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Novel substrates for monitoring of catalytic activity resulting in hydrolytic release of products which can be readily detected by spectrophotometric or fluorometric means are provided. The substrates have the general formula: ##STR## wherein B is a nucleotide base capable of assisting in hydrolysis

Novel substrates for monitoring of catalytic activity resulting in hydrolytic release of products which can be readily detected by spectrophotometric or fluorometric means are provided. The substrates have the general formula: ##STR## wherein B is a nucleotide base capable of assisting in hydrolysis of the phosphate ester at the 3'-position; R is a moiety selected from the group consisting of umbelliferonyl, 4-methylumbelliferonyl, 3-flavonyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl, phenylsulfonate, phenylsulfonyl and phenylsulfoxide; R' is a moiety selected from the group consisting of hydrogen, alkyl, alkenyl cycloalkyl, aryl, araalkyl, acyl, oxaalkyl, thioalkyl, oxacycloalkyl, and thiocycloalkyl and R" is hydrogen or a cation selected from the group consisting of calcium, barium, lithium, sodium, ammonium, substituted ammonium and pyridinium. The substrates are capable of undergoing catalytic-induced hydrolysis of the phosphate ester at the 3'-position to yield a species capable of being monitored spectrophotometrically or fluorometrically.

대표청구항 ▼

1. A substrate having the following structural formula: ##STR## wherein B is a nucleotide base capable of assisting in hydrolysis of the phosphate ester at the 3'-position, R is a moiety selected from the group consisting of umbelliferonyl, 4-methyl umbelliferonyl, 3-flavonyl, o-nitrophenyl, m-nitro

1. A substrate having the following structural formula: ##STR## wherein B is a nucleotide base capable of assisting in hydrolysis of the phosphate ester at the 3'-position, R is a moiety selected from the group consisting of umbelliferonyl, 4-methyl umbelliferonyl, 3-flavonyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl, phenylsulfonate, phenylsulfonyl and phenylsulfoxide, R' is a moiety selected from the group consisting of hydrogen, alkyl, alkenyl cycloalkyl, aryl, araalkyl, acyl, oxaalkyl, thioalkyl, oxacycloalkyl, and thiocycloalkyl, and R" is hydrogen or a cation selected from the group consisting of calcium, barium, lithium, sodium, ammonium, substituted ammonium and pyridinium, said substrate being capable of undergoing enzymatic-induced hydrolysis of the phosphate ester at the 3'-position to yield a species capable of being monitored spectrophotometrically or fluorometrically. 2. The substrate of claim 1 wherein B is a pyrimidine analog. 3. The substrate of claim 2 wherein B is a member selected from the group consisting of uracil, dihydrouracil, cytosine, dihydrocytosine and halogenated uracils. 4. The substrate of claim 2 wherein B is a uracil. 5. The substrate of claim 1 wherein R is umbelliferonyl. 6. The substrate of claim 1 wherein R is 4-methyl umbelliferonyl. 7. The substrate of claim 1 wherein R is flavonyl. 8. The substrate of claim 1 wherein R' is acetyl. 9. The substrate of claim 1 wherein R" is calcium. 10. The substrate of claim 1 wherein R" is a member selected from the group consisting of ammonium, or substituted ammonium. 11. A substrate at least essentially stable towards medium-induced hydrolysis of the phosphate ester at the 3'-position and having the following structural formula: ##STR## wherein B is a nucleotide base capable of assisting, after deblocking of the substrate, in hydrolysis of the phosphate ester at the 3'-position, R is a moiety selected from the group consisting of umbelliferonyl, 4-methyl umbelliferonyl, 3-flavonyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl, phenylsulfonate, phenylsulfonyl and phenylsulfoxide, R' is a moiety selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, araalkyl, acyl, oxaalkyl and oxacycloalkyl, and R" is hydrogen or a cation selected from the group consisting of calcium, barium, lithium, sodium, ammonium, substituted ammonium and pyridinium and R'" is a blocking group capable of at least essentially preventing medium-induced hydrolysis of the phosphate ester at the 3' position, R'" being capable of being removed to provide a substrate characterized by the ability to undergo catalytic induced hydrolysis of the phosphate ester at the 3' position to yield a species capable of being monitored spectrophotometrically or fluorometrically. 12. The substrate of claim 11 wherein the moiety at the 5' position is R'". 13. The substrate of claim 12 wherein R'" is a member selected from the group consisting of tetrahydropyranyl, 4-methoxytetrahydropyranyl, 1-ethoxyethyl and t-butyl dimethylsilyl. 14. The substrate of claim 13 wherein R'" is t-butyl dimethylsilyl. 15. The substrate of claim 11 wherein R' is acetyl and R'" is tetrahydropyran-2-yl.