| 摘要 |
The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of the general formula: <IMAGE> 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)nCH3, 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)mCH3, 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 DEG 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.
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