# US 5,445,943 A · Method for the colorimetric determination of an analyte by means of benzyl alcohol dehydrogenase and a chromogenic redox indicator

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- Number: US 5,445,943 A
- Document ID: US5445943A
- Status: Granted
- Kind: A
- Application number: 08/225,722
- Filed: 1994-04-08
- Granted: 1995-08-29
- Earliest claimed: 1993-04-08
- Assignees: Boehringer Mannheim GmbH
- Inventors: Joachim Hoenes
- Web page: https://patentgrep.com/patent/US5445943A

## CPC

- C07D 471/04: Ortho-condensed systems
- C07C 255/24: containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the same saturated acyclic carbon skeleton
- C07D 231/46: Oxygen atom in position 3 or 5 and nitrogen atom in position 4
- C07D 487/04: Ortho-condensed systems
- C07D 513/04: Ortho-condensed systems
- C07F 9/6561: containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C12Q 1/32: involving dehydrogenase
- Y10T 436/173845: Amine and quaternary ammonium
- Y10T 436/174614: Tertiary amine
- Y10T 436/175383: Ammonia
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The invention concerns a method for the colorimetric determination of NADH by means of enzymatic oxidation with benzyl alcohol dehydrogenase whereby an aromatic nitrosoaniline compound of the general formula I is reduced to a colored quinone diimine ##STR1## in which R.sup.1 denotes hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkyl or alkylene substituted if desired by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, amino substituted if desired once or twice by alkyl which in turn can be substituted if desired by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy and R denotes a residue which forms a color by electron conjugation with a quinone diimiine system. The invention also concerns a corresponding agent as well as a chromogenic nitroso compound of formula I as well as its use for the enzymatic colorimetric determination of an analyte.

## Claims

### Claim 1

A method for the colorimetric determination of NAD(P)H or an analyte which forms NAD(P)H, comprising the steps of
a) reacting NAD(P)H with benzyl alcohol dehydrogenase in the presence of a nitrosoaniline compound of the formula I: ##STR45## wherein said nitrosoaniline compound is an electron acceptor, b) enzymatically reducing the nitrosoaniline compound to a colored quinone diimiine, and
c) measuring any color produced as an indication of the amount of NAD(P)H,
wherein
R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which produces a color by electron conjugation with a quinone diimiine system.

### Claim 2 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula III ##STR46## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy,
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 h.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.4 and R.sup.5 form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylalkyl or polyalkoxyalkyl residue each of which in turn is unsubstituted or substituted in the alkyl moiety by a hydroxy residue.

### Claim 3 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula III ##STR47## wherein R.sup.2 forms a --CH.sub.2 --CH--, --CH.sub.2 --O--, --CR.sup.12 R.sup.13 --, --O--, --S-- or --NH-- bridge with the residue R.sup.1 in which NH can be unsubstituted or substituted by an alkyl residue and R.sup.12 and R.sup.13 are selected from the group consisting of carboxy and alkyl which are unsubstituted or substituted by hydroxy, carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.4 and R.sup.5 form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalky, alkoxycarbonylalkyl, dioxanylalkyl or polyalkoxyalkyl residue each of which in turn is unsubstituted or substituted in the alkyl moiety by a hydroxy residue.

### Claim 4 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula III: ##STR48## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy,
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.2 is in an ortho position in relation to NR.sup.4 R.sup.5, and R.sup.4 or R.sup.5 together with R.sup.2 form an alkylene residue.

### Claim 5 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula III ##STR49## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy or can form a --CH.sub.2 --CH--, --CH.sub.2 --O--, --CR.sup.12 R.sup.13 --, --O--, --S-- or --NH-- bridge with the residue R.sup.1 in which NH can be unsubstituted or substituted by an alkyl residue and R.sup.12 and R.sup.13 are selected from the group consisting of carboxy and alkyl which are unsubstituted or substituted by hydroxy, carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H,
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5, and
R.sup.4 and R.sup.5 together form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylalky or polyalkoxyalkyl residue, each of which is unsubstituted or substituted in the alkyl moiety by a hydroxy residue, wherein when R.sup.2 is in ortho position in relation to NR.sup.4 R.sup.5, and R.sup.4 or R.sup.5 together R.sup.2 form an alkylene residue.

