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US 4,952,495 AGranted
Hydrolyzable compounds which release electron transfer agents and analytical use of same
AssigneeEastman Kodak Company
InventorsRobert T. Belly, Patricia M. Scensny, Annie L. Wu, Chung-yuan Chen
Life of this patent
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Abstract
Hydrolyzable substrates comprise blocked moieties which, when cleaved from the substrate during hydrolysis, provide electron transfer agents. The released electron transfer agents can be recycled between a reductant and a reducible compound that upon reduction provides a detectable species. Alternatively, they can be recycled between an oxidant and an oxidizable compound that upon oxidation provides a detectable species. These substrates are useful in analytical compositions, elements and methods for the determination of hydrolytic analytes, such as hydrolytic enzymes or biological cells containing such enzymes.
Claims14
A compound containing a non-functioning electron transfer agent which can be enzymatically hydrolyzed to release a functioning electron transfer agent, said compound represented by the formula:
- BLOCK-ETA
- wherein BLOCK is an enzymatically hydrolyzable group selected from the group consisting of:
- --CO--R* wherein R* is unsubstituted alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group,
- thioxophosphono or a salt thereof,
- a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide,
- or a mono-, oligo- or polysaccharide linked in a glycosidic bond to ETA,
- and ETA is said non-functioning electron transfer agent that when released provides a functioning electron transfer agent selected from the group consisting of phenazine, naphthazine, phenothiazine, a phenazonium compound or a p-benzo- or naphthoquinone electron transfer agent in its reduced form, said BLOCK and ETA being connected through an oxy in the 1- or 4- ring positions where said functioning electron transfer agent is a p-benzo- or naphthoquinone, and an imino linking group from a ring nitrogen where said functioning electron transfer agent is a phenazine, naphthazine, phenothiazine or phenazonium compound,
- provided that when BLOCK is thioxophosphono or a salt thereof, or a mono-, oligo- or polysaccharide, ETA provides a p-benzoquinone or naphthoquinone electron transfer agent in its reduced form,
- said p-benzo- or naphthoquinone having the structure:
- ##STR13## wherein R.sup.7 and R.sup.8 are independently hydrogen, hydroxy, halo, cyano, nitro, carboxy, carboxyalkyl, carboxamido, sulfonamido, trihaloalkyl, sulfonyl, carboxaldehyde, carbonylalkyl, alkyl, alkenyl, alkoxy, hydroxyalkyl, hydroxyalkoxy, alkoxyalkyl, alkoxyalkoxy, acetoxyalkyl, acetoxyalkoxy, aryl, alkaryl, heterocycle or heteroalkyl,
- R.sup.9 and R.sup.10 are independently selected from the groups defined for R.sup.7 and R.sup.8, or taken together supply the atoms needed to complete a 4- to 8-membered fused carbocyclic or heterocyclic ring,
- provided at least one of R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is not hydrogen.
9 more elements
An analytical composition comprising:
- (a) a reducible compound which is an organic compound which provides a detectable signal in response to reduction by an electron transfer agent in its reduced form, and (b) a compound containing a non-functioning ETA which can be enzymatically hydrolyzed to release a functioning ETA, said compound represented by the formula:
- BLOCK-ETA
- wherein BLOCK is an enzymatically hydrolyzable group selected from the group consisting of:
- --CO--R* wherein R* is unsubstituted alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group.
- phosphono or thioxophosphono or a salt thereof,
- a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide,
- or a mono-, oligo- or polysaccharide linked in a glycosidic bond to ETA,
- and ETA is said non-functioning electron transfer agent that when released provides a functioning electron transfer agent selected from the group consisting of phenazine, naphthazine, phenothiazine, phenazonium compound or an o- or p- benzo- or naphthoquinone electron transfer agent in its reduced form which is capable of reducing said reducible compound, said BLOCK and ETA being connected through an oxy in the 1-, 2- or 4- ring positions where said functioning electron transfer agent is an o- or p-benzo- or naphthoquinone, and an imino linking group from a ring nitrogen where said functioning electron transfer agent is a phenazine, naphthazine, phenothiazine or phenazonium compound,
- provided that when BLOCK is phosphono or thioxophosphono or a salt thereof, or a mono-, oligo- or polysaccharide, ETA provides a p-benzoquinone or naphthoquinone electron transfer agent in its reduced form.
- said p-benzo- or naphthoquinone having the structure:
- ##STR14## wherein R.sup.7 and R.sup.8 are independently hydrogen, hydroxy, halo, cyano, nitro, carboxy, carboxyalkyl, carboxamido, sulfonamido, trihaloalkyl, sulfonyl, carboxaldehyde, carbonylalkyl, alkyl, alkenyl, alkoxy, hydroxyalkyl, hydroxyalkoxy, alkoxyalkyl, alkoxyalkoxy, acetoxyalkyl, acetoxyalkoxy, aryl, alkaryl, heteroxycle or heteroalkyl,
- R.sup.9 and R.sup.10 are independently selected from the groups defined for R.sup.7 and R.sup.8, or taken together supply the atoms needed to complete a 4- to 8-membered fused carbocyclic or heterocyclic ring,
- provided at least one of R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is not hydrogen.
