Photocleavable derivatives of hydroxyprenesulfonic acids

• US 5,514,710 A
• Filed: 01/11/1994
• Issued: 05/07/1996
• Est. Priority Date: 01/11/1994
• 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., Gee, Kyle R.
• Primary Examiner(s): Hollrah, Glennon H.
• Assistant Examiner(s): TILL, MARY C
• Application Number:
  US08/180,543
• Time in Patent Office: 847 Days
• Field of Search: 562/45, 562/46, 562/70, 562/71, 562/72, 558/270, 558/271, 514/512, 514/576, 514/577, 549/453, 549/454
• US Class Current: 514/512
• CPC Class Codes: C07C 309/43 having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system

Abstract

The invention describes the synthesis and use of photoactivated (or caged) fluorescent dyes. Upon illumination at less than about 400 nm the caged dyes release highly fluorescent, water soluble hydroxypyrenesulfonic acid dyes according to the following equation: ##STR1## X, Y, and Z are independently sulfonic acid, a sulfonic acid salt, a hydroxyl group, or hydrogen, with at least one of X, Y, and Z being a sulfonic acid or sulfonic acid salt. LINK is either an ether linkage or a carbonate linkage. BLOCK is a caging group whose photolysis results in liberation of a free hydroxypyrenesulfonic acid dye.

The caged fluorescent dyes are useful for application in aqueous solutions, including fluids of biological origin. The caged dyes of the present invention are especially useful for flow tagging velocimetry.

Claims

1. A compound of the formula ##STR13## wherein at least one of X, Y, and Z is --SO₃ H or --SO₃ --SALT;

the remainder of X, Y and Z are independently --SO₃ H, --SO₃ --SALT, --OH, or H;

where SALT is an alkali metal cation, ammonium, alkylammonium, polyalkylammonium, alkoxyammonium, or polyalkoxyammonium ion, the alkyl portions of each independently containing 1-6 carbon atoms;

LINK is either --O-- or --O--(C═

O)--O--;

and BLOCK is described by the formula ##STR14## where R¹ is hydrogen, carboxylate (--CO₂ H), an alkyl group having 1-6 carbons, or ARYL;

wherein ARYL is an unsubstituted phenyl, or phenyl substituted independently one or more times by --NO₂, --OR³, where R³ is an alkyl having 1-6 carbons;

or --(CH)_n --CO₂ --M, where n=0-3 and M is Li⁺, Na⁺, K⁺, or NH₄⁺ ;

the remaining positions on the aromatic ring A are optionally independently substituted one or more times by --NO₂, or --OR³ ;

or two adjacent positions on aromatic ring A, taken in combination, form a fused 6 membered ring to give a naphthyl structure;

or substituents on two adjacent positions on aromatic ring A, taken in combination, form a methylenedioxy structure;

or BLOCK is described by the formula ##STR15## where the remaining positions on aromatic rings B and C are optionally independently substituted one or more times by halogen, --NO₂, --OR⁴, and --NR⁵ R⁶, where R⁴, R⁵ and R⁶ are independently alkyl groups having 1-6 carbons;

or LINK is --O-- and BLOCK is described by the formula ##STR16## wherein R⁷ is H or Me; and

R² is H, --OH, --OR⁴, or --NR⁵ R⁶.

2. A compound according to claim 1, wherein BLOCK is described by the formula ##STR17## where R^l is hydrogen, carboxylate (--CO₂ H), an alkyl group having 1-6 carbons, or ARYL;

wherein ARYL is an unsubstituted phenyl, or phenyl substituted independently one or more times by --NO₂, --OR³, where R³ is an alkyl having 1-6 carbons;

or --(CH)_n --CO₂ --M, where n=0-3 and M is Li⁺, Na⁺, K⁺, or NH₄⁺ ;

the remaining positions on the aromatic ring A are optionally independently substituted one or more times by --NO₂, --OR³, or --(CH)_n --CO₂ --M;

or two adjacent positions on aromatic ring A, taken in combination, form a fused 6 membered ring to give a naphthyl structure;

or substituents on two adjacent positions on aromatic ring A, taken in combination, form a methylenedioxy structure.

