Fluorescent entity comprising a fluorophore covalently attached to at least one oligonucleotide and comprising at least one functional group, and uses thereof

• US 20050239073A1
• Filed: 06/06/2003
• Published: 10/27/2005
• Est. Priority Date: 06/06/2002
• Status: Abandoned Application

• Current Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Original Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Inventors: Bazin, Herve, Mathis, Gerard, Maurin, Fabrice, Trinquet, Eric
• Application Number:
  US10/516,744
• Publication Number:
  US 20050239073A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

The invention relates to a fluorescent entity comprising a fluorophore, with the exception of a rare earth metal cryptate, covalently attached to one or more oligonucleotide(s) or oligonucleotide analog(s) and comprising at least one functional group, introduced or generated on the fluorophore or one of the oligonucleotides or oligonucleotide analogs, allowing it to be coupled with a carrier molecule. The invention also relates to a fluorescent conjugate consisting of a fluorescent entity covalently attached to a carrier molecule.

Claims

1. A fluorescent entity comprising a fluorophore, with the exception of a rare earth metal cryptate, covalently attached to one or more oligonucleotide(s) or oligonucleotide analog(s), characterized in that it comprises at least one functional group, introduced or generated on the fluorophore or one of the oligonucleotides or oligonucleotide analogs.

2. The entity as claimed in claim 1, characterized in that the oligonucleotide or the oligonucleotide analog comprises from 2 to 60 nucleotide units.

3. The entity as claimed in claim 1, characterized in that the functional group can be attached to said entity via a spacer arm.

4. The entity as claimed in claim 1, characterized in that the fluorophore comprises one or more aromatic rings and has a high molecular extinction coefficient, greater than 20 000, preferably greater than 50 000.

5. The entity as claimed in claim 1, characterized in that the fluorophore is chosen from rhodamines, cyanins, squaraines, bodipys, fluoresceines and their derivatives.

6. The entity as claimed in claim 1, characterized in that the functional group is chosen from the groups:

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom.

7. The fluorescent entity as claimed in claim 1, of formula (I):

in which;

A represents a group chosen from;

—

N(R₃)_r

r=2 or 3

the dashed lines each represent the carbon atoms required to form 1 to 3 fused rings, the groups R₃ being attached to these rings;

X and Y each represent N, C═

O, O, S or C(CH₃)₂

m has a value 1, 2, 3 or 4;

q has a value 1, 2 or 3;

(R₃)_q represents q groups R₃, which may be identical or different;

the groups R₁, R₂ and R₃ are identical or different and are chosen from hydrogen;

a group —

(CH₂)_s-Z in which s ranges from 0 to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above;

a functional group is chosen from the groups;

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxy-succinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1 and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom; and

an oligonucleotide or oligonucleotide analog optionally comprising a functional group as said;

R₄ is chosen from;

H;

OH;

CH₃;

Cl and the groups of formula;

the substituents R₄ in the allylic position possibly forming, with the polyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms, which may or may not be saturated, said rings possibly containing one or more atoms of O, N and S, and possibly being optionally substituted with an oxo group.

8. The fluorescent entity as claimed in claim 1, of formula (II) or (III):

or

in which

the dashed lines each represent the carbon atoms required to form 1 to 3 fused rings, the groups R₃ being attached to these rings;

X represents N, C═

O, O, S or C(CH₃)₂;

m has a value 1, 2, 3 or 4;

q has a value 1, 2 or 3;

(R₃)₂ represents q groups R₃, which may be identical or different;

the groups R₁ and R₃, which may be identical or different, are chosen from hydrogen;

a group —

(CH₂)_s-Z in which s ranges from 0 to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above;

a functional group is chosen from the groups;

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxy-succinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom;

an oligonucleotide or oligonucleotide analog optionally comprising a functional group as said;

R₄ is chosen from;

H;

OH;

CH₃;

Cl and the groups of formula;

the substituents R₄ in the allylic position possibly forming, with the polyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms, which may or may not be saturated, said rings possibly containing one or more atoms of O, N and S, and possibly being optionally substituted with an oxo group.

9. The fluorescent entity as claimed in claim 1, of formula (IV), (V), (VI) or (VII):

in which R₁, R₂, R₃ and R₅ are identical or different and are chosen from hydrogen;

a group —

(CH₂)_s-Z in which s ranges from 0 to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above;

a functional group is chosen from the groups;

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxy-succinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom; and

an oligonucleotide or oligonucleotide analog optionally comprising a functional group as said;

q has a value 1, 2 or 3.