### Claim 6 (depends on 1)

The method according to claim 1, wherein R is selected from the group consisting of a N,N-dihydroxyethylaniline, N,N-dimethylaniline and N,N-diethylaniline residue.

### Claim 7 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula IV: ##STR50## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7,
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkytthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, or PO.sub.3 H.sub.2,
R.sup.8 is selected from the group consisting of alkyl, thioalkyl or aralkyl which is unsubstituted or substituted by hydroxy, carboxy, SO.sub.3 H or PO.sub.3 H.sub.2, and an amino which is unsubstituted or substituted by one or two alkyl groups which in turn can be substituted by hydroxy, carboxy, SO.sub.3 H, dialkylphosphinyl or PO.sub.3 H.sub.2,
wherein at least one of R.sup.7 and R.sup.8 is an amino group, and
R.sup.9 is an alkyl or aralkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO.sub.3 H and PO.sub.3 H.sub.2,
as well as corresponding tautomeric forms and their salts.

### Claim 8 (depends on 7)

The method according to claim 7, wherein R.sup.7 is an amino which is substituted by 2 alkyl residues and the alkyl residues are linked to form a ring, wherein apart from the N atom of the amino group, said ring is uninterrupted or interrupted by oxygen, sulphur, a further nitrogen atom or an amino group which is unsubstituted or substituted by one or two acyl groups, alkoxy groups, aralkoxycarbonyl groups, H.sub.2 N--CO, alkyl, aralkyl, or arylcarbamoyl groups.

### Claim 9 (depends on 7)

The method according to claim 7, wherein R is selected from the group consisting of the following residues: ##STR51## wherein R.sup.10, R.sup.11, R.sup.12 and R.sup.13 are individually selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, alkylthio, aralkyl, aryl, carboxy, alkoxycarbonyl, carboxamido, cyano, halogen, and an amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl residues, and
R.sup.14 is selected from the group consisting of alkyl or aralkyl which is unsubstituted or substituted by a residues selected from the group consisting of hydroxy, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2 an dialkylphosphinyl.

### Claim 10 (depends on 9)

The method according to claim 9, wherein two adjacent residues form an alkylene group which in turn is unsubstituted or substituted by aryl or is anellated.

### Claim 11 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula IV: ##STR52## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkylthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2,
wherein R.sup.8 and R.sup.9 represent a saturated or unsaturated chain with 3 or 4 members individually selected from the group consisting of nitrogen atoms, carbon atoms and sulphur atoms, wherein the carbon atoms are unsubstituted or substituted by a residue selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen, and an amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl residues, and
wherein any nitrogen atoms which are not bound via a double bond are substituted by a residue selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by SO.sub.3 H, PO.sub.3 H.sub.2, COOH or dialkylphosphinyl, and an aralkyl,
as well as corresponding tautomeric forms and their salts.

### Claim 12 (depends on 1)

The method according to claim 1, wherein R is a residue of the general formula IV: ##STR53## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkylthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2,
wherein R.sup.8 and R.sup.9 represent a saturated or unsaturated chain with 3 or 4 members composed of nitrogen atoms, carbon atoms and/or sulphur atoms, wherein the carbon atoms are unsubstituted or substituted by a residue selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen, and an amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl residues, and
wherein two adjacent chain substituents forman alkylene group which in turn is unsubstituted or substituted by aryl, and wherein any nitrogen atoms which are not bound via a double bond are substituted by a residue selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by SO.sub.3 H, PO.sub.3 H.sub.2, COOH or dialkylphosphinyl, and an aralkyl,
as well as corresponding tautomeric forms and their salts.

### Claim 13

A nitrosoaniline compound of the formula I, ##STR54## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which produces a color by electron conjugation with a quinone diimiine system.

