Patent US5242805A successfully refreshed from Google Patents.
US5242805A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Long wavelength lipophilic fluorogenic glycosidase substrates
Assignee: Molecular Probes Inc
•
Filing: 1991-08-23
•
Granted: 1993-09-07
•
Expires: 2011-08-23
Abstract
Abstract The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of the general formula: ##STR1## wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; where at least one of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 is a lipophilic residue of the formula --L(CH 2 ) n CH 3 , where n is greater than 3 and less than 22, and where L is a methylene --CH 2 --, an amide --NHCO--, a sulfonamide --NHSO 2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--; and where the remainder of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 , which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH 2 ) m CH 3 , where m is less than 22, and where L' is a methylene --CH 2 --, an amide --NHCO--, a sulfonamide --NHSO 2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--. A preferred embodiment of the invention is a non-fluorescent substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, an orange to red fluorescent detection product which is retained inside a viable cell more than about 2 hours at greater than about 15° C. and which is non-toxic to the cell. The substrates are used for evaluating a glycosidic enzyme in living plant or animal cells whether the enzyme is present endogenously; present as a result of manipulation of the cell's genome, or added to the cell exogenously, such as by covalently binding the enzyme to a protein to form an enzyme-protein complex that enters the cell.
Inventors
- 👤 John J. Naleway
- 👤 Yu-zhong Zhang
- 👤 Richard P. Haugland
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 17:21
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C07; C07H; C07H17/00; C12Q2334/00; G; G01; G01N; G01N2333/00; G01N2333/90; G01N2333/914; G01N2333/924
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
204 words
A glycosidase substrate comprising a resorufin derivative of the formula: ##STR7## wherein Gly is a carbohydrate bonded through an anomeric carbon atom to the derivative in a glycosidic linkage; where at least one of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 is a first lipophilic residue of the formula --L(CH 2 ) n CH 3 , where n is greater than 3 and less than 22, and where L is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and where the remainder of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH 2 ) m CH 3 , where m is less than 22, and where L' is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--.
Claim 2
Dependent Claim
204 words
A glycosidase substrate comprising a resorufin derivative of the formula: ##STR7## wherein Gly is a carbohydrate bonded through an anomeric carbon atom to the derivative in a glycosidic linkage; where at least one of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 is a first lipophilic residue of the formula --L(CH 2 ) n CH 3 , where n is greater than 3 and less than 22, and where L is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and where the remainder of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH 2 ) m CH 3 , where m is less than 22, and where L' is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--.
Claim 3
Dependent Claim
12 words
A substrate, as claimed in claim 1, wherein Gly is a monosaccharide.
Claim 4
Dependent Claim
32 words
A substrate, as claimed in claim 1, wherein Gly is a sialic acid, a glucose, galactose, galactose, mannose, fructose, glucosamine, glucuronic acid, fucose, rhamnose, galactosamine, an arabinose, iduronic acid, or a cellobiose.
Claim 5
Dependent Claim
19 words
A substrate, as claimed in claim 3, wherein the Gly is a galactose, a glucuronic acid, or a cellobiose.
Claim 6
Dependent Claim
27 words
A substrate, as claimed in claim 4, wherein Gly is linked to the resorufin derivative in a β-glycosidic linkage through an anomeric carbon atom of said carbohydrate.
Claim 7
Dependent Claim
11 words
a substrate, as claimed in claim 5, wherein Gly is β-D-galactose.
Claim 8
Dependent Claim
12 words
A substrate, as claimed in claim 5, wherein Gly is β-D-glucuronic acid.
Claim 9
Dependent Claim
11 words
A substrate, as claimed in claim 5, wherein Gly is β-D-glucose.
Claim 10
Dependent Claim
11 words
A substrate, as claimed in claim 5, wherein Gly is β-cellobiose.
Claim 11
Dependent Claim
19 words
A substrate, as claimed in claim 1, wherein L is methylene --CH 2 --, amide --NHCO--, or carboxamide --CONH--.
Claim 12
Dependent Claim
11 words
A substrate, as claimed in claim 10, wherein L is methylene.
Claim 13
Dependent Claim
20 words
A substrate, as claimed in claim 1, wherein R 2 is the lipophilic residue and n is less than 11.
Claim 14
Dependent Claim
25 words
A substrate, as claimed in claim 1, wherein R 2 is the lipophilic residue and n is greater than 9 and less than about 18.
Claim 15
Dependent Claim
29 words
A substrate, as claimed in claim 13, wherein 1-3 of substituents R 4 , R 6 , and R 8 are halogens, which may be the same or different.
Claim 16
Dependent Claim
59 words
A substrate, as claimed in claim 1, wherein Gly is β-D-galactose, β-D-glucuronic acid, β-D-glucose, or β-cellobiose; R 2 is an lipophilic residue --L(CH 2 ) n CH 3 , where n is greater than 8 and less than 18, and L is methylene --CH 2 --, amide --NHCO--, urethane --NHCOO--, urea --NHCONH--, thiourea --NHCSNH--, or sulfonamide --NHSO 2 --.
Claim 17
Dependent Claim
30 words
A substrate, as claimed in claim 15, wherein R 2 is --L(CH 2 ) 10 CH 3 , and L is alkyl --CH 2 --, amide --NHCO--, or carboxamide --CONH--.
Claim 18
Dependent Claim
75 words
A nonfluorescent resorufin substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, a fluorescent resorufin detection product excitable at between about 460 nm and 570 nm and with fluorescence emission maximum at a wavelength longer than about 550 nm, which fluorescent detection product is retained inside a viable cell more than about 2 hours at greater than about 15° C. and which is non-toxic to the cell.
Claim 19
Dependent Claim
75 words
A nonfluorescent resorufin substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, a fluorescent resorufin detection product excitable at between about 460 nm and 570 nm and with fluorescence emission maximum at a wavelength longer than about 550 nm, which fluorescent detection product is retained inside a viable cell more than about 2 hours at greater than about 15° C. and which is non-toxic to the cell.
Claim 20
Independent Claim
280 words
A method for evaluating a glycosidic enzyme in a sample comprising: a) contacting the sample to be evaluated with a non-fluorescent resorufin substrate of the formula ##STR8## wherein Gly is a carbohydrate attached through its anomeric carbon in a glycosidic linkage that is specifically hydrolyzable by said enzyme to yield a fluorescent phenoxazine analog; where at least one of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 is a first lipophilic residue of the formula --L(CH 2 ) n CH 3 , where n is greater than 3 and less than 22, and where L is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and where the remainder of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH 2 ) m CH 3 , where m is less than 22, and where L' is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; b) removing a first portion of said sample; c) exciting said first portion of the sample with a radiation source at a wavelength of between about 460 nm and about 570 nm; and d) observing the first portion in conjunction with means for the detecting fluorescence intensity of the fluorescent analog.
Claim 21
Independent Claim
280 words
A method for evaluating a glycosidic enzyme in a sample comprising: a) contacting the sample to be evaluated with a non-fluorescent resorufin substrate of the formula ##STR8## wherein Gly is a carbohydrate attached through its anomeric carbon in a glycosidic linkage that is specifically hydrolyzable by said enzyme to yield a fluorescent phenoxazine analog; where at least one of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 is a first lipophilic residue of the formula --L(CH 2 ) n CH 3 , where n is greater than 3 and less than 22, and where L is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and where the remainder of substituents R 1 , R 2 , R 4 , R 6 , R 8 , and R 9 which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH 2 ) m CH 3 , where m is less than 22, and where L' is a methylene --CH 2 --, an amide --NHCO--, sulfonamide --NHSO 2 --, carboxamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; b) removing a first portion of said sample; c) exciting said first portion of the sample with a radiation source at a wavelength of between about 460 nm and about 570 nm; and d) observing the first portion in conjunction with means for the detecting fluorescence intensity of the fluorescent analog.
Claim 22
Dependent Claim
43 words
A method, as claimed in claim 18, for evaluating a glycosidic enzyme in living cells and wherein contacting the sample with the substrate comprises incubating the cells in a medium containing said substrate for sufficient time, such that the substrate enters the cells.
Claim 23
Dependent Claim
48 words
A method, as claimed in claim 19, for evaluating two glycosidic enzymes, wherein the incubation of cells in a medium containing said substrate further comprises adding a substrate that yields a separately detectable fluorescent reaction product, and incubating for sufficient time, such that both substrates enter the cells.
References Cited (Backward Citations)
US3731222A
Cited
US4812409A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-09-06 | 1997-03-08 | 1997-09-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-09-06 | 2001-03-08 | 2001-09-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-09-06 | 2005-03-08 | 2005-09-07 | $7700.00 | Window Closed / Lapsed |