Method for the colorimetric determination of an analyte with a PQQ-dependent dehydrogenase

• US 5,484,708 A
• Filed: 04/08/1994
• Issued: 01/16/1996
• Est. Priority Date: 04/08/1993
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Unkrig, Volker, Hoenes, Joachim
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/224,869
• Time in Patent Office: 648 Days
• Field of Search: 435/14, 435/4, 435/26, 435/25, 435/118, 435/119, 435/120, 435/133, 435/28, 436/63
• US Class Current: 435/14
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 513/04 Ortho-condensed systems
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose

Abstract

The invention concerns a method for the colorimetric determination of an analyte by means of enzymatic oxidation with a PQQ-dependent dehydrogenase in the presence of an electron acceptor from the group of the electron-rich aromatic nitroso compounds by enzymatic reduction of the nitroso compound to an imino compound and detection of the imino compound by color formation. In addition the invention concerns a corresponding agent containing a PQQ-dependent dehydrogenase, an electon-rich aromatic nitroso compound as well as, if desired, a detection reagent for imino compounds. The invention in addition concerns novel nitrosoamino compounds which form colored quinone diimines on reduction as well as their use for the colorimetric enzymatic determination of an analyte.

Claims

1. A method for the colorimetric determination of an analyte, comprising,

enzymatically oxidizing an analyte with an oxidoreductase in the presence of a direct electron acceptor, wherein said direct electron acceptor is an electron-rich aromatic nitroso compound,

wherein the electron-rich aromatic nitroso compound is reduced enzymatically in the presence of a pyrrolo-quinoline quinone (PQQ)-dependent dehydrogenase to an imino compound with concomitant oxidation of the analyte without further enzymatic reduction to an aromatic amine,

reacting said imino compound with a non-oxidative chromogenic detection reagent, and

measuring any color formation.

2. The method according to claim 1, wherein said aromatic nitroso compound is a compound of formula I:

##STR41## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂, or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, or an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H, or CO₂ H, and an amino group NR³ R⁴ wherein said alkoxy, aryloxy arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group, and

R³ and R⁴ are individually selected from the group consisting of hydrogen, an aryl group, an alkyl group, an alkoxy group, a hydroxyalkoxy group, a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO₃ H₂, SO₃ H, COOH, or a salt form of PO₃ H₂, SO₃ H or COOH, and an amino group which is unsubstituted or substituted at least once by an alkyl group, wherein said aryl group and said alkyl group are unsubstituted or substituted by a hydroxy.

3. The method according to claim 2, wherein said aromatic nitroso compound is a compound selected from the group consisting of the compounds of formula III-XII:

##STR42## wherein X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶, R⁵ is selected from the group consisting of hydrogen, an alkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl, R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, dialkylphosphinyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which in turn are unsubstituted or substituted by at least one hydroxy, carboxy, or alkoxycarbonyl residues, wherein said alkyl, alkenyl, alkoxy, alkythio, aryl, arylthio, and aralkyl groups are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

R⁹, R¹⁰, R¹¹ and R¹² 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 hydroxy, carboxy or alkoxycarbonyl residues;

wherein two adjacent residues form an alkylene group which is unsubstituted or substituted by aryl or is anellated, and

R¹³ is selected from the group consisting of alkyl and aralkyl wherein said alkyl and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂ or dialkylphosphinyl.

4. The method according to claim 1, wherein said aromatic nitroso compound is a compound of formula I:

##STR43## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂, or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, or an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H or CO₂ H, and an amino group NR³ R⁴ wherein said alkoxy aryloxy arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group, and

R³ and R⁴ form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted by a residue selected from the group consisting of an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylylalkyl and a polyalkoxyalkyl residue each of which is unsubstituted or substituted by a hydroxy residue in the alkyl moiety.