3. A compound according to claim 2, wherein LINK is --O--.

4. A compound according to claim 2, wherein X, Y, and Z are --SO₃ H or --SO₃ --SALT.

5. A compound according to claim 1, wherein BLOCK is described by the formula ##STR18## where R¹ is hydrogen, carboxylate (--CO₂ H), an alkyl group having 1-6 carbons, or ARYL;

wherein ARYL is an alkoxy or nitro-substituted phenyl.

6. A compound according to claim 1 of the formula ##STR19## wherein LINK is --O--or --O--(C═

O)--O--; and

X, Y, and Z are --SO₃ H or --SO₃ --SALT.

7. A compound according to claim 1, wherein BLOCK is described by the formula ##STR20## where the remaining positions on aromatic rings B and C are optionally independently substituted one or more times by halogen, --NO₂, --OR⁴, and --NR⁵ R⁶, where R⁴, R⁵ and R⁶ are independently alkyl groups having 1-6 carbons.

8. A compound according to claim 7, wherein LINK is --O--and X, Y, and Z are --SO₃ H or --SO₃ --SALT.

9. A compound according to claim 7, wherein BLOCK is described by the formula ##STR21##

10. A compound according to claim 1, wherein X, Y, and Z are --SO₃ H or --SO₃ --SALT;

LINK is --O--; and

BLOCK is described by the formula ##STR22## wherein R⁷ is H or Me; and

R² is H, --OH, --OR⁴, or --NR⁵ R⁶.

11. A mixture comprising an aqueous solution of a photoactive compound of the formula ##STR23## wherein at least one of X, Y, and Z is --SO₃ H or --SO₃ --SALT;

the remainder of X, Y and Z are independently --SO₃ H, --SO₃ --SALT, --OH, or H;

where SALT is an alkali metal cation, ammonium, alkylammonium, polyalkylammonium, alkoxyammonium, or polyalkoxyammonium ion, the alkyl portions of each independently containing 1-6 carbon atoms;

LINK is either --O-- or --O----(C═

O)--O--;

and BLOCK is described by the formula ##STR24## where R¹ is hydrogen, carboxylate (--CO₂ H), an alkyl group having 1-6 carbons, or ARYL;

wherein ARYL is an unsubstituted phenyl, or phenyl substituted independently one or more times by --NO₂, --OR³, where R³ is an alkyl having 1-6 carbons;

or --(CH)_n --CO₂ --M, where n=0-3 and M is Li⁺, Na⁺, K⁺, or NH₄⁺ ;

the remaining positions on the aromatic ring A are optionally independently substituted one or more times by --NO₂, --OR³, or --(CH)_n --CO₂ --M;

or two adjacent positions on aromatic ring A, taken in combination, form a fused 6 membered ring to give a naphthyl structure;

or substituents on two adjacent positions on aromatic ring A, taken in combination, form a methylenedioxy structure;

or BLOCK is described by the formula ##STR25## where the remaining positions on aromatic rings B and C are optionally independently substituted one or more times by halogen, --NO₂, --OR⁴, and --NR⁵ R⁶, where R⁴, R⁵ and R⁶ are independently alkyl groups having 1-6 carbons;

or LINK is --O-- and BLOCK is described by the formula ##STR26## wherein R⁷ is H or Me; and

R² is H, --OH, --OR⁴, or --NR⁵ R⁶ ;

wherein the concentration of said compound is greater than 10 nanogram per milliliter and less than 10 milligrams per milliliter.

12. A mixture according to claim 11, further comprising a fluorescent dye of the formula ##STR27## wherein the fluroescent dye is the photolysis product of said photoactive compound, and X, Y, and Z are as defined for said photoactive compound.

13. A mixture according to claim 12, wherein the fluorescent dye is formed by photolysis of said photoactive compound using light having a wavelength of less than about 400 nanometers.

14. A mixture according to claim 13, wherein the fluorescent dye, when illuminated by light at a wavelength greater than about 425 nanometers, fluoresces at a wavelength greater than about 480 nanometers.

15. A mixture according to claim 14, wherein the fluorescence of said fluorescent dye is detected at a site that is remote from the site of the photolysis of said photoactive compound and is performed at a time that is at least 1 microsecond subsequent to the time of photolysis.

16. A mixture according to claim 12, wherein the aqueous solution is a biological fluid.

17. A mixture according to claim 16, wherein the biological fluid is present within a living organism or cell.