Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-08-14
2001-03-27
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S019000, C435S021000, C549S283000, C549S362000
Reexamination Certificate
active
06207365
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel fluorescent enzyme substrate and a method for determining an enzyme activity by means of the enzyme substrate.
BACKGROUND ART
Techniques are known to determine the activity of a specified enzyme with high sensitivity by measuring its fluorescence. Among the techniques, there is a method known where a 4-methylumbelliferone derivative is employed as a substrate (see, the scheme below). Since the enzyme reaction product (4-methylumbelliferone) from the substrate is a phenol derivative in this method, the enzyme activity in the reaction is inhibited by a phenolate ion that has increased in the reaction solution. Thus, there is the possibility that it will become difficult to determine the enzyme activity consistently within a sufficient period of reaction time.
Also, U.S. Pat. Nos. 5,316,906 and 5,443,986 disclose an enzyme reaction substrate having a certain substituent that is released by an enzyme reaction and emits strong fluorescence as well as a method for determining an enzyme activity by means of the substrate. In this case, the substrate has weak fluorescence even before the enzyme reaction takes place and further, it generates a precipitate after the enzyme reaction (see, the scheme below).
DISCLOSURE OF THE INVENTION
The present inventors have developed novel enzyme substrates and methods for determining enzyme activities, and have discovered novel fluorescent enzyme substrates characterized by the following: (1) they do not display any fluorescence before enzyme reactions, but their reaction products display strong fluorescence after the reactions; and (2) the enzyme reaction products have chemically inert structures (aromatic compounds) that are not phenol derivatives. Furthermore, the present inventors have succeeded in establishing methods for determining enzyme activities by means of such enzyme substrates and accomplished this invention. The scheme as shown below illustrates the concept of the invention.
Namely, the novel enzyme substrates according to this invention yield products having coumarin skeletons (coumarin derivatives) that display strong fluorescence after the enzyme reactions; therefore, they are not provided with functional groups such as phenolic hydroxyl and are biochemically inert, also being not susceptible to aqueous solutions having a wide range of pH.
Furthermore, the methods for determining enzyme activities according to this invention determine the enzyme activities with high sensitivity by employing the above-mentioned substrates through fluorescence measurement.
Specifically, this invention, in a general sense, provides an enzyme substrate represented by the following formula:
(BLOCK-O)-X
cu
wherein BLOCK (or BLOCK group) is any one blocking group selected from the group consisting of: a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt biologically exchangeable with the foregoing groups; a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl; and a monovalent blocking group derivable by removal of any one hydroxyl from a monosaccharide or a polysaccharide, and said BLOCK is cleaved from said substrate by the action of a specified enzyme to yield a HO—X
cu
product, and further, said HO—X
cu
product intramolecularly forms a lactone ring to provide a coumarin derivative;
wherein X
cu
has a structure represented by the following formula and is covalently bound to oxygen O at C
1
carbon,
wherein L represents H, NH
4
, alkyl having 1 to 4 carbons, tetraalkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal; and further,
wherein the coumarin derivative has a structure represented by the following formula:
Moreover, the invention provides the above-mentioned enzyme substrate wherein the C
3
-C
4
bond forms a 5- or 6-membered aromatic ring; H is bound to C
5
, C
6
, and C
7
; and H or CH
3
is bound to C
8
.
Also, the invention provides the above-mentioned enzyme substrate wherein the X
cu
has a structure represented by the following formula:
Also, the invention provides the above-mentioned enzyme substrate wherein dialkylamino having 1 to 3 carbons is bound to C
5
; hydrogen is bound to C
3
, C
4
, C
6
, and C
7
; and H or CH
3
is bound to C
8
.
Furthermore, the invention provides the above-mentioned enzyme substrate wherein the X
cu
has a structure represented by the following formula:
Still further, the invention provides the above-mentioned enzyme substrate wherein alkyloxy having 1 to 3 carbons is bound to C
3
and C
5
; H is bound to C
4
, C
6
, and C
7
; and H or CH
3
is bound to C
8
.
Also, the invention provides the above-mentioned enzyme substrate wherein the X
cu
has a structure represented by the following formula:
In addition, the invention provides the above-mentioned enzyme substrate wherein the BLOCK is a phosphoric acid group (PO
3
−2
), D-galactopyranoside, or acetyl (CH
3
CO).
Moreover, the invention provides a method for determining an enzyme activity, said method comprising the following two steps ((A)and (B)) of:
(A) treating a sample containing an enzyme to be detected, with an enzyme substrate represented by the following formula:
(BLOCK-O)-X
cu
wherein BLOCK is any one blocking group selected from the group consisting of: a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt biologically exchangeable with the foregoing groups; a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl; and a monovalent blocking group derivable by removal of any one hydroxyl from a monosaccharide or a polysaccharide, and said BLOCK is cleaved from said substrate by the action of a specified enzyme to yield a HO—X
cu
product, and further, said HO—X
cu
product intramolecularly forms a lactone ring to provide a coumarin derivative;
wherein X
cu
has a structure represented by the following formula, the C
7
-C
2
bond and the C
8
-C
9
bond have cis-configuration with respect to the C
7
═C
8
double bond, and the X
cu
is covalently bound to oxygen O at C
1
carbon,
wherein L represents H, NH
4
, alkyl having 1 to 4 carbons, tetralkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal; and further,
wherein said coumarin derivative has a structure represented by the following formula:
and
(B) detecting said coumarin derivative.
Also, the invention provides a method for determining an enzyme activity, said method comprising the following two steps ((A) and (B)) of:
(A) treating a sample containing an enzyme to be detected, under irradiation conditions, with an enzyme substrate represented by the following formula:
(BLOCK-O)-X
cu
wherein BLOCK is any one blocking group selected from the group consisting of: a monovalent blocking group derivable by removal of one hydroxyl from a phosphoric acid group, a sulfuric acid group, or a salt biologically exchangeable with the foregoing groups; a monovalent blocking group derivable by removal of a hydroxyl from an aliphatic carboxyl, an aromatic carboxyl, an amino acid carboxyl, or a peptide carboxyl; and a monovalent blocking group derivable by removal of any one hydroxyl from a monosaccharide or a polysaccharide, and said BLOCK is cleaved from said substrate by the action of a specified enzyme to yield a HO—X
cu
product, and further, said HO—X
cu
product intramolecularly forms a lactone ring to provide a coumarin derivative;
wherein X
cu
has a structure represented by the following formula, the C
7
-C
2
bond and the C
8
-C
9
bond have trans-configuration with respect to the C
7
═C
8
double bond, and the X
cu
is covalently bound to oxygen O at C
1
carbon,
wherein L represents H, NH
4
, alkyl having 1 to 4 carbons, tetralkylammonium having 1 to 4 carbons, or an alkaline metal or an alkaline earth metal; a
Nohta Hitoshi
Shiono Hirofumi
Utsuyama Chika
Gitomer Ralph
Laboratory of Molecular Biophotonics
Leydig , Voit & Mayer, Ltd.
Moran Marjorie A.
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