Phenyl xanthene dyes

• US 9,090,775 B2
• Filed: 02/24/2014
• Issued: 07/28/2015
• Est. Priority Date: 05/09/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y., Menchen, Steven, Zou, Ruiming, Benson, Scott
• Primary Examiner(s): Leeser, Erich A
• Application Number:
  US14/188,309
• Publication Number:
  US 20140242665A1
• Time in Patent Office: 519 Days
• Field of Search: 549/382
• US Class Current: 1/1
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• 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 11/12 without any OH group bound to an aryl nucleus
• CPC Class Codes: C09B 11/22 containing OH groups bound to an aryl nucleus and their ethers and 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: C09B 11/26 Triarylmethane dyes in which at least one of the aromatic nuclei is heterocyclic phthaleins C09B11/24
• CPC Class Codes: C09B 13/00 Oxyketone dyes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 62/0025 Specific dyes not provided for in groups C09B62/004 - C09B62/018

Abstract

Fluorescent phenyl xanthene dyes are described that comprise any fluorescein, rhodamine or rhodol comprising a particular C9 phenyl ring. One or both of the ortho groups on the lower C9 phenyl ring is ortho substituted with a group selected from alkyl, heteroalkyl, alkoxy, halo, haloalkyl, amino, mercapto, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno, sulfinyl, and sulfino. In one embodiment, halo and/or hydroxy groups are used. Optimal dyes contain a lower C9 phenyl ring in which both ortho groups are the same and the lower ring exhibits some form a symmetry relative to an imaginary axis running from the phenyl rings point of attachment to the remainder of the xanthene dye through a point para to the point of attachment. The phenyl xanthene dyes may be activated. Furthermore, the phenyl xanthene dyes may be conjugated to one or more substances including other dyes. The phenyl xanthene dyes are useful for a number of purposes, including labels for use in automated DNA sequencing as well the formation of fluorescent “bar codes” for polymeric particles used in the multiplexed analysis of analytes.

Claims

1. A lipid soluble fluorescent phenyl xanthene dye having a structure of formula III:

2. The lipid soluble phenyl xanthene dye of claim 1 wherein the phenyl ring substituents are selected from the group consisting of hydrogen, alkoxy, halo, and haloalkyl groups.

3. The lipid soluble phenyl xanthene dye of claim 1 wherein R¹¹ and R¹⁵ are identical.

4. The lipid soluble phenyl xanthene dye of claim 1 wherein said dye is a fluorescein.

5. The lipid soluble phenyl xanthene dye of claim 1 further comprising one or more lipophilic groups selected from (C4-C20) alkyls, (C5-C40) aryls, and/or (C6-C40) arylalkyls.

6. The lipid soluble phenyl xanthene dye of claim 5 wherein said dye contains one or more linkers that contain a linking group capable of forming a covalent or non-covalent linkage to a second substance.

7. The lipid soluble phenyl xanthene dye of claim 6 wherein said second substance is selected from a biomolecule, a glass substrate, a metal substrate and a polymer substrate.

8. The lipid soluble phenyl xanthene dye of claim 6 wherein said second substance is another dye and the phenyl xanthene dye is part of an energy transfer network.

9. A fluorescent phenyl xanthene dye having a structure of the following formula:

11. The phenyl xanthene dye of claim 9 wherein —

S is a direct bond or a spacing moiety.

12. The phenyl xanthene dye of claim 9 wherein -LG is an electrophilic group selected from activated esters, acrylamides, acyl azides, acyl halides, acyl nitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boronates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters, and sulfonyl halides.

13. The phenyl xanthene dye of claim 9 wherein -LG is a nucleophilic group selected from amines, anilines, alcohols, phenols, thiols, hydrazines, and hydroxylamines.

14. The phenyl xanthene dye of claim 9 wherein —

CS is a polymer, glass substrate, metal substrate, biomolecule or second dye.

15. The phenyl xanthene dye of claim 14 wherein the biomolecule is selected from peptides, polypeptides, proteins, amino acids, enzymes, receptors, antibodies, hormones, polysaccharides, carbohydrates, oligonucleotides, polynucleotides, nucleosides, and nucleic acids.

16. The phenyl xanthene dye of claim 14 wherein the second dye is selected from phenyl xanthene dyes, cyanine dyes, phthalocyanine dyes, squarine dyes, acridine dyes, alizarene dyes, azo dyes, anthraquinine dyes, BODIPY®

dyes, coumarin dyes, lanthanide complexes, oxazine dyes, phenazathionium dyes, phenazoxonium dyes, porphyrin dyes, pyrene dyes, pyrilium dyes, perylene dyes, pheoxazine dyes and phenezine dyes.

10. A phenyl xanthene having a structure of formula (III):

17. The phenyl xanthene dye of claim 10 wherein —

S is a direct bond or a spacing moiety.

18. The phenyl xanthene dye of claim 10 wherein -LG is an electrophilic group selected from activated esters, acrylamides, acyl azides, acyl halides, acyl nitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boronates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters, and sulfonyl halides.

19. The phenyl xanthene dye of claim 10 wherein -LG is a nucleophilic group selected from amines, anilines, alcohols, phenols, thiols, hydrazines, and hydroxylamines.

20. The phenyl xanthene dye of claim 10 wherein —

CS is a polymer, glass substrate, metal substrate, biomolecule or second dye.

21. The phenyl xanthene dye of claim 20 wherein the biomolecule is selected from peptides, polypeptides, proteins, amino acids, enzymes, receptors, antibodies, hormones, polysaccharides, carbohydrates, oligonucleotides, polynucleotides, nucleosides, and nucleic acids.

22. The phenyl xanthene dye of claim 20 wherein the second dye is selected from phenyl xanthene dyes, cyanine dyes, phthalocyanine dyes, squarine dyes, acridine dyes, alizarene dyes, azo dyes, anthraquinine dyes, BODIPY®

dyes, coumarin dyes, lanthanide complexes, oxazine dyes, phenazathionium dyes, phenazoxonium dyes, porphyrin dyes, pyrene dyes, pyrilium dyes, perylene dyes, pheoxazine dyes and phenezine dyes.