MARKING REAGENTS BEARING DIAZO AND NITRO FUNCTIONS, METHODS FOR THE SYNTHESIS OF SUCH REAGENTS AND METHODS FOR DETECTING BIOLOGICAL MOLECULES

• US 20100136538A1
• Filed: 06/09/2008
• Published: 06/03/2010
• Est. Priority Date: 06/11/2007
• Status: Active Grant

• Current Assignee: CNRS, Biomerieux B.V. (Compagnie Merieux Alliance SAS), Universite De Strasbourg (Financière Meeschaert SA)
• Original Assignee: CNRS, Biomerieux B.V. (Compagnie Merieux Alliance SAS), Universite De Strasbourg (Financière Meeschaert SA)
• Inventors: Laayoun, Ali, Kotera, Mitsuharu, Laurent, Alain
• Granted Patent: US 8,309,695 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The present invention relates to a labeling reagent of formula:

in which:

• • R₁ represents at least one detectable label,
  • L and A are each a linker arm,
  • n is an integer equal to 1, and
  • u is an integer between 0 and 2.
    The present invention also describes a method of synthesizing said markers and also applications for the labeling of biological molecules, more particularly nucleic acids, with a labeling reagent bearing diazo and nitro functions. The invention is particularly suitable for use in the diagnostics field.

Claims

1. A labeling reagent of formula (A):

in which;

R₁ represents a detectable label or at least two detectable labels linked to one another by at least one multimeric structure,

R₂ and R₃ represent, independently of one another;

H, NO₂, Cl, Br, F, I, R₄-(L)_n-Y—

X—

, OR, SR, NR², R, NHCOR, CONHR or COOR, with R being alkyl or aryl, and R₄ represents a detectable label or at least two detectable labels linked to one another by at least one multimeric structure,

L is a linker arm comprising a linear chain of at least two covalent bonds,

n is an integer equal to 1,

A is a linker arm comprising at least one covalent double bond allowing the conjugation of the diazo function with the aromatic ring, and

u is an integer between 0 and 2.

2. A labeling reagent as claimed in claim 1, of formula (C):

in which;

R₁ represents a detectable label or at least two detectable labels linked to one another by at least one multimeric structure,

R₂ and R₃ represent, independently of one another;

H, NO₂, Cl, Br, F, I, R₄-(L)_n-Y—

X—

, OR, SR, NR², R, NHCOR, CONHR or COOR, with R being alkyl or aryl, and R₄ represents a detectable label or at least two detectable labels linked to one another 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 1.

3. A labeling reagent as claimed in claim 1, of formula (E):

4. A reagent as claimed in claim 1, wherein the nitro group is in meta or para position.

5. A reagent as claimed in claim 1, wherein R₁ is composed of a D-biotin residue of formula (F):

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

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

b) said cyclic precursor is subsequently opened with a halogen acid to form a halogenated aromatic ketone,

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

d) said protected precursor is subjected to an amination reaction (Gabriel reaction) to form an aminated precursor,

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

f) the labeled precursor is subjected to a reaction for deprotection of the carbonyl function, to form a labeled and carbonylated precursor, and lastly

g) the labeled and carbonylated precursor is converted into a labeling reagent as claimed in any of claims 1 to 5 by conversion of the carbonyl function into a diazo function (Bamford Stevens reaction).

7. A method of labeling a biological molecule, comprising contacting a biological molecule, in homogeneous solution in a substantially aqueous buffer, with a reagent as claimed in claim 1.

8. A labeled biological molecule obtainable by the method as claimed in claim 7.

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

fragmenting the nucleic acid;

attaching a label to at least one of the fragments via a labeling reagent selected from the reagents as claimed in claim 1,

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

10. The method as claimed in claim 9, wherein fragmenting and labeling are performed in two steps.

11. The method as claimed in claim 9, wherein fragmenting and labeling are performed in one step.

12. The method as claimed in claim 9, wherein labeling is performed in substantially aqueous homogeneous solution.

13. The method as claimed in claim 9, wherein fragmenting is performed enzymatically, physically or chemically.

14. A labeled nucleic acid obtainable by the method as claimed in claim 9.

15. A kit for detecting a target nucleic acid, comprising a labeled nucleic acid as claimed in claim 14.

16. A solid support on which is bound a reagent as claimed in claim 1.

17. A method of capturing nucleic acids, comprising the following steps:

providing a solid support on which is bound, directly or indirectly, at least one biological molecule as claimed in claim 8, the biological molecule comprising a diazomethyl function,

contacting said support with a biological sample which may contain free nucleic acids, and

washing the solid support where the molecule or molecules is/are bound covalently at least to a nucleic acid.