Certain tricyclic and pentacyclic-hetero nitrogen rhodol dyes

• US 5,227,487 A
• Filed: 04/16/1990
• Issued: 07/13/1993
• Est. Priority Date: 04/16/1990
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard P., Whitaker, James E.
• Primary Examiner(s): Rotman, Alan L.
• Application Number:
  US07/509,360
• Time in Patent Office: 1,184 Days
• Field of Search: 544/150, 544/70, 544/230, 546/15, 546/196, 546/62, 546/47, 548/525, 548/407, 549/223, 549/225
• US Class Current: 546/15
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C09B 11/10 Amino derivatives of triarylmethanes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials

Abstract

Novel fluorescent dyes based on the rhodol structure are provided. The new reagents contain functional groups capable of forming a stable fluorescent product with functional groups typically found in biomolecules or polymers including amines, phenols, thiols, acids, aldehydes and ketones. Reactive groups in the rhodol dyes include activated esters, isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides, aldehydes, alcohols, acrylamides and haloacetamides. The products are detected by their absorbance or fluorescence properties. The spectral properties of the fluorescent dyes are sufficiently similar in wavelengths and intensity to fluorescein or rhodamine derivatives as to permit use of the same equipment. The dyes, however, show less spectral sensitivity to pH in the physiological range than does fluorescein, have higher solubility in non-polar solvents and have improved photostability and quantum yields.

Claims

1. A fluorescent dye having the structure:

##STR2## wherein R¹ is H, and R² is H, CF₃ CO, CH₃, or C₂ H₅ ;

or R¹ in combination with R² is (CH₂)₃ ;

R³ is H, CH₃, or C₂ H₅, and R⁴ is H;

or R³ in combination with R⁴ is (CH₂)₃ ;

R⁵ is H, CH₂ NH₂, CH₂ NHCOCH₂ Cl, CH₂ NHCOCH₂ l, CH₂ NHCOCH;

CH₂, or CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is H, Cl, Br, CH₃, or C₂ H₅, or (CH₂)_n CH₃ where n is an integer from 0 to 15, or (CH₂)_n CO₂ H,

where n is an integer from 0 to 11; and

R⁷ is H, CO₂ H, ##STR3## NH₂, NCS, CH₂ NHCOCH₂ Cl, or CH₂ NHCOCH₂ l;

such that R⁷ is not H if R⁵ is H.

2. A fluorescent dye as recited in claim 1 wherein R¹, R³, R⁴, R⁵, and R⁶ are H;

R² is selected from the group consisting of H and CF₃ CO; and

R⁷ is selected from the group consisting of CO₂ H and ##STR4##

3. A fluorescent dye as recited in claim 1 wherein R¹ is CH₃ ;

R² is C₂ H₅ ;

R³, R⁴, and R⁵ are H;

R⁶ is selected from the group consisting of H, Cl, Br, (CH₂)_n CH₃ wherein n is an integer from 0 to 15, and (CH₂)_n CO₂ H, where n is an integer from 0 to 11; and

R⁷ is selected from the group consisting of CO₂ H and ##STR5##

4. A fluorescent dye as recited in claim 1 wherein R¹, R⁴, and R⁵ are H;

R² and R³ are independently selected from the group consisting of CH₃ and C₂ H₅ ;

R⁶ is selected from the group consisting of H, Cl, Br, CH₃ and C₂ H₅ ; and

R⁷ is selected from the group consisting of CO₂ H and ##STR6##

5. A fluorescent dye as recited in claim 1 wherein R¹ in combination with R² is (CH₂)₃ ;

R³, R⁴, and R⁵ are H;

R⁶ is selected from the group consisting of H, Cl, Br, (CH₂)_n CH₃ where n is an integer from 0 to 15, and (CH₂)_n CO₂ H, where n is an integer from 0 to 11; and

R⁷ is selected from the group consisting of CO₂ H and ##STR7##

6. A fluorescent dye as recited in claim 1 wherein R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is selected from the group consisting of H, CH₂ NH₂, CH₂ NHCOCH₂ Cl, CH₂ NHCOCH₂ l, CH₂ NHCOCH;

CH₂, and CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is selected from the group consisting of H, Cl, Br, (CH₂)_n CH₃ where n is an integer from 0 to 15, and (CH₂)_n CO₂ H where n is an integer from 0 to 11; and

R⁷ is selected from the group consisting of CO₂ H and ##STR8##

7. A fluorescent dye as recited in claim 1 wherein R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁶ is Cl; and

R⁷ is selected from the group consisting of NH₂, NCS, CH₂ NHCOCH₂ Cl and CH₂ NHCOCH₂ l.

8. A fluorescent dye as recited in claim 1 having the formula:

##STR9## wherein R⁵ is selected from the group consisting of H, CH₂ NH₂, CH₂ NHCOCH₂ Cl, CH₂ NHCOCH₂ l, CH₂ NHCOCH;

CH₂, and CH₂ NHCOC₆ H₄ N₃ ;

R⁶ is selected from the group consisting of H, Cl, Br, (CH₂)_n CH₃ where n is an integer from 0 to 15, and (CH₂)_n CO₂ H where n is an integer from 0 to 11; and

R⁷ is selected from the group consisting of CO₂ H and ##STR10##

9. A fluorescent dye as recited in claim 1 having the formula:

##STR11## wherein R⁵ is H;

R⁶ is Cl; and

R⁷ is selected from the group consisting of NH₂, NCS, CH₂ NHCOCH₂ Cl and CH₂ NHCOCH₂ l.

10. A fluorescent dye having the structure:

##STR12## wherein R¹ and R⁴ are independently hydrogen, alkyl, carboxyalkyl, aminoalkyl, halogen or alkoxy, the alkyl portions of which contain less than 4 carbons;

R² and R³ are independently hydrogen, methyl or ethyl, or trifluoroacetyl; and

R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁶ is H, Cl, Br, CH₃, or CH₂ H₅, and R⁹ is COCH₃ ;

or R⁶ and R⁹ in combination are (CH₂)₂ CO; and

R⁷ is H, CO₂ H or ##STR13## such that R⁷ is not H if R⁹ is COCH₃.

11. A fluorescent dye as recited in claim 10 wherein R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁶ is selected from the group consisting of H, Cl, Br, CH₃, and CH₂ H₅ ;

R⁷ is selected from the group consisting of CO₂ H and ##STR14## and R⁹ is COCH₃.

12. A fluorescent dye as recited in claim 10 wherein R¹ in combination with R², or R³ in combination with R⁴ are (CH₂)₃ ;

R⁵ is H;

R⁷ is selected from the group consisting of H, CO₂ H and ##STR15## and R⁶ and R⁹ in combination are (CH₂)₂ CO.

13. A fluorescent dye having the structure:

##STR16## wherein R⁵ is H;

R⁶ is H, Cl, Br, CH₃, or CH₂ H₅, and R⁹ is COCH₃ ;

or R⁶ and R⁹ in combination are (CH₂)₂ CO; and

R⁷ is H, CO₂ H or ##STR17## such that R⁷ is not H if R⁹ is COCH₃.

14. A fluorescent dye as recited in claim 13 wherein R⁵ is H;

R⁶ is selected from the group consisting of H, Cl, Br, CH₃, and CH₂ H₅ ;

R⁷ is selected from the group consisting of CO₂ H and ##STR18## and R⁹ is COCH₃.

15. A fluorescent dye as recited in claim 13 wherein R⁵ is H;

R⁷ is selected from the group consisting of H, CO₂ H and ##STR19## and R⁶ and R⁹ in combination are (CH₂)₂ CO.