Labeling reagents, methods for the synthesis of such reagents and methods for the detection of biological molecules

• US 7,691,635 B2
• Filed: 03/24/2005
• Issued: 04/06/2010
• Est. Priority Date: 03/26/2004
• Status: Expired due to Fees

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Laayoun, Ali, Bernal-Mendez, Eloy
• Primary Examiner(s): Saeed; Kamal A
• Assistant Examiner(s): BARKER, MICHAEL P
• Application Number:
  US10/590,973
• Publication Number:
  US 20080032288A1
• Time in Patent Office: 1,839 Days
• Field of Search: 436/56, 436/63, 436/501, 436/544, 436/546, 535/6, 536/23.1, 548/304.1, 514/393
• US Class Current: 436/56
• 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 (0):

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, NO₂, Cl, Br, F, I, R²-(L)_n-Y—X—, OR, SR, NR₂, R, NHCOR, CONHR, COOR, —CO—NH—(CH₂)₃—(O—CH₂—CH₂)₃—CH₂—NH—R², —CO—NH—(CH₂)₃—(O—CH₂—CH₂)₄—CH₂—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 or 1,
  • —Y—X— represents —CONH—, —NHCO—, —CH₂O—, —CH₂S—,
  • —Z— represents —NH—, —NHCO—, —CONH— or —O—,
  • m is an integer between 1 and 10, preferably between 1 and 3, and
  • p is an integer between 1 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.

Claims

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

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

3. The reagent according to claim 2, wherein p is less than or equal to m.

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

5. The reagent, according to claim 4, of formula (3):

6. The reagent according to claim 5, wherein R² consists of a D-biotin residue of formula (4):

7. The reagent according to claim 6, wherein R¹ is CH₃, and R³ and R⁴ each represent H.

8. The reagent according to claim 4, in which the structure -(L)_n- consists of:

spermine or N,N′

-bis(3-aminopropyl)-1,4-diaminobutane;

NH₂—

(CH₂)₃—

NH—

(CH₂)₄—

NH—

(CH₂)₃—

NH₂, or

spermidine or N-(3-aminopropyl)-1,4-butanediamine;

H₂N—

(CH₂)₄—

NH—

(CH₂)₃—

NH₂, or

a derivative containing an alanine motif;

NH₂—

CH₂—

CH₂—

COOH.

11. The reagent according to claim 1, wherein:

L comprises a motif —

(O—

CH₂—

CH₂)—

, repeated from 1 to 20 times, and

—

Z—

is —

NH—

, —

NHCO—

or —

CONH—

.

13. A method for the synthesis of a labeling reagent according to claim 1, comprising the following steps:

a) providing a label or a label precursor having a reactive function R⁶,

b) providing a linker arm of formula (8);

R⁷—

(Z—

(CH₂)_p)_m—

R⁸

in which;

—

Z—

represents —

NH—

, —

NHCO—

, or —

CONH—

,

m is an integer between 2 and 10,

p is an integer between 1 and 10,

R⁷ and R⁸ represent two reactive functions which may be identical or

different,

c) reacting together the reactive function R⁶ of said label or label precursor and the function R⁷ the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, R⁶ and R⁷ being complementary,

d) providing a derivative of formula (9);

15. The method of synthesis according to claim 13, further comprising:

an additional step consisting of protection of the ketone or aldehyde function of compound (9), and

a subsequent additional step consisting of deprotection of said ketone or aldehyde function.

17. A method for the labeling of a biological molecule, comprising bringing into contact, in a homogeneous solution in a substantially aqueous buffer, the biological molecule and a reagent according to claim 1.

19. A labeled biological molecule which can be obtained by the method according to claim 17.

21. A method for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid, the method comprising:

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 claim 1,

said reagent coupling covalently and predominantly on at least one phosphate of said fragment.

23. The method according to claim 21, wherein the labeling reagent is chosen from the compounds of formula (3):

25. The method according to claim 23, wherein the fragmentation and the labeling are carried out in two steps.

27. The method according to claim 23, wherein the fragmentation and the labeling are carried out in one step.

29. The method according to claim 25, wherein the labeling is carried out in a substantially aqueous homogeneous solution.

30. The method according to claim 27, wherein the labeling is carried out in a substantially aqueous homogeneous solution.

32. The method according to claim 25, wherein the fragmentation is carried out by an enzymatic, physical, or chemical process.

34. A labeled nucleic acid obtained by the method according to claim 21.

36. A kit for the detection of a target nucleic acid, comprising a labeled nucleic acid according to claim 34.

38. A solid support to which is attached a reagent according to claim 1.

40. A method for the capture of nucleic acids, comprising:

providing a solid support to which is directly or indirectly attached at least one biological molecule according to claim 19, 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 a nucleic acid.

9. A temperature-stable labeling reagent of formula (6):

10. The labeling reagent, according to claim 9, of formula (7):

12. The reagent according to claim 9, wherein:

L comprises a motif —

(O—

CH₂—

CH₂)—

, repeated from 1 to 20 times, and

—

Z—

is —

NH—

, —

NHCO—

or —

CONH—

.

14. A method for the synthesis of a labeling reagent according to claim 9, comprising the following steps:

a) providing a label or a label precursor having a reactive function R⁶,

b) providing a linker arm of formula (8);

R⁷—

(Z—

(CH₂)_p)_m—

R⁸

in which;

—

Z—

represents —

NH—

, —

NHCO—

, or —

CONH—

,

m is an integer between 2 and 10,

p is an integer between 1 and 10,

R⁷ and R⁸ represent two reactive functions which may be identical or different,

c) reacting together the reactive function R⁶ of said label or label precursor and the function R⁷ the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, R⁶ and R⁷ being complementary,

d) providing a derivative of formula (9);

16. The method of synthesis according to claim 14, further comprising:

an additional step consisting of protection of the ketone or aldehyde function of compound (9), and

a subsequent additional step consisting of deprotection of said ketone or aldehyde function.

18. A method for the labeling of a biological molecule, comprising bringing into contact, in homogeneous solution in a substantially aqueous buffer, a biological molecule and a reagent according to claim 9.

20. A labeled biological molecule which can be obtained by the method according to claim 18.

22. A method for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid, the method comprising:

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 claim 9,

said reagent coupling covalently and predominantly on at least one phosphate of said fragment.

24. The method according to claim 22, wherein the labeling reagent is chosen from the compounds of formula (3):

26. The method according to claim 24, wherein the fragmentation and the labeling are carried out in two steps.

28. The method according to claim 24, wherein the fragmentation and the labeling are carried out in one step.

31. The method according to claim 26, wherein the labeling is carried out in a substantially aqueous homogeneous solution.

33. The method according to claim 26, wherein the fragmentation is carried out by an enzymatic, physical, or chemical process.

35. A labeled nucleic acid obtained by the method according to claim 22.

37. A kit for the detection of a target nucleic acid, comprising a labeled nucleic acid according to claim 35.

39. A solid support to which is attached a reagent according to claim 9.

41. 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 biological molecule according to claim 20, 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 a nucleic acid.