10. The fluorescent entity as claimed in claim 1, of formula (VIII):

in which the substituents R₆ to R₁₂ are chosen from;

hydrogen;

a halogen;

an alkyl;

a cycloalkyl;

aryl;

arylalkyl;

acyl;

sulfo;

a functional group is chosen from the groups;

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1 and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom; and

an oligonucleotide or oligonucleotide analog optionally comprising a functional group chosen from those mentioned as said.

11. The entity as claimed in claim 7, of formula (IX):

in which R₁, R₂, R₃, R₄, X, Y, m and q are as defined above.

12. The entity as claimed in claim 11, in which X and Y each represent a group C(CH₃)₂.

13. The entity as claimed in claim 11, in which

R₁ and R₂ represent an alkyl comprising from 1 to 4 carbon atoms or a group of formula below, at least one of the groups R₁ and R₂ representing a group of formula below:

R₄ represents hydrogen

q=1, m=2

R₃ represents hydrogen;

a group CH₂)_s-Z in which s ranges from 0 to 4 and Z represents a group CH₃, SO₃H, OH or N⁺R₁R₂R₃ in which R₁, R₂ and R₃ are as defined above;

a functional group is chosen from the groups;

maleimide, carboxylic acid, haloacetamide, alkyl halide, azido, hydrazido, aldehyde, ketone, amino, sulfhydryl, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazide, azidonitrophenyl, azidophenyl, azide, 3-(2-pyridyldithio)proprionamide and glyoxal, and groups of formula;

where n ranges from 0 to 8 and p is equal to 0 or 1, and Ar is a 5- or 6-membered heterocycle comprising 1 to 3 hetero atoms, optionally substituted with a halogen atom;

an oligonucleotide or oligonucleotide analog optionally comprising a functional group as said;

R₄ is chosen from;

H;

OH;

CH₃;

Cl and the groups of formula;

the substituents R₄ in the allylic position possibly forming, with the polyethylenic chain, 1 to 3 fused rings containing from 4 to 14 atoms, which may or may not be saturated, said rings possibly containing one or more atoms of O, N and S, and possibly being optionally substituted with an oxo group.

14. The entity as claimed in claim 1, characterized in that the fluorophore is covalently attached to the oligonucleotide either directly or via a spacer arm.

15. The entity as claimed in claim 14, characterized in that the fluorophore is attached to the oligonucleotide via a spacer arm consisting of a divalent organic radical chosen from linear or branched C₁-C₂₀ alkylene groups optionally containing one or more double bonds or triple bonds and/or optionally containing one or more hetero atoms, such as oxygen, nitrogen, sulfur, phosphorus, or one or more carbamoyl or carboxamido group(s);

C₅-C₈ cycloalkylene groups and C₆-C₁₄ arylene groups, said alkylene, cycloalkylene or arylene groups optionally being substituted with alkyl, aryl or sulfonate groups.

16. The entity as claimed in claim 15, characterized in that the spacer arm is chosen from the groups:

in which n₁ and n₂ are between 2 and 6.

17. The entity as claimed in claim 1, characterized in that the oligonucleotide comprises from 5 to 60, in particular 5 to 20, preferably from 5 to 15, nucleotide units.

18. The entity as claimed in claim 17, characterized in that the oligonucleotide consists of a series of ribonucleotide or deoxyribonucleotide units attached to one another via bonds of the phosphodiester type.

19. The entity as claimed in claim 17, characterized in that the oligonucleotide consists of a series of ribonucleotide or deoxyribonucleotide units or of nucleotide analog units modified on the sugar or on the base, attached to one another by natural internucleotide bonds of the phosphodiester type, some of the internucleotide bonds being optionally replaced with phosphonate, phosphoramide or phosphorothioate bonds.

20. The entity as claimed in claim 17, characterized in that the oligonucleotide consists of a series comprising both ribonucleotide or deoxyribonucleotide units attached to one another by bonds of the phosphodiester type and nucleoside analog units attached to one another by amide bonds.

21. The entity as claimed in claim 17, characterized in that the oligonucleotide consists of a series of ribonucleotide or deoxyribonucleotide units attached to one another by bonds of the phosphodiester type and of nucleoside analog units attached to one another by amide bonds, said oligonucleotide comprising at least 5 internucleotide bonds of the phosphodiester type at the end intended to be attached to the fluorophore.

22. The entity as claimed in claim 1, characterized in that the functional group is an amine function of a nucleotide unit of the oligonucleotide or of the oligonucleotide analog, or results from the reaction of a free amine function of a nucleotide unit of the oligonucleotide or the oligonucleotide analog, with a group chosen from the groups:

ester, carboxylic acid, isothiocyanate, aldehyde, carbonyl, sulfonyl halide, alkyl halide, azide, hydrazide, dichlorotriazine, anhydride, haloacetamide, maleimide and sulfhydryl.

23. The entity as claimed in claim 1, characterized in that the functional group results from the reaction of a free amine function of a nucleotide unit of the oligonucleotide or of the oligonucleotide analog, with an N-hydroxysuccinimidyl ester.

24. The entity as claimed in claim 1, characterized in that the functional group(s) is (are) attached to the fluorophore and/or to the oligonucleotide by a spacer arm consisting of a divalent organic radical, chosen from linear or branched C₁-C₂₀ alkylene groups optionally containing one or more double bonds or triple bonds and/or optionally containing one or more hetero atoms, such as oxygen, nitrogen, sulfur, phosphorus, or one or more carbamoyl or carboxamido group(s);

C₅-C₈ cycloalkylene groups and C₆-C₁₄ arylene groups, said alkylene, cycloalkylene or arylene groups being optionally substituted with alkyl, aryl or sulfonate groups.

25. The entity as claimed in claim 24, characterized in that the spacer arm is chosen from the groups:

in which n, and n₂ are between 2 and 6.

26. A fluorescent conjugate consisting of an entity as claimed in claim 1 covalently attached to a carrier molecule.

27. The conjugate as claimed in claim 26, characterized in that the fluorophore of the fluorescent entity is cyanin-5, the oligonucleotide of said entity has the sequence A₁₅ and the carrier molecule is cAMP.

28. The conjugate as claimed in claim 26, characterized in that the final molar ratio is greater than 0 and less than 100, preferably greater than 0 and less than 20.

29. The fluorescent conjugate as claimed in claim 26, characterized in that the carrier molecule is an antibody, an antigen, an intracellular messenger, an intercellular messenger, a protein, a peptide, a hapten, a lectin, biotin, avidin, streptavidin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a nucleic acid, a hormone, a vitamin, a medicinal product, a polymer, a polymeric particle, glass, a particle of glass or a surface made of glass or of a polymer.

30. The fluorescent conjugate as claimed in claim 29, characterized in that the carrier molecule is an antibody or streptavidin.

31. The use of a fluorescent entity as claimed in claim 1, as a fluorescent tracer.

32. The use as claimed in claim 31, for detecting and/or determining, by fluorescence, an analyte in a medium liable to contain it.

33. The use as claimed in claim 32, for determining an interaction between biomolecules;

or for determining a biological activity such as;

an enzyme activity, the activation of a membrane-bound receptor, the transcription of a gene, a membrane transport or a variation in membrane polarization.

34. The use as claimed in claim 31, in a method for screening medicinal products.

35. The use as claimed in claim 34, in which a fluorescent conjugate is used as an acceptor fluorescent compound in the presence of a donor fluorescent compound.

36. The use as claimed in claim 35, in which a fluorescent conjugate is used as a donor fluorescent compound in the presence of an acceptor fluorescent compound.

37. The use as claimed in claim 35, in fluorescence microscopy, in flow cytometry, in fluorescence polarization or in fluorescence correlation.

38. The use of a conjugate as claimed in claim 26, as a contrast agent for optical imaging in vivo.

39. A method for increasing the fluorescence intensity of a fluorophore attached to a carrier molecule, characterized in that a fluorescent entity as claimed in claim 1 is used as a fluorophore.

40. A method for decreasing the phenomenon of aggregation at the surface of a carrier molecule attached to a fluorophore, characterized in that a fluorescent entity as claimed in claim 1 is used in place of said fluorophore.

41. A method for increasing the quantum yield of a fluorophore attached to a carrier molecule, characterized in that a fluorescent entity as claimed in claim 1 is used as a fluorophore.