Fluorophore chelated lanthanide luminescent probes with improved quantum efficiency

• US 9,221,759 B2
• Filed: 01/13/2011
• Issued: 12/29/2015
• Est. Priority Date: 01/13/2010
• Status: Active Grant

• Current Assignee: New Jersey Institute of Technology, Rutgers University
• Original Assignee: New Jersey Institute of Technology, Rutgers University
• Inventors: Mustaev, Arkady, Kozlov, Maxim, Marras, Salvatore, Krasnoperov, Lev, Wirpsza, Laura, Pillai, Shyamala
• Primary Examiner(s): Hartley, Michael G
• Assistant Examiner(s): SAMALA, JAGADISHWAR RAO
• Application Number:
  US13/522,043
• Publication Number:
  US 20130202536A1
• Time in Patent Office: 1,811 Days
• Field of Search: 424/9.6, 424/130.1, 546/5, 546/82, 546/83, 546/86, 546/90, 546/157
• US Class Current: 1/1
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0028 Oxazine dyes
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07D 215/227 only one oxygen atom which is attached in position 2
• CPC Class Codes: C07D 215/233 only one oxygen atom which is attached in position 4
• CPC Class Codes: C07D 215/38 Nitrogen atoms nitro radicals C07D215/18
• CPC Class Codes: C07D 257/02 not condensed with other rings
• 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: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention relates to novel luminescent compositions of matter containing a fluorophore, synthetic methods for making the compositions, macromolecular conjugates of the compositions, and the use of the compositions in various methods of detection. The invention also provides kits containing the compositions and their conjugates for use in the methods of detection.

Claims

1. A composition comprising:

(i) a fluorophore of Formula (I) or Formula (II); and

(ii) a chelating moiety covalently joined, to the fluorophore,

wherein Formula (I) is;

2. The composition of claim 1, wherein R₂ is selected from the group consisting of CF₃, CH₃, and O—

CH₂CH₃.

3. The composition of claim 1, wherein R₃ is selected from the group consisting of O—

CH₂CH₃ and O—

(CH₂)₃—

N₃.

4. The composition of claim 1, wherein R₄ is selected from the group consisting of H, CH₂C(O)NH—

(CH₂)₄—

N═

C═

S, and CH₂C(O)NH—

(CH₂)₄—

NHC(O)CH₂Br.

5. The composition of claim 1, wherein R₅ is selected from the group consisting of alkylene, heteroalkylene and alkylarylene groups, which further contain a crosslinking group selected from the group consisting of maleimide, disulfide, activated ester, azide and acetylene.

6. The compostion of claim 5, wherein R₅ is

7. The composition of claim 1, wherein R₆ is selected from the group consisting of alkylene, heteroalkylene and alkylarylene groups, which further contain a crosslinking group selected from the group consisting of maleimide, disulfide, activated ester, azide and acetylene.

8. The composition of claim 1, wherein R₇ is selected from the group consisting of alkylene, heteroalkylene and alkylarylene groups, which further contain a crosslinking group selected from the group consisting of maleimide, disulfide, activated ester, azide and acetylene.

9. The composition of claim 1, wherein the fluorophore has the formula:

10. The composition of claim 1, wherein the fluorophore has the formula:

11. The composition of claim 1, wherein the fluorophore has the formula:

12. The compostion of claim 11, wherein X is

13. The luminescent probe composition of claim 1, wherein the chelating moiety is selected from the group consisting of EDTA, DTPA, TTHA, DOTA, TAGA, DOTP, DTPA-BMA, DO2P, and HP-DO3A.

14. The composition of claim 1 further comprising a metal ion chelated to the chelating moiety, wherein the metal is a lanthanide selected from the group consisting of:

Gd(III), Dy(III), Ho(III), Er(III), Eu(III), Tb(III), Sm(III), Ce(III), Pr(III), Yb(III), Tm(III), Nd(III), and Tb(IV).

15. The composition of claim 1, wherein the composition is conjugated to a macromolecule, wherein the macromolecule is selected from the group consisting of a polypeptide and an aptamer with binding affinity to a predetermined peptide target.

16. The composition of claim 15, wherein the macromolecule is a polypeptide selected from the group consisting of an antibody or antigen-binding fragment thereof, a ligand for a cellular receptor, avidin, and streptavidin.

17. The composition of claim 15, wherein the macromolecule is a nucleic acid.

18. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

19. A luminescently labeled hairpin-forming oligonucleotide consisting of:

(a) the luminescent composition of claim 1;

wherein the R₁ chelating moiety is selected from the group consisting of EDTA, DTPA, TTHA, DOTA, TAGA, DOTP, DTPA-BMA, DO2P, and HP-DO3A; and

wherein the luminescent composition further comprises a lanthanide chelated to the chelating moiety, the lanthanide selected from the group consisting of;

Gd(III), Dy(III), Ho(III), Er(III), Eu(III), Tb(III), Sm(III), Ce(III), Pr(III), Yb(III), Tm(III), Nd(III), and Tb(IV);

the luminescent composition covalently conjugated to a hairpin-forming oligonucleotide; and

(b) a quencher moiety capable of quenching the fluorescence of the fluorophore or the luminescence of the lanthanide moiety, wherein the quencher moiety is covalently conjugated to the hairpin-forming oligonucleotide;

the oligonucleotide having a closed conformation including a single-stranded loop and a stem duplex formed by complementary 3′ and

5′

arms, wherein the quencher moiety is in a quenching relationship to at least one of the lanthanide moiety or the fluorophore;

wherein, when excited at the maximum excitation wavelength of the fluorophore, emission at the maximum emission wavelength of the fluorophore is substantially suppressed relative to the unquenched magnitude and emission at the maximum emission wavelength of the fluorophore; and

the oligonucleotide having an open conformation, not including the stem duplex, in which the quencher moiety is not in a quenching relationship with the lanthanide or the fluorophore;

wherein, when excited at the maximum excitation wavelength of one or both of the first and second sensitizer moieties, the luminescence of the lanthanide moiety increases due to fluorescence resonance energy transfer from the fluorophore.

20. The oligonucleotide of claim 19, wherein hybridization of said loop to a target nucleotide sequence causes the oligunucleotide to assume its open configuration.

21. The oligonucleotide of claim 20, wherein the quencher moiety is selected from the group consisting of BHQ, DABCYL, and variants of DABCYL.

22. The oligonucleotide of claim 20, wherein the single-stranded loop and one strand of the stem duplex is complementary to the target strand, whereby the oligonucleotide is capable of serving as a primer for DNA polymerase.

23. The oligonucleotide of claim 19, wherein said oligonucleotide includes a terminal extension capable of serving as a priming region for a DNA polymerase when the olignucleotide is in its closed conformation.