### Claim 14 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula III: ##STR55## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy,
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.4 and R.sup.5 are alkylene residues which are uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted with an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylalkyl or polyalkoxyalkyl residue each of which in turn is unsubstituted or substituted in the alkyl moiety by a hydroxy residue.

### Claim 15 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula III: ##STR56## wherein R.sup.2 forms a --CH.sub.2 --CH--, --CH.sub.2 --O--, --CR.sup.12 R.sup.13 --, --O--, --S-- or --NH-- bridge with the residue R.sup.1 in which NH can be unsubstituted or substituted by an alkyl residue and R.sup.12 and R.sup.13 are selected from the group consisting of carboxy and alkyl which are unsubstituted or substituted by hydroxy, carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.4 and R.sup.5 are alkylene residues which are uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylalkyl or polyalkoxyalkyl residue each of which in turn is unsubstituted or substituted in the alkyl moiety by a hydroxy residue.

### Claim 16 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula III: ##STR57## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2 , SO.sub.3 H or carboxy,
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group which is unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5,
wherein R.sup.4 and R.sup.5 are individually selected from the group consisting of hydrogen; an alkyl group which is unsubstituted or substituted by a hydroxy; alkoxy; hydroxyalkoxy; a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2 , SO.sub.3 H, or COOH as such or in a salt form; and an amino group which is unsubstituted or substituted at least once by an alkyl,
wherein R.sup.2 is in an ortho position in relation to NR.sup.R.sup.5, and R.sup.4 or R.sup.5 together with R.sup.2 represents an alkylene residue.

### Claim 17 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula III: ##STR58## wherein R.sup.2 is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy or arylthio, an alkyl or alkylene unsubstituted or substituted by carbox-y, PO.sub.3 H.sub.2 or SO.sub.3 H, an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy
R.sup.3 is selected from the group consisting of a hydroxy group, alkyl, alkoxy, aryl, aryloxy, arylthio or alkylthio group wherein the alkyl residue is unsubstituted or substituted by a hydroxy group, an alkoxy group, an amino group unsubstituted or substituted at least once by alkyl, PO.sub.3 H.sub.2, SO.sub.3 H or CO.sub.2 H, an amino group in a salt form selected from the group consisting of ammonium, alkali and alkaline earth salts, and an amino group NR.sup.4 R.sup.5, and
R.sup.4 and R.sup.5 together form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylalkyl or polyalkoxyalkyl residue, each of which is unsubstituted or substituted in the alkyl moiety by a hydroxy residue,
wherein when R.sup.2 is in an ortho position in relation to N.sup.4 R.sup.5, R.sup.4 or R.sup.5 together with R.sup.2 form an alkylene residue.

### Claim 18 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula IV: ##STR59## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7,
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkylthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2,
R.sup.8 is selected from the group consisting of alkyl, thioalkyl or aralkyl which is unsubstituted or substituted by hydroxy, carboxy, SO.sub.3 H or PO.sub.3 H.sub.2, and an amino which is unsubstituted or substituted by one or two alkyl groups which in turn can be substituted by hydroxy, carboxy, SO.sub.3 H, dialkylphosphinyl or PO.sub.3 H.sub.2,
wherein at least one of R.sup.7 and R.sup.8 is an amino group, and
R.sup.9 is an alkyl or aralkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO.sub.3 H and PO.sub.3 H.sub.2,
as well as corresponding tautomeric forms and their salts.

### Claim 19 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R.sup.1 is an amino group which is substituted by 2 alkyl residues and the alkyl residues are linked to form a ring, wherein apart from the N atom of the amino group, said ring is uninterrupted or interrupted by oxygen, sulphur, a further nitrogen atom or an amino which is unsubstituted or substituted by one or two acyl groups, alkoxy groups, aralkoxycarbonyl groups, H.sub.2 N--CO, alkyl, aralkyl, or arylcarbamoyl groups.

