Rhodamine-based fluorophores useful as labeling reagents

• US 6,750,357 B1
• Filed: 06/25/1999
• Issued: 06/15/2004
• Est. Priority Date: 06/25/1999
• Status: Expired due to Term

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Syngen Incorporated
• Inventors: Chiarello, Ronald H., Yokobata, Kathy E., Liu, Wing
• Primary Examiner(s): SOLOLA, TAOFIQ A
• Application Number:
  US09/344,226
• Time in Patent Office: 1,817 Days
• Field of Search: 549/394, 436/172, 436/800
• US Class Current: 549/394
• CPC Class Codes: C07D 311/80 Dibenzopyrans; Hydrogenated dibenzopyrans
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• 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: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1007 Non-condensed systems
• CPC Class Codes: C09K 2211/1048 with oxygen
• CPC Class Codes: C09K 2211/1088 containing oxygen as the only heteroatom
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Fluorescent dyes based on rhodamine are derivatized to form labeled conjugates that fluoresce upon excitation with light of an appropriate wavelength. Particularly preferred embodiments are certain single isomer form rhodamine phosphoramidites. These rhodamine phosphoramidites enhance the efficiency of synthesizing rhodamine-labeled oligonucleotides by solid phase methods. Conjugate embodiments of the invention are prevented from being converted to a non-fluorescent lactam form due to having a fully substituted amide linkage derived from the 3-position carboxylate.

Claims

1. A fluorophore conjugate comprising:

a conjugated substance and a fluorophore, the conjugated substance being an amino acid, peptide, protein, nucleotide, oligonucleotide, or nucleic acid to which is attached one or more fluorophores, the fluorophore conjugate having the structure illustrated by Formula 1

where R₁ and R₁₀ taken alone are hydrogen or halogen;

R₂, R₅, R₆ and R₉ taken alone are hydrogen, alkyl, carboxyalkyl, aminoalkyl, alkylether, alkylthioether, halogen or alkoxy;

R₃, R₄, R₇ and R₈ taken alone are hydrogen, an alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, or aryl;

R₂ and R₃ taken together are alkyl chains each having from 2 to 5 carbon atoms connecting the 2′

carbon to the nitrogen attached to the 3′

carbon;

R₉ and R₈ taken together are alkyl chains each having from 2 to 5 carbon atoms connecting the 7′

carbon to the nitrogen attached to the 6′

carbon;

R₄ and R₅ taken together are alkyl, each having from 2 to 5 carbon atoms connecting the 4′

carbon to the nitrogen attached to the 3′

carbon;

R₆ and R₇ taken together are alkyl, each having from 2 to 5 carbon atoms connecting the 5′

carbon to the nitrogen attached to the 6′

carbon;

R₃ and R₄ taken together form an alkyl or alkylene chain containing up to 5 atoms in the principal chain, consisting of carbon and one or more heteroatoms from the group consisting of nitrogen or oxygen, with both terminal valence bonds of said chain being attached to the nitrogen attached to the 3′

carbon;

R₇ and R₈ taken together form an alkyl or alkylene chain containing up to 5 atoms in the principal chain, consisting of carbon and one or more heteroatoms from the group consisting of nitrogen or oxygen, with both terminal valence bonds of said chain being attached to the nitrogen attached to the 6′

carbon;

R₁₁, R₁₂, R₁₃, and R₁₄, are each hydrogen or halogen, where R₈ is an alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, and aryl, having from 1 to 10 carbon atoms, and Z represents a linker plus the conjugated substance, wherein said conjugated substance lacks a lactam ring.

2. The conjugate as in claim 1 wherein the conjugated substance is bound to the fluorophore through an amide, ester, ether, disulfide, or thioether linkage.

3. The conjugate as in claim 1 wherein the linkage between the fluorophore and conjugated substance has a phosphate ester.

4. The fluorescent conjugate as in claim 1 wherein the conjugated substance is attached to a solid support.

5. The fluorescent conjugate as in claim 4 wherein the solid support is controlled pore glass.

6. The fluorescent conjugate as in claim 4 wherein the solid support is a polymer support.

7. The fluorescent conjugate as in claim 1 wherein the conjugated substance is part of a cell membrane.

8. The fluorescent conjugate as in claim 1 wherein the conjugated substance is part of a viral coat.

9. The fluorescent conjugate as in claim 1 wherein the fluorophore is derived from tetramethylrhodamine.

10. The fluorescent conjugate as in claim 1 wherein the fluorophore is derived from rhodamine 101.

11. The fluorescent conjugate as in claim 1 wherein the fluorophore is derived from rhodamine B.