Fluorescent dyes and methods of use thereof

• US 10,106,573 B2
• Filed: 07/08/2016
• Issued: 10/23/2018
• Est. Priority Date: 10/11/2011
• Status: Active Grant

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Li, Zaiguo, Pande, Praveen
• Primary Examiner(s): Oh, Taylor V
• Application Number:
  US15/205,438
• Publication Number:
  US 20160311846A1
• Time in Patent Office: 837 Days
• Field of Search: 546 37, 546 48, 5483037, 548525, 549223, 549227, 549394, 536 2532
• CPC Class Codes: C07D 311/88 Nitrogen atoms
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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: C09B 11/02 derived from diarylmethanes
• CPC Class Codes: C09B 11/06 Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
• 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: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D

Abstract

Provided are methods for labeling target molecules, such as nucleic acids, with fluorescent dye compounds having the formula

One method embodiment includes contacting reactive group Z of the fluorescent dye compound with the target molecule such that reactive group Z reacts with the target molecule to form a covalent bond between the group and the target molecule. Another method embodiment includes contacting a fluorescent dye compound that further includes a first member of a binding pair, with a target molecule that includes a second member of the binding pair. Also provided are target molecules labeled with the fluorescent dye compounds.

Claims

1. A method for fluorescently labeling a target molecule, comprising the steps of:

providing a fluorescent dye compound having the formula;

2. The method of claim 1, wherein -A-B—

Z is

3. The method of claim 2, wherein n is 1-4.

4. The method of claim 2, wherein -A-B—

Z is

5. The method of claim 1, wherein -A-B—

Z is

6. The method of claim 1, wherein the fluorescent dye compound has the structure

7. The method of claim 6, wherein R⁶ and R⁹ are both H or CH₃.

8. The method of claim 6, wherein X and Y are

(a) OH and O, respectively;

(b) NHCH₂CH₃ and NCH₂CH₃, respectively;

or

(c) N(CH₃)₂ and N⁺(CH₃)₂, respectively.

9. The method of claim 1, wherein the fluorescent dye compound is selected from the group consisting of:

10. The method of claim 9, wherein the fluorescent dye compound is selected from the group consisting of:

11. The method of claim 1, wherein the fluorescent dye compound is selected from the group consisting of:

12. The method of claim 1, wherein the fluorescent dye compound is selected from the group consisting of:

13. The method of claim 1, wherein the target molecule is a nucleic acid.

14. The method of claim 1, wherein the target molecule is a peptide nucleic acid.

15. The method of claim 1, wherein the target molecule is a nucleotide.

16. The method of claim 1, wherein the carbon lengths for said alkenyl, alkynyl, and alkyl groups are in the range of 1 to 10.

17. The method of claim 13, wherein the carbon lengths for said alkenyl, alkynyl, and alkyl groups are in the range of 1 to 10.