LABELLING REAGENTS HAVING A PYRIDINE NUCLEUS BEARING A DIAZOMETHYL FUNCTION, PROCESS FOR SYNTHESIS OF SUCH REAGENTS AND PROCESSES FOR DETECTION OF BIOLOGICAL MOLECULES

• US 20110111514A1
• Filed: 07/28/2009
• Published: 05/12/2011
• Est. Priority Date: 07/29/2008
• Status: Active Grant

• Current Assignee: Biomerieux Chemin De L'Orme, CNRS, Universite De Strasbourg (Financière Meeschaert SA)
• Original Assignee: CNRS, Biomerieux B.V. (Compagnie Merieux Alliance SAS), Universite De Strasbourg (Financière Meeschaert SA)
• Inventors: Kotera, Mitsuhara, Laayoun, Ali, Laurent, Alain
• Granted Patent: US 9,266,902 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C07D 213/24 with substituted hydrocarbon radicals attached to ring carbon atoms
• CPC Class Codes: C07D 233/32 One oxygen atom
• CPC Class Codes: C07D 333/24 Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/10 Characterised by chemical treatment
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A process for synthesis of a labelling reagent, a process for the labelling of a biological molecule, a labelled biological molecule obtained by the process, a process for labelling and fragmentation of a single or double strand nucleic acid, a labelled nucleic acid capable of being obtained by the process, a kit for detection of a target nucleic acid containing a labelled nucleic acid, a solid support onto which is attached a reagent and a process for capture of nucleic acids.

Claims

1. 1. Labelling reagent of formula (C):

3. Labelling reagent according to claim 1, of formula (E):

5. Labelling reagent according to claim 1, of formula (G):

7. Reagent according to claim 1, wherein R₁ is made up of a D-biotin residue of formula (I):

8. Process for synthesis of a labelling reagent according to claim 1, comprising the following stages:

a) an aromatic carboxylic acid derivative is reacted with the enolate of a lactone (Claisen type reaction) to form a cyclic precursor,

b) which cyclic precursor is then opened with a halo acid to form an aromatic halo ketone,

c) the carbonyl function of the aromatic halo ketone is protected by a protective group to form a protected precursor,

d) which protected precursor is subjected to an amination reaction (of the Gabriel type) to form an aminated precursor,

e) which aminated precursor is deprotected to liberate the amine function, the said amine function being reacted with a detectable label the carboxyl function whereof is activated to form a precursor containing a detectable label,

f) the labelled precursor is subjected to a reaction of deprotection of the carbonyl function to form a labelled and carbonyl-containing precursor, and finally

g) the labelled and carbonyl-containing precursor is transformed into a labelling reagent, such as described above, by transformation of the carbonyl function into a diazo function.

10. Process for the labelling of a biological molecule, comprising the contacting of a biological molecule and a reagent according to claim 1 in homogeneous solution, in an appreciably aqueous buffer.

11. Labelled biological molecule capable of being obtained by the process according to claim 10.

12. Process for labelling and fragmentation of a single or double strand nucleic acid comprising the following stages:

fragmenting the nucleic acid,

attaching a label onto at least one of the fragments by means of a labelling reagent selected from the reagents according to claim 1,

the said reagent coupling covalently and mainly onto at least one phosphate of the said fragment.

13. Process according to claim 12, wherein the fragmentation and the labelling are effected in two stages.

14. Process according to claim 12, wherein the fragmentation and the labelling are effected in one stage.

15. Process according claim 12, wherein the labelling is effected in appreciably aqueous homogeneous solution.

16. Process according to claim 12, wherein the fragmentation is effected by an enzymatic, physical or chemical route.

17. Labelled nucleic acid capable of being obtained by the process according to claim 12.

18. Kit for detection of a target nucleic acid containing a labelled nucleic acid according to claim 17.

19. Solid support onto which is attached a reagent according to claim 1.

20. Process for capture of nucleic acids comprising the following stages:

a solid support is used onto which there is directly or indirectly attached at least one biological molecule according to claim 11, the biological molecule or the nucleic acid containing a diazomethyl function,

it is contacted with a biological sample capable of containing free nucleic acids, and

the solid support where the molecule(s) are covalently attached to at least one nucleic acid is washed.

2. Labelling reagent of formula (D):

4. Labelling reagent according to claim 2, of formula (F):

6. Labelling reagent according to claim 2, of formula (H):

9. Process for synthesis of a labelling reagent according to claim 2, comprising the following stages:

a) an aromatic dicarboxylic acid is esterified to form a diester,

b) this diester is regioselectively substituted with a compound derived from mono-protected aminated ethylene glycol to form a precursor protected on the carboxyl function and the amine function,

c) which precursor is then reduced to the alcohol and oxidised to the aldehyde,

d) the aldehyde synthesised in stage c) is subjected to an acid treatment in order concomitantly to protect this function while deprotecting the amine function, to lead to an aminated acetal,

e) which aminated acetal is reacted with a detectable label the carboxyl function whereof is activated to form a precursor containing a detectable label,

f) the labelled precursor is subjected to a reaction of deprotection of the carbonyl function to form a labelled and carbonyl-containing precursor, and finally

g) the labelled and carbonyl-containing precursor is transformed into a labelling reagent, such as described above, by transformation of the carbonyl function into a diazo function by a Bamford Stevens type reaction.