US4378458A
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US
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Novel chromogenic and/or fluorogenic substrates for monitoring catalytic or enzymatic activity
Assignee: Roche Diagnostics Corp
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Filing: 1981-03-30
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Granted: 1983-03-29
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Expires: 2001-03-30
Abstract
Abstract 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: <IMAGE> 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.
Inventors
- 👤 James R. Gohlke
- 👤 Eddie Hedaya
- 👤 Jemo Kang
- 👤 Jeanette D. Mier
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 09:00
CPC Classifications
C; C07; C07H; C07H21/00; C07H19/00; C07H19/02; C07H19/04
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
121 words
A substrate having the following structural formula: ##STR9## 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.
Claim 2
Dependent Claim
121 words
A substrate having the following structural formula: ##STR9## 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.
Claim 3
Dependent Claim
11 words
The substrate of claim 1 wherein B is a pyrimidine analog.
Claim 4
Dependent Claim
23 words
The substrate of claim 2 wherein B is a member selected from the group consisting of uracil, dihydrouracil, cytosine, dihydrocytosine and halogenated uracils.
Claim 5
Dependent Claim
10 words
The substrate of claim 2 wherein B is a uracil.
Claim 6
Dependent Claim
9 words
The substrate of claim 1 wherein R is umbelliferonyl.
Claim 7
Dependent Claim
10 words
The substrate of claim 1 wherein R is 4-methyl umbelliferonyl.
Claim 8
Dependent Claim
9 words
The substrate of claim 1 wherein R is flavonyl.
Claim 9
Dependent Claim
9 words
The substrate of claim 1 wherein R' is acetyl.
Claim 10
Dependent Claim
9 words
The substrate of claim 1 wherein R" is calcium.
Claim 11
Dependent Claim
20 words
The substrate of claim 1 wherein R" is a member selected from the group consisting of ammonium, or substituted ammonium.
Claim 12
Dependent Claim
172 words
A substrate at least essentially stable towards medium-induced hydrolysis of the phosphate ester at the 3'-position and having the following structural formula: ##STR10## 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.
Claim 13
Dependent Claim
172 words
A substrate at least essentially stable towards medium-induced hydrolysis of the phosphate ester at the 3'-position and having the following structural formula: ##STR10## 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.
Claim 14
Dependent Claim
14 words
The substrate of claim 11 wherein the moiety at the 5' position is R'".
Claim 15
Dependent Claim
22 words
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.
Claim 16
Dependent Claim
10 words
The substrate of claim 13 wherein R'" is t-butyl dimethylsilyl.
Claim 17
Dependent Claim
13 words
The substrate of claim 11 wherein R' is acetyl and R'" is tetrahydropyran-2-yl.
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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) | 1986-03-28 | 1986-09-27 | 1987-03-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-03-28 | 1990-09-27 | 1991-03-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-03-28 | 1994-09-27 | 1995-03-29 | $7700.00 | Window Closed / Lapsed |