Labeling reagents, methods for synthesizing such reagents and methods for detecting biological molecules

• US 7,338,805 B2
• Filed: 05/03/2002
• Issued: 03/04/2008
• Est. Priority Date: 05/04/2001
• Status: Expired due to Fees

• Current Assignee: Bio Merieux, Centre National De La Recherche Scientifique, Universite Joseph Fourier, Universite Joseph Fourier Domaine Universitaire St Martin D'Heres
• Original Assignee: Bio Merieux, Centre National De La Recherche Scientifique, Universite Joseph Fourier
• Inventors: Laayoun, Ali, Kotera, Mitsuharu, Bernal Mendez, Eloy, Lhomme, Jean, Bourget, Cecile, Trevisiol, Emmanuelle, Menou, Lionel
• Primary Examiner(s): Gakh; Yelena G.
• Application Number:
  US10/137,454
• Publication Number:
  US 20030186448A1
• Time in Patent Office: 2,132 Days
• Field of Search: 436/56, 436/63, 436/501, 436/544, 436/546, 535/6, 536/23.1
• US Class Current: 436/56
• CPC Class Codes: C07C 245/14 having diazo groups bound to acyclic carbon atoms of a carbon skeleton
• CPC Class Codes: C07C 245/18 the carbon skeleton being further substituted by carboxyl groups
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a temperature-stable labeling reagent of formula:

in which

• • R¹ represents H or an alkyl, aryl or substituted aryl group,
  • R² represents a detectable marker or at least two detectable markers linked together by at least one multimeric structure,
  • L is a linking arm containing a linear succession of at least two covalent bonds and n an integer equal to 0 or 1,
  • R³ and R⁴ represent independently of each other: H, NO₂, Cl, Br, F, I, R²-(L)_n-Y—X—, OR, SR, NR₂, R, NHCOR, CONHR, COOR with R=alkyl or aryl,
  • A is a linking arm containing at least one covalent double bond allowing conjugation of the diazo functional group with the aromatic ring and u is an integer between 0 and 2, preferably equal to 0 or 1, and
  • —Y—X— represents —CONH—, —NHCO—, —CH₂O—, —CH₂S—.

The present invention also describes a method for synthesizing said markers as well as applications for labeling biological molecules, in particular nucleic acids, with a labeling reagent carrying the diazomethyl functional group.

The invention finds a preferred application in the field of diagnosis.

Claims

1. A temperature-stable labeling reagent of formula (0):

2. The labeling reagent as claimed in claim 1 of formula (1):

3. The reagent as claimed in claim 1 of formula (2):

4. The reagent as claimed in claim 1 of formula (3):

5. The reagent as claimed in claim 1 of formula (4):

6. The reagent as claimed in claim 1 of formula (21):

7. The reagent as claimed in claim 1 of formula (22):

8. The reagent as claimed in claim 1 of formula (23):

9. The reagent as claimed in claim 1, wherein R³ and R⁴ represent independently of each other:

H, NO₂, OCH₃, —

CO—

NH—

(CH₂)₃—

(O—

CH₂—

CH₂)₃—

CH₂—

NH—

R², —

CO—

NH—

(CH₂)₃—

(O—

CH₂—

CH₂)₄—

CH₂—

NH—

R².

10. The reagent as claimed in claim 1 of formula (2′

);

11. A method for synthesizing a labeling reagent as claimed in claim 10, comprising the following steps:

a) providing a derivative of formula (17);

12. The reagent as claimed in claim 1 of formula (24):

13. The reagent as claimed in claim 1, in which the structure R²-(L)_n- is of formula (5):

14. The reagent as claimed in claim 1 of formula (6):

15. The reagent as claimed in claim 1 of formula (25):

16. The reagent as claimed in claim 1 of formula (14):

17. The reagent as claimed in claim 1 of formula (26):

18. The reagent as claimed in claim 1 of formula (15):

19. The reagent as claimed in claim 1 of formula (27):

20. The reagent as claimed in claim 1, wherein L comprises a unit —

(O—

CH₂—

CH₂)—

, repeated from 1 to 20 times.

21. A method for synthesizing a labeling reagent, as claimed in claim 1, comprising the following steps:

a) providing a derivative of formula (16a);

22. The method of synthesis as claimed in claim 21, which comprises:

an additional step of protecting the ketone or aldehyde functional group of compound (16a), and

an additional subsequent step of deprotecting said ketone or aldehyde functional group.

23. The method of synthesis as claimed in claim 21, which consists in carrying out the following steps:

a) providing a derivative of formula (16);

24. The method of synthesis as claimed in claim 21, wherein M in R⁶=COOM is methyl or ethyl.

25. A method for synthesizing a labeling reagent, as claimed in claim 1, comprising the following steps:

a) providing a derivative of formula (16a);

26. The method of synthesis as claimed in claim 25, wherein M in R⁶=COOM is methyl or ethyl.

27. A method for labeling a biological molecule, in particular a nucleic acid, comprising bringing a biological molecule and a reagent as claimed in claim 1 into contact in a homogeneous solution, in a substantially aqueous buffer.

28. A labeled biological molecule obtained by the method as claimed in claim 27.

29. A method for labeling and fragmenting a single- or double-stranded nucleic acid, comprising the following steps:

fragmenting the nucleic acid,

attaching a marker to at least one of the fragments via a labeling reagent chosen from the compounds of formula of claim 1,

wherein;

said reagent is covalently and predominantly coupled to at least one phosphate of said fragment.

30. The method as claimed in claim 29, wherein the labeling reagent is chosen from the compounds of formula (1) of claim 2.

31. The method as claimed in claim 29, wherein the fragmentation and the labeling are carried out in two steps.

32. The method as claimed in claim 29, wherein the fragmentation and the labeling are carried out in one step.

33. The method as claimed in claim 29, wherein the labeling is carried out in a substantially aqueous homogeneous solution.

34. The method as claimed in claim 29, wherein the fragmentation is carried out by the enzymatic, physical or chemical route.

35. A labeled nucleic acid obtained by the method as claimed in claim 29.

36. A kit for detecting a target nucleic acid comprising a labeled nucleic acid as claimed in claim 35.

37. A solid support onto which a reagent as claimed in claim 1 is attached.

38. The reagent as claimed in claim 1, wherein the reagent is stable for at least 15 days at a temperature of at least −

20°

C.