US5049673A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Fluorescent indicator dyes for calcium working at long wavelengths
Assignee: University of California
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Filing: 1987-10-30
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Granted: 1991-09-17
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Expires: 2007-10-30
Abstract
Abstract The present invention discloses a new class of calcium specific fluorescent indicator dyes having visible excitation and emission wavelengths. The new fluorescent indicator dyes combine at least one tricyclic chromophore with a tetracarboxylate parent CA 2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of BAPTA to give a rhodamine-like or fluorescein-like fluorophore. Binding of calcium 2+ increases the fluorescence of the new compounds by up to 40-fold. The calcium 2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators give better resolution of high [CA 2+ ] levels than were previously obtainable with predecessor compounds such as quin-2 or fluo-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescent microscopy and flow cytometry than the UV required by previous indicators.
Inventors
- 👤 Roger Y. Tsien
- 👤 Akwasi Minta
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 02:18
CPC Classifications
C; C09; C09B; C09B11/00; C09B11/04; C09B11/10; C09B11/24; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/84
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
220 words
A chemical compound having the general formula: ##STR14## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: E 1 and E 2 are independently H, CH 3 , C 2 H 5 , CH 2 OH, COOH, or CH 2 COOH, or E 1 and E 2 together are --(CH 2 ) m --V--CH 2 ) n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH 2 --, --O--, --NH--, --NMe--, --S--, and --S--S--; W is H, OH, or COOH; X is H, Me, COOH, F, Cl, Br, I, or NO 2 ; Y is --O--, --NMe--, --S--, --CH 2 --, --CMe 2 --, --CF 2 --, ##STR15## or a direct sigma bond making a five-membered central ring; Z 1 , Z 2 , Z 3 , and Z 4 are independently H, F, Cl, Br, I, or Me, and Q 1 , Q 2 equal R 1 R 2 N - , ##STR16## or HO - , O.sup.═ or R 1 R 2 N--, O.sup.═, where R 1 and R 2 are independently selected from the group consisting of H, Me, and Et; or Z 1 , Q 1 , Z 3 together are ##STR17## and Z 2 , Q 2 , Z 4 together are ##STR18##
Claim 2
Dependent Claim
220 words
A chemical compound having the general formula: ##STR14## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: E 1 and E 2 are independently H, CH 3 , C 2 H 5 , CH 2 OH, COOH, or CH 2 COOH, or E 1 and E 2 together are --(CH 2 ) m --V--CH 2 ) n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH 2 --, --O--, --NH--, --NMe--, --S--, and --S--S--; W is H, OH, or COOH; X is H, Me, COOH, F, Cl, Br, I, or NO 2 ; Y is --O--, --NMe--, --S--, --CH 2 --, --CMe 2 --, --CF 2 --, ##STR15## or a direct sigma bond making a five-membered central ring; Z 1 , Z 2 , Z 3 , and Z 4 are independently H, F, Cl, Br, I, or Me, and Q 1 , Q 2 equal R 1 R 2 N - , ##STR16## or HO - , O.sup.═ or R 1 R 2 N--, O.sup.═, where R 1 and R 2 are independently selected from the group consisting of H, Me, and Et; or Z 1 , Q 1 , Z 3 together are ##STR17## and Z 2 , Q 2 , Z 4 together are ##STR18##
Claim 3
Dependent Claim
13 words
The compound of claim 1 wherein said tetraacetic acid esters are alpha-acyloxyalkyl esters.
Claim 4
Dependent Claim
12 words
The compound of claim 1 wherein said alpha-acyloxyalkyl esters are acetoxymethyl esters.
Claim 5
Dependent Claim
221 words
A chemical compound having the general formula: ##STR19## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: E 1 and E 2 are independently H, CH 3 , C 2 H 5 , CH 2 OH, COOH, or CH 2 COOH, or E 1 and E 2 together are --(CH 2 ) m --V--CH 2 ) n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH 2 --, --O--, --NH--, --NMe--, --S--, and --S--S--; W is H, OH, or COOH; X is H, Me, COOH, F, Cl, Br, I, or NO 2 ; Y is --O--, --NMe--, --S--, --CH 2 --, --CMe 2 --, --CF 2 --, ##STR20## or a direct sigma bond making five-membered central ring; Z 1 , Z 2 , Z 3 , and Z 4 are independently H, F, Cl, Br, I, or Me, and Q 1 , Q 2 equal R 1 R 2 N - , ##STR21## or HO - , O.sup.═ or R 1 R 2 N - , O.sup.═, where R 1 and R 2 are independently selected from the group consisting of H, Me, and Et; or Z 1 , Q 1 , Z 3 together are ##STR22## and Z 2 , Q 2 , Z 4 together are ##STR23##
Claim 6
Dependent Claim
221 words
A chemical compound having the general formula: ##STR19## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein: E 1 and E 2 are independently H, CH 3 , C 2 H 5 , CH 2 OH, COOH, or CH 2 COOH, or E 1 and E 2 together are --(CH 2 ) m --V--CH 2 ) n -- where m and n are independently 1 or 2 and V is selected from the group consisting of --CH 2 --, --O--, --NH--, --NMe--, --S--, and --S--S--; W is H, OH, or COOH; X is H, Me, COOH, F, Cl, Br, I, or NO 2 ; Y is --O--, --NMe--, --S--, --CH 2 --, --CMe 2 --, --CF 2 --, ##STR20## or a direct sigma bond making five-membered central ring; Z 1 , Z 2 , Z 3 , and Z 4 are independently H, F, Cl, Br, I, or Me, and Q 1 , Q 2 equal R 1 R 2 N - , ##STR21## or HO - , O.sup.═ or R 1 R 2 N - , O.sup.═, where R 1 and R 2 are independently selected from the group consisting of H, Me, and Et; or Z 1 , Q 1 , Z 3 together are ##STR22## and Z 2 , Q 2 , Z 4 together are ##STR23##
Claim 7
Dependent Claim
13 words
The compound of claim 4 wherein said tetraacetic acid esters are alpha-acyloxyalkyl esters.
Claim 8
Dependent Claim
12 words
The compound of claim 4 wherein said alpha-acyloxyalkyl esters are acetoxymethyl esters.
Claim 9
Dependent Claim
18 words
A fluorescent, calcium binding compound comprised of (9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 10
Dependent Claim
18 words
A fluorescent, calcium binding compound comprised of (9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 11
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of (9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 12
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of (9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-dimethylamino-3H-xanthen-3-ylidene)dimethylammonium and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 13
Dependent Claim
18 words
A fluorescent, calcium binding compound comprised of 9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 14
Dependent Claim
18 words
A fluorescent, calcium binding compound comprised of 9-(6-hydroxy-4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy) phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 15
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 16
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 17
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
Claim 18
Dependent Claim
17 words
A fluorescent, calcium binding compound comprised of 9-(4-bis(carboxymethyl)amino-3-(2-(2-bis(carboxymethyl)amino-5-methylphenoxy)ethoxy)phenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-one and the pharmaceutically acceptable non-toxic salts and esters thereof.
References Cited (Backward Citations)
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-09-16 | 1995-03-18 | 1995-09-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-09-16 | 1999-03-18 | 1999-09-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-09-16 | 2003-03-18 | 2003-09-17 | $7700.00 | Window Closed / Lapsed |