Heterocycle-substituted xanthene dyes

• US 10,053,484 B2
• Filed: 12/12/2016
• Issued: 08/21/2018
• Est. Priority Date: 12/04/2009
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Mao, Fei, Leung, Wai-Yee, Cheung, Ching-Ying, Hoover, Hye Eun
• Primary Examiner(s): Rozof, Timothy R
• Application Number:
  US15/375,649
• Publication Number:
  US 20170247402A1
• Time in Patent Office: 617 Days
• CPC Class Codes: A61K 49/0017 Fluorescence in vivo
• CPC Class Codes: C07D 311/88 Nitrogen atoms
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• 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: C07D 491/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07H 15/26 Acyclic or carbocyclic radicals, substituted by hetero rings
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention relates to fluorescent dyes in general. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.

Claims

1. A compound of Formula 1a:

2. The compound of claim 1, wherein X is O.

3. The compound of claim 1, wherein said fused saturated or unsaturated ring formed by R₁ and R₉ and/or R₄ and R₅ ring is substituted by -L-PO₃²⁻

or -L-SO₃⁻

.

4. The compound of claim 1, wherein said fused saturated or unsaturated ring formed by R₁ and R₉ and/or R₄ and R₅ is substituted by at least one alkyl.

5. The compound of claim 1, wherein at least one of R₂ and R₃ is -L-SO₃⁻

.

6. The compound of claim 5, wherein at least one of R₂ and R₃ is —

SO₃⁻

.

7. The compound of claim 1, wherein R₇ is pyrazolyl substituted with -L-R_x, and optionally further substituted with halogen, CO₂⁻

, CN, alkyl, alkenyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminosulfonyl, dialkylaminosulfonyl, aminosulfonyl, aryl, heteroaryl, -L-PO₃²⁻

, L-SO₃⁻

, or -L-R_x.

8. The compound of claim 1, wherein R₇ is a pyrazolyl substituted with -L-R_x, and an aryl.

9. The compound of claim 8, wherein the aryl is substituted with one or more substituent selected from the group consisting of —

OH, oxo, halo, alkoxy, dialkylamino, —

PO₃²⁻

, —

SO₃⁻

, —

CO₂⁻

, a reactive group, and a heterocyclyl.

10. The compound of claim 8, wherein the aryl is substituted with a halo and a —

SO₃⁻

.

11. The compound of claim 10, wherein R_x is a carboxylic acid.

12. The compound of claim 1, wherein at least one or more pair of R₂ and R_1a, R₃ and R_4a, R₄ and R₅, R₅ and R₆, R₈ and R₉, or R₉ and R₁, together with the atoms to which they are attached form one or more fused saturated or unsaturated rings that are optionally substituted by at least one halogen, CN, alkyl, alkenyl, alkoxy, alkylthio, alkylamino, dialkylamino, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminosulfonyl, dialkylaminosulfonyl, aminosulfonyl, aryl, heteroaryl, -L-PO₃²⁻

, L-SO₃⁻

, or -L-R_x.

13. A kit comprising:

i) a compound of claim 1; and

ii) instructions instructing the use of the compound.

14. The kit of claim 13, wherein the kit additionally comprises a buffer and optionally materials or devices for purifying conjugation products.

15. A biomolecule comprising a label having a structure of a compound of claim 1, comprising at least one reactive moiety, wherein the at least one reactive moiety of the compound has undergone a reaction which attaches the label to the biomolecule.

16. The biomolecule of claim 15, wherein the biomolecule comprises a polynucleotide or a polypeptide.

17. An immunoglobin comprising a label having a structure of a compound of claim 1, comprising at least one reactive moiety, wherein at least one reactive moiety of the compound has undergone a reaction which attaches the label to the immunoglobin.