Energy transfer dyes with enhanced fluorescence

• US 6,849,745 B2
• Filed: 10/29/2001
• Issued: 02/01/2005
• Est. Priority Date: 05/03/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Spurgeon, Sandra L., Rosenblum, Barnett
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/014,743
• Publication Number:
  US 20020086985A1
• Time in Patent Office: 1,191 Days
• Field of Search: 435/6, 536/24.33, 536/25.3, 536/26.66, 549/223
• US Class Current: 549/223
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/04 derived from triarylmethanes , i.e. central C-atom is substituted by amino, cyano, alkyl
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R₂₁Z₁C(O)R₂₂R₂₈ where R₂₁ is a C_1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R₂₈ includes a functional group which attaches the linker to the acceptor dye.

Claims

1. An energy transfer dye comprising:

a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;

a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and

a non-nucleosidic linker linking the 5- or 6-ring position of the donor dye to the 5- or 6-ring position of the acceptor dye.

2. The energy transfer dye of claim 1 in which the donor dye is a fluorescein dye.

3. The energy transfer dye of claim 1 in which the linker has a backbone that is less than 9 atoms in length.

4. The energy transfer dye of claim 1 in which the linker comprises a functional group selected from an alkene, a diene, an alkyne, a five membered ring having at least one unsaturated bond, a six membered ring having at least one unsaturated bond and a fused ring structure.

5. The energy transfer dye of claim 1 which further comprises a linking group suitable for attaching the energy transfer dye to another substance.

6. The energy transfer dye of claim 5 in which the linking group is attached to the 4′

-position of the 4,7-dichlororhodamine acceptor dye.

7. The energy transfer dye of claim 1 which comprises the structure:

wherein

R¹, R², R³ and R⁴ are each, independently of one another, selected from hydrogen and alkyl, or alternatively R¹ and R⁵, R² and R⁶, R³ and R⁸ and/or R⁴ and R⁹ may be taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring;

R⁵, R⁶, R⁷, R⁹ and R¹⁰ are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitric, alkoxy, phenyl and substituted phenyl, or alternatively, R⁶ and R⁷ and/or R⁹ and R¹⁰ may be taken together with the atoms to which they are bonded to form a benzo group;

R⁸ is selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl and linking group;

X¹ and X³ are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy;

L is the linker linking the donor and acceptor dyes;

R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R¹² and R¹³ and/or R¹⁵ and R¹⁶ may be taken together with the atoms to which they are bonded to form a benzo group;

R¹⁴ is selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl and linking group; and

X¹¹, X¹², X¹³ and X¹⁵ are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy.