Long wavelength lipophilic fluorogenic glycosidase substrates

• US 5,242,805 A
• Filed: 08/23/1991
• Issued: 09/07/1993
• Est. Priority Date: 08/23/1991
• 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., Naleway, John J., Zhang, Yu-zhong
• Primary Examiner(s): Griffin, Ronald W.
• Assistant Examiner(s): Webber, Pamela S.
• Application Number:
  US07/749,255
• Time in Patent Office: 746 Days
• Field of Search: 536/1.1, 544/102, 435/4, 435/18, 436/3, 436/35, 372/53
• US Class Current: 435/18
• CPC Class Codes: C07H 17/00 Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 2334/00 O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)

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 R₁, R₂, R₄, R₆, R₈, and R₉ is a lipophilic residue of the formula --L(CH₂)_n CH₃, where n is greater than 3 and less than 22, and where L is a methylene --CH₂ --, an amide --NHCO--, a sulfonamide --NHSO₂ --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--; and

where the remainder of substituents R₁, R₂, R₄, R₆, R₈, and R₉, 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'"'"'(CH₂)_m CH₃, where m is less than 22, and where L'"'"' is a methylene --CH₂ --, an amide --NHCO--, a sulfonamide --NHSO₂ --, 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.

Claims

1. 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 R₁, R₂, R₄, R₆, R₈, and R₉ is a first lipophilic residue of the formula --L(CH₂)_n CH₃, where n is greater than 3 and less than 22, and where L is a methylene --CH₂ --, an amide --NHCO--, sulfonamide --NHSO₂ --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and

where the remainder of substituents R₁, R₂, R₄, R₆, R₈, and R₉ 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'"'"'(CH₂)_m CH₃, where m is less than 22, and where L'"'"' is a methylene --CH₂ --, an amide --NHCO--, sulfonamide --NHSO₂ --, carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--.

2. A substrate, as claimed in claim 1, wherein Gly is a monosaccharide.

3. 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.

4. A substrate, as claimed in claim 3, wherein the Gly is a galactose, a glucuronic acid, or a cellobiose.

5. 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.

6. a substrate, as claimed in claim 5, wherein Gly is β

-D-galactose.

7. A substrate, as claimed in claim 5, wherein Gly is β

-D-glucuronic acid.

8. A substrate, as claimed in claim 5, wherein Gly is β

-D-glucose.

9. A substrate, as claimed in claim 5, wherein Gly is β

-cellobiose.

10. A substrate, as claimed in claim 1, wherein L is methylene --CH₂ --, amide --NHCO--, or carboxamide --CONH--.

11. A substrate, as claimed in claim 10, wherein L is methylene.

12. A substrate, as claimed in claim 1, wherein R₂ is the lipophilic residue and n is less than 11.

13. A substrate, as claimed in claim 1, wherein R₂ is the lipophilic residue and n is greater than 9 and less than about 18.

14. A substrate, as claimed in claim 13, wherein 1-3 of substituents R₄, R₆, and R₈ are halogens, which may be the same or different.

15. A substrate, as claimed in claim 1, wherein

Gly is β

-D-galactose, β

-D-glucuronic acid, β

-D-glucose, or β

-cellobiose;

R₂ is an lipophilic residue --L(CH₂)_n CH₃, where n is greater than 8 and less than 18, and L is methylene --CH₂ --, amide --NHCO--, urethane --NHCOO--, urea --NHCONH--, thiourea --NHCSNH--, or sulfonamide --NHSO₂ --.

16. A substrate, as claimed in claim 15, wherein R₂ is --L(CH₂)₁₀ CH₃, and L is alkyl --CH₂ --, amide --NHCO--, or carboxamide --CONH--.

17. 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.

18. 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 R₁, R₂, R₄, R₆, R₈, and R₉ is a first lipophilic residue of the formula --L(CH₂)_n CH₃, where n is greater than 3 and less than 22, and where L is a methylene --CH₂ --, an amide --NHCO--, sulfonamide --NHSO₂ --, carboxamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or thiourea --NHCSNH--; and

where the remainder of substituents R₁, R₂, R₄, R₆, R₈, and R₉ 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'"'"'(CH₂)_m CH₃, where m is less than 22, and where L'"'"' is a methylene --CH₂ --, an amide --NHCO--, sulfonamide --NHSO₂ --, 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.

19. 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.

20. 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.