### Claim 20 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula IV: ##STR60## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkylthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2,
wherein R.sup.8 and R.sup.9 represent a saturated or unsaturated chain with 3 or 4 members individually selected from the group consisting of nitrogen atoms, carbon atoms and sulphur atoms, wherein the carbon atoms are unsubstituted or substituted by a residue selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen, an amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl residues, and
wherein any nitrogen atoms which are not bound via a double bond are substituted by a residue selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by SO.sub.3 H, PO.sub.3 H.sub.2, COOH or dialkylphosphinyl, and an aralkyl,
as well as corresponding tautomeric forms and their salts.

### Claim 21 (depends on 13)

The nitrosoaniline compound according to claim 13, wherein R is a residue of the general formula IV: ##STR61## wherein X-Y is selected from the group consisting of NR.sup.6 --CO and N.dbd.CR.sup.7
R.sup.6 is selected from the group consisting of H; an alkyl unsubstituted or substituted by hydroxy; carboxy; SO.sub.3 H; PO.sub.3 H.sub.2 ; and dialkylphosphinyl,
R.sup.7 is selected from the group consisting of H, alkyl, alkenyl, alkoxy, alkylthio, aryl, aralkyl, a salt of one of these acid residues, an alkoxycarbonyl, carboxy, alkoxycarbonyl, carboxamido, halogen and an amino which is unsubstituted or substituted by one or two alkyl residues which is substituted or unsubstituted with at least one residue selected from the group consisting of hydroxy, carboxy and alkoxy-carbonyl residues, wherein the alkyl, alkenyl, alkoxy, alkylthio, aryl, and aralkyl residues are unsubstituted or substituted by hydroxy, dialkylphosphinyl, carboxy, SO.sub.3 H, PO.sub.3 H.sub.2,
wherein R.sup.8 and R.sup.9 represent a saturated or unsaturated chain with 3 or 4 members individually selected from the group consisting of nitrogen atoms, carbon atoms and sulphur atoms, wherein the carbon atoms are unsubstituted or substituted by a residue selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen, an amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl residues,
wherein two adjacent chain substituents forman alkylene group which in turn is unsubstituted or substituted by aryl or is anellated, and wherein any nitrogen atoms which are not bound via a double bond are substituted by a residue selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by SO.sub.3 H, PO.sub.3 H.sub.2, COOH or dialkylphosphinyl, and an aralkyl,
as well as corresponding tautomeric forms and their salts.

### Claim 22

A reagent for the colorimetric determination of NAD(P)H comprising benzyl alcohol dehydrogenase and a nitrosoaniline compound of formula I: ##STR62## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.

### Claim 23

A reagent for the colorimetric determination of benzyl alcohol dehydrogenase comprising NAD(P)H and a direct chromogenic electron acceptor, wherein the electron acceptor is a nitrosoaniline compound of formula I: ##STR63## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.

### Claim 24

A reagent for the colorimetric determination of a NAD(p).sup.+ -dependent dehydrogenase comprising a substrate of the dehydrogenase to be determined, NAD(P).sup.+, an NAD(P)H-converting dehydrogenase and a direct chromogenic electron acceptor,
wherein the NAD(P)H-converting dehydrogenase is benzyl alcohol dehydrogenase and wherein the electron acceptor is a nitrosoaniline compound of formula I: ##STR64## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.

### Claim 25

A reagent for the colorimetric determination of an analyte comprising an analyte-specific NAD(P)-dependent dehydrogenase, NAD(P), an NAD(P)H-oxidizing enzyme and a direct chromogenic electron acceptor, wherein said NAD(P)H-oxidizing enzyme is benzyl alcohol dehydrogenase and said direct chromogenic electron acceptor is a nitrosoaniline compound of formula I: ##STR65## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.

### Claim 26

A process for the production of a nitrosoaniline compound of formula I: ##STR66## wherein R.sup.1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO.sub.3 H.sub.2, dialkylphosphinyl or SO.sub.3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO.sub.3 H.sub.2, SO.sub.3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system,
comprising reacting a p-nitrosophenyl ether of formula XVII: ##STR67## with an amino compound of formula XVIII: ##STR68##