Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer

• US 6,008,373 A
• Filed: 06/07/1995
• Issued: 12/28/1999
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Carnegie Mellon University
• Original Assignee: Carnegie Mellon University
• Inventors: Mujumdar, Ratnakar B., Waggoner, Alan S., Mujumdar, Swati R.
• Primary Examiner(s): Higel, Floyd D.
• Application Number:
  US08/476,880
• Time in Patent Office: 1,665 Days
• Field of Search: 544/212, 544/328, 546/272, 546/273, 548/427, 548/455, 535/6, 536/800, 436/6
• US Class Current: 548/427
• CPC Class Codes: C09B 11/22 containing OH groups bound to an aryl nucleus and their ethers and esters
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/086 more than five >CH- groups
• CPC Class Codes: C09B 23/10 The polymethine chain containing an even number of >CH- groups styryl dyes C09B23/14, C09B23/14 takes precedence
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The present invention provides low molecular weight fluorescent labeling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials. The complexes may also contain functional groups permitting covalent reaction with materials containing reactive groups.

Claims

1. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a first fluorochrome having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome when said complex is excited by light to said second fluorochrome;

a linker for covalently attaching said first and second fluorochromes capable of transfer of resonance energy between said first and second fluorochromes;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons; and

at least one group capable of forming a covalent bond with said target material.

2. The complex recited in claim 1 wherein said first fluorochrome has an extinction coefficient greater than 20,000 Liters per mole centimeter.

3. The complex recited in claim 1 wherein said second fluorochrome has a fluorescence quantum yield greater than or equal to 0.05.

6. The complex recited in claim 1 wherein said group is a reactive group that reacts with said target material and said target material has functional groups selected from the group consisting of amino, hydroxyl, sulfhydryl, carboxyl and carbonyl.

7. The complex recited in claim 6 wherein said reactive group is selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide, sulfonyl halide, alkylimidoester, arylimidoester substituted hydrazines, substituted hydroxylamines, carbodiimides, acid halide, and phosphoramidite.

8. The complex recited in claim 1 wherein the combined molecular weight of said first and second fluorochromes and said linker is within the range of about 500 to about 10,000 Daltons.

19. The complex recited in claim 1 wherein said linker is a linker of about 2 to 20 bond lengths.

20. The complex recited in claim 1 wherein said group capable of forming covalent bonds is a functional group capable of covalently binding to a reactive group on the target material, said functional group being selected from the group consisting of amino, sulfhydryl, carbonyl, carboxyl and hydroxy groups and said reactive group being selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide, sulfonyl halide, alkylimidoester, arylimidoester substituted hydrazines, substituted hydroxylamines, carbodiimides, acid halide, and phosphoramidite.

4. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a first fluorochrome having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome to said second fluorochrome when said complex is excited by light;

a linker for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons;

at least one group capable of forming a covalent bond with said target material; and

,

water solubilizing constituents, said water solubilizing constituents being unreactive with said group capable of forming a covalent bond with said target material.

5. The complex recited in claim 4 wherein said solubilizing constituents are selected from the group consisting of amide, sulfonate, sulfate, phosphate, quaternary ammonium, hydroxyl and phosphonate.

9. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a first fluorochrome having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome when said complex is excited by light to said second fluorochrome;

a linker for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons;

at least one group capable of forming a covalent bond with said target material; and

,

a third fluorochrome having third absorption and emission spectra covalently attached through a linker to said second fluorochrome;

the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said second fluorochrome, and a portion of the emission spectrum of said second fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome for transferring energy absorbed from said first fluorochrome to said second fluorochrome to said third fluorochrome.

10. The complex recited in claim 9 wherein said first fluorochrome is selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulfonate, bispyrromethene boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

11. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a plurality of first fluorochromes, each having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of each said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of each said first fluorochrome for transfer of energy absorbed by each said first fluorochrome to said second fluorochrome when said complex is excited by light;

a plurality of linkers for covalently attaching said first fluorochromes to said second fluorochrome for transfer of resonance energy from each said first fluorochromes to said second fluorochrome upon excitation with light;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of one of said first fluorochromes, said second fluorochrome and said linker is less than about 20,000 Daltons; and

,

at least one group capable of forming a covalent bond with said target material.

13. The complex recited in claim 11 wherein said group capable of forming covalent bonds is a reactive group that reacts with said target material and said target material has functional groups selected from the group consisting of amino, hydroxyl, sulfhydryl, carbonyl and carboxyl.

14. The complex recited in claim 13 wherein said reactive group is selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide, sulfonyl halide, alkylimidoester, arylimidoester substituted hydrazines, substituted hydroxylamines, carbodiimides, acid halide, and phosphoramidite.

12. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a plurality of first fluorochromes, each having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of each said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of each said first fluorochrome for transfer of energy absorbed by each said first fluorochrome to said second fluorochrome when said complex is excited by light;

a plurality of linkers for covalently attaching said first fluorochromes to said second fluorochrome for transfer of resonance energy from each said first fluorochromes to said second fluorochrome upon excitation with light;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of one of said first fluorochromes, said second fluorochrome and said linker is less than about 20,000 Daltons;

at least one group capable of forming a covalent bond with said target material; and

,

water solubilizing constituents, said water solubilizing constituents being unreactive with said group capable of forming a covalent bond with said target material.

15. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a first fluorochrome having first absorption and emission spectra;

a plurality of second fluorochromes each having second absorption and emission spectra, the wavelength of the emission maximum of each said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of each said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome and each being capable of accepting energy from said first fluorochrome when said first fluorochrome is excited by light;

a plurality of linkers for covalently attaching said first fluorochrome to each of said second fluorochromes for transfer of resonance energy from said first fluorochrome to each of said second fluorochromes;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons; and

at least one group capable of forming a covalent bond with said target material.

17. The complex recited in claim 15 wherein said group capable of forming covalent bonds is a reactive group that reacts with said target material and said target material has functional groups selected from the group consisting of amino, hydroxyl, sulfhydryl, carbonyl and carboxyl.

18. The complex recited in claim 17 wherein said reactive group is selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, iodoacetamide, dichlorotriazine, maleimide, sulfonyl halide, alkylimidoester, arylimidoester, substituted hydrazines, substituted hydroxylamines, carbodiimides, acid halide, and phosphoramidite.

16. A low molecular weight fluorescent labeling complex for labeling a target material consisting of:

a first fluorochrome having first absorption and emission spectra;

a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome when said complex is excited by light to said second fluorochrome;

a linker for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;

at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons;

at least one group capable of forming a covalent bond with said target material; and

,

water solubilizing constituents, said water solubilizing constituents being unreactive with said group capable of forming a covalent bond with said target material.