10 more elements
A dry analytical element for the determination of a hydrolytic enzyme or living cells containing same comprising an absorbent carrier material, and containing a compound containing a non-functioning ETA which can be enzymatically hydrolyzed to release a functioning ETA, said compound represented by the formula:
- BLOCK-ETA
- wherein BLOCK is an enzymatically hydrolyzable group selected from the group consisting of:
- --CO--R* wherein R* is alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, phosphono or thioxophosphono or a salt thereof,
- a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide,
- or a mono-, oligo- or polysaccharide linked in a glycosidic bond to ETA,
- and ETA is said non-functioning electron transfer agent that when released provides a functioning electron transfer agent selected from the group consisting of phenazine, naphthazine, phenothiazine, phenazonium compound or an o- or p-benzo- or naphthoquinone electron transfer agent in its reduced form, said BLOCK and ETA being connected through an oxy in the 1-, 2- or 4- ring positions where said functioning electron transfer agent is an o- or p-benzo- or naphthoquinone, and an imino linking group from a ring nitrogen where said functioning electron transfer agent is a phenazine, naphthazine, phenothiazine or phenazonium compound,
- provided than when BLOCK is phosphono or thioxophosphono or a salt thereof, or a mono- oligo- or polysaccharide, ETA provides a p-benzoquinone or naphthoquinone electron transfer agent in its reduced form,
- said p-benzo- or naphthoquinone having the structure:
- ##STR16## wherein R.sup.7 and R.sup.8 are independently hydrogen, hydroxy, halo, cyano, nitro, carboxy, carboxyalkyl, carboxamido, sulfonamido, trihaloalkyl, sulfonyl, carboxyaldehyde, carbonylalkyl, alkyl, alkenyl, alkoxy, hydroxyalkyl, hydroxyalkoxy, alkoxyalkyl, alkoxyalkoxy, acetoxyalkyl, acetoxyalkoxy, aryl, alkaryl, heterocycle or heteroalkyl,
- R.sup.9 and R.sup.10 are independently selected from the groups defined for R.sup.7 and R.sup.8, or taken together supply the atoms needed to complete as 4- to 8-membered fused carbocyclic or heterocyclic ring,
- provided at least one of R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is not hydrogen.
8 more elements
A method for the determination of a hydrolytic enzyme or living cells containing same comprising the steps of;
- A. under hydrolyzing conditions, contacting a sample of a liquid suspected of containing a hydrolytic enzyme or an organism containing same with a compound containing a non-functioning ETA which can be enzymatically hydrolyzed to release a functioning ETA, said compound represented by the formula:
- BLOCK-ETA
- wherein BLOCK is an enzymatically hydrolyzable group selected from the group consisting of:
- --CO--R* wherein R* is alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, phosphono or thioxophosphono or a salt thereof,
- a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide,
- or a mono-, oligo- or polysaccharide linked in a glycosidic bond to ETA,
- and ETA is said non-functioning electron transfer agent that when released provides a functioning electron transfer agent selected from the group consisting of phenazine, naphthazine, phenothiazine, phenazonium compound or an o- or p-benzo- or naphthoquinone electron transfer agent in its reduced form, said BLOCK and ETA being connected through an oxy in the 1-, 2- or 4- ring position where said functioning electron transfer agent is an o- or p-benzo- or naphthoquinone, and an imino linking group in a ring nitrogen where said functioning electron transfer agent is a phenazine, naphthazine, phenothiazine or phenazonium compound,
- provided that when the BLOCK is phosphono or thioxophosphono or a salt thereof, or a mono-, oligo- or polysaccharide, ETA provides a p-benzoquinone or naphthoquinone electron transfer agent in its reduced form,
- said p-benzo- or naphthoquinone having the structure:
- ##STR18## wherein R.sup.7 and R.sup.8 are independently hydrogen, hydroxy, halo, cyano, nitro, carboxy, carboxyalkyl, carboxamido, sulfonamido, trihaloalkyl, sulfonyl, carboxyaldehyde, carbonylalkyl, alkyl, alkenyl, alkoxy, hydroxyalkyl, hydroxyalkoxy, alkoxyalkyl, alkoxyalkoxy, acetoxyalkyl, acetoxyalkoxy, aryl, alkaryl, heterocycle or heteroalkyl,
- R.sup.9 and R.sup.10 are independently selected from the groups defined for R.sup.7 or R.sup.8, or taken together supply the atoms needed to complete a 4- to 8-membered fused carbocyclic or heterocyclic ring,
- provided at least one of R.sup.7, R.sup.8,R.sup.9 and R.sup.10 is not hydrogen
- said contacting being carried out in the presence of a reducible compound which can be reduced by said electron transfer agent to provide a detectable species, and
- B. determining said detectable species provided from the reduction of said reducible compound.
11 more elements
Dependencies are inferred from the claim text.