synthesis of a labeling reagent as claimed in any one of claims I to 17, comprising the following steps: a) a label or a label precursor having a reactive function R 6 is provided, b) a linker arm of formula (8): R--(Z-(CH 2 )p)m-R 25 is provided, in which formula: 42 * -Z- represents-NH-, * m is an integer between 1 and 10, e p is an integer between I and 3, e R 7 and R' represent two reactive functions which may be identical or different, 5 c) the reactive function R 6 of said label or label precursor and the function R 7 of the linker arm of formula (8) are reacted together in the presence of at least one coupling agent so as to form a covalent bond, R 6 and R 7 being complementary, d) a derivative of formula (9): 0 R R3 R4 R-(L)"-Y -x 10 is provided, in which formula: " R' represents H or an alkyl, aryl or substituted aryl group, * L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 15 e R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L)n-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 3 -CH 2 -NH-R 2 , CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 4 -CH 2 -NH-R 2 with R = alkyl or aryl, * -Y-X- represents -CONH-, -NHCO-, -CH 2 0-, -CH 2 S-, e A is a linker arm comprising at least one covalent double bond enabling the conjugation of 20 the diazomethyl function with the aromatic ring and u is an integer equal to 0 or 1, and e R 9 represents a reactive function complementary to R 8 , e) the reactive function R 9 of the derivative of formula (9) and the function R 8 of the linker arm of formula (8) are reacted together in the presence of at least one coupling agent so as to form a covalent bond, 43 f) the hydrazine or one of its derivatives is reacted with the ketone or aldehyde function so as to form a hydrazone, and g) the hydrazone is converted to a diazomethyl function by means of an appropriate treatment. 5
16. 19. The method as claimed in claim 18, wherein m is an integer between I and 3.
17. 20. The method of synthesis as claimed in claim 19, wherein it comprises: " an additional step consisting of protection of the ketone or aldehyde function of compound 10 (9), and * a subsequent additional step consisting of deprotection of said ketone or aldehyde function.
18. 21. A temperature-stable labeling reagent of formula (1) as defined in claim 1, substantially as herein described with reference to structures 13, 14 or 15 of Figure 1/2. 15
19. 22. A method for the synthesis of a labeling reagent of claim 21, which method is substantially as herein described with reference to Example 2.
20. 23. A method for the labeling of a biological molecule, comprising bringing into 20 contact, in homogeneous solution, in a substantially aqueous buffer, a biological molecule and a reagent, obtained as claimed in any one of claims 18 to 20 or 22.
21. 24. A method according to claim 23, wherein the biological molecule is a nucleic acid. 25
22. 25. A labeled biological molecule when obtained by means of the method as claimed in claims 23 or 24.
23. 26. A method for the labeling and fragmentation of a single-stranded or double 30 stranded nucleic acid, comprising the following steps: * fragmenting the nucleic acid, 44 e attaching a label to at least one of the fragments by means of a labeling reagent chosen from the reagents obtained as claimed in any one of claims 18 to 20, said reagent coupling covalently and predominantly on at least one phosphate of said fragment. 5 27. The method as claimed in claim 26,wherein the labeling reagent is chosen from the compounds of formula (3): R3 R R-(NH-CH 2 -CH 2 ) 3 -NH-CO-CH 2 -CH 2 -CO-NR 4R N2 in which: e R' represents H or an alkyl, aryl or substituted aryl group, 10 * R 2 represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, and e R 3 and R 4 represent, independently of one another: H, NO 2 . Cl, Br, F, I, R 2 -(L),-Y-X-, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, -CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 3 -CH 2 -NH-R 2 , CO-NH-(CH 2 ) 3 -(O-CH 2 -CH 2 ) 4 -CH 2 -NH-R 2 with R = alkyl or aryl. 15
24. 28. The method as claimed in claim 26 or claim 27, wherein the fragmentation and the labeling are carried out in two steps.
25. 29. The method as claimed in claim 26 or claim 27, wherein the fragmentation and 20 the labeling are carried out in one step.
26. 30. The method as claimed in any one of claims 26 to 29, wherein the labeling is carried out in a substantially aqueous homogeneous solution. 25 31. The method as claimed in any one of claims 26 to 30, wherein the fragmentation is carried out by an enzymatic, physical or chemical process. 45
27. 32. A method for the labeling and fragmentation of a single-stranded or double stranded nucleic acid, comprising the following steps: * fragmenting the nucleic acid, e attaching a label to at least one of the fragments by means of a labeling reagent chosen 5 from the reagents obtained as claimed in claim 22, said reagent coupling covalently and predominantly on at least one phosphate of said fragment.
28. 33. A labeled nucleic acid when obtained by means of the method as claimed in any one of claims 26 to 32. 10
29. 34. A solid support to which is attached a reagent as claimed in any one of claim I to 17.
30. 35. A method for the capture of nucleic acids, comprising the following steps: 15 e providing a solid support to which is directly or indirectly attached at least one biological molecule as claimed in claim 25, or a nucleic acid as claimed in claim 33, the biological molecule or the nucleic acid comprising a diazomethyl function, " bringing into contact a biological sample which may contain free nucleic acids, and e washing the solid support where the molecule(s) is (are) covalently attached at least to a 20 nucleic acid.
31. 36. The use of a labeling reagent obtained by the method of any one of claims 18 to 20 or 22 for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid comprising: 25 e fragmenting the nucleic acid, e attaching a label to at least one of the fragments by means of said labeling reagent, said reagent coupling covalently and predominantly on at least one phosphate of said fragment. 30