Labelling reagents having a pyridine nucleus bearing a diazomethyl function, process for synthesis of such reagents and processes for detection of biological molecules

• US 9,266,902 B2
• Filed: 07/28/2009
• Issued: 02/23/2016
• 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: Laurent, Alain, Laayoun, Ali, Kotera, Mitsuhara
• Primary Examiner(s): Andres, Janet L
• Assistant Examiner(s): CARCANAGUE, DANIEL R
• Application Number:
  US13/001,712
• Publication Number:
  US 20110111514A1
• Time in Patent Office: 2,401 Days
• Field of Search: 514/338, 546/274.4, 436/94
• US Class Current: 1/1
• 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. A labelling reagent of formula (D):

2. The labelling reagent according to claim 1, of formula (F):

3. The labelling reagent according to claim 1, of formula (H):

4. The labelling reagent according to claim 1, wherein R₁ is a D-biotin residue of formula (I):

5. A process for synthesizing the labelling reagent according to claim 1, comprising:

a) esterifying an aromatic dicarboxylic acid to form a diester,

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

c) reducing the precursor to an alcohol and then oxidizing the alcohol to an aldehyde,

d) acid treating the aldehyde to concomitantly protect the aldehyde function while deprotecting the amine function, to form an aminated acetal,

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

f) deprotecting the carbonyl function of the labelled precursor to form a labelled and carbonyl-containing precursor, and then

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

6. A process for labelling a biological molecule, comprising contacting the biological molecule with the labelling reagent according to claim 1 in a homogeneous solution, in an aqueous buffer that contains at least 50% water.

7. A labelled biological molecule obtained by the process according to claim 6, wherein the biological molecule is selected from the group consisting of nucleic acids, antigens, antibodies, polypeptides, and proteins.

8. A process for labelling and fragmenting a single or double strand nucleic acid comprising:

fragmenting the nucleic acid,

attaching a label onto at least one of the fragments with the labelling reagent according to claim 1,

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

9. The process according to claim 8, wherein the labelling is effected in an aqueous homogeneous solution that contains at least 50% water.

10. A labelled nucleic acid obtained by the process according to claim 8.

11. A kit for detection of a target nucleic acid containing the labelled nucleic acid according to claim 10.

12. A solid support onto which is attached the labelling reagent according to claim 1.

13. A process for capturing nucleic acids comprising:

attaching directly or indirectly to a solid support at least one biological molecule according to claim 7,

contacting the solid support with a biological sample capable of containing free nucleic acids, and then

washing the solid support.

14. A labelling reagent of formula (D):

15. A process for synthesizing the labelling reagent according to claim 14, comprising:

a) esterifying an aromatic dicarboxylic acid to form a diester,

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

c) reducing the precursor to an alcohol and then oxidizing the alcohol to an aldehyde,

d) acid treating the aldehyde to concomitantly protect the aldehyde function while deprotecting the amine function, to form an aminated acetal,

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

f) deprotecting the carbonyl function of the labelled precursor to form a labelled and carbonyl-containing precursor, and then

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

16. A process for labelling a biological molecule, comprising contacting the biological molecule with the labelling reagent according to claim 14 in a homogeneous solution, in an aqueous buffer that contains at least 50% water.

17. A labelled biological molecule obtained by the process according to claim 16.

18. A process for labelling and fragmenting a single or double strand nucleic acid comprising:

fragmenting the nucleic acid,

attaching a label onto at least one of the fragments with the labelling reagent according to claim 14,

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

19. The process according to claim 18, wherein the labelling is effected in an aqueous homogeneous solution that contains at east 50% water.

20. A labelled nucleic acid obtained by the process according to claim 18.

21. A kit for detection of a target nucleic acid containing the labelled nucleic acid according to claim 20.

22. A solid support onto which is attached the labelling reagent according to claim 14.

23. A process for capturing nucleic acids, comprising:

attaching directly or indirectly to a solid support at least one biological molecule according to claim 17,

contacting the solid support with a biological sample capable of containing free nucleic acids, and then

washing the solid support.