5. The method according to claim 1, wherein said aromatic nitroso compound is a compound of formula I:

##STR44## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂, or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, or an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H, or CO₂ H, and an amino group NR³ R⁴, wherein said alkoxy, aryloxy, arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group,

R¹ is in an ortho position in relation to NR³ R⁴, and

R³ or R⁴ together with R¹ form an alkylene residue.

6. The method according to claim 1, wherein said aromatic nitroso compound is a compound of formula II:

##STR45## wherein X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, dialkylphosphinyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which in turn are unsubstituted or substituted by at least one hydroxy, carboxy, or alkoxycarbonyl residues, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl groups are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

R⁷ is selected from the group consisting of alkyl, thioalkyl, aralkyl, an amino group which is unsubstituted or substituted by one or two alkyl groups which in turn are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, dialkylphosphinyl or PO₃ H₂, wherein said alkyl, thioalkyl and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H or PO₃ H₂,

wherein at least one of R⁶ and R⁷ is an amino group and

R⁸ is selected from the group consisting of an alkyl or aralkyl group which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H or PO₃ H₂,

as well as corresponding tautomeric forms and their salts.

7. The method according to claim 6, wherein R⁶ is an amino substituted by 2 alkyl residues, and wherein these residues are linked to form a ring which 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 groups selected from the group consisting of an alkyl group, an alkoxy group, an aralkoxycarbonyl group, H₂ N--CO, an aralkyl group, an arylcarbamoyl group, hydrogen, carboxy, alkoxycarbonyl, carboxamido and halogen.

8. The method according to claim 1, wherein said aromatic nitroso compound is a compound of formula II:

##STR46## wherein X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶ ,

R⁵ is selected from the group consisting of hydrogen, an alkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, dialkylphosphinyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which in turn are unsubstituted or substituted by at least one hydroxy, carboxy, or alkoxycarbonyl residues, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl groups are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

R⁷ and R⁸ form a saturated or unsaturated chain with 3 or 4 members selected from the group consisting of nitrogen atoms, carbon atoms and sulphur atoms, wherein said carbon atoms are unsubstituted or substituted by 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 hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms which are not bound via a double bond are substituted by alkyl, aralkyl or dialkylphosphinyl;

wherein two adjacent chain substituents form an alkylene group which in turn is unsubstituted or substituted by aryl or is anellated,

as well as corresponding tautomeric forms and their salts.

9. The method according to claim 1, wherein said non-oxidative chromogenic detection reagent is a coupling reagent for imino compounds.

10. The method according to claim 9, wherein said coupling reagent is selected from the group consisting of a phenol, naphthol, aniline and naphthylamine derivative.

11. The method according to claim 9, wherein said coupling reagent is selected from the group consisting of 2,4,6-tribromo-3-hydroxybenzoic acid, 2,4-dibromo-1-naphthol and 1-naphthol-4-sulfonic acid.

12. The method according to claim 1, wherein the imino compound is detected by oxidation of a leuco dye to a dye.

13. The method according to claim 12, wherein said leuco dye is selected from the group consisting of diarylimidazoles, triarylimidazoles and naphthylamines.

18. The method according to claim 1, wherein said aromatic nitrosoaniline compound is a compound of the general formula XIII:

##STR48## wherein R¹ is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy, arylthiol, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, PO₃ H₂ or SO₃ H, an amino which is unsubstituted or substituted at least once by an alkyl which in turn is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or carboxy,

X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which are in turn unsubstituted or substituted with at least one hydroxy, carboxy, or alkoxycarbonyl residue;

wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

A is a saturated or unsaturated chain consisting of three members selected from the group consisting of nitrogen, sulphur and carbon, wherein the carbon atoms are unsubstituted or substituted by 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 in turn are substituted or unsubstituted with at least one hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms that are not bound via a double bond are substituted by hydrogen, aralkyl, or an alkyl which is unsubstituted or substituted by SO₃ H₂, PO₃ H₂, carboxy or dialkylphosphinyl, wherein two adjacent chain substituents form an alkylene group which is unsubstituted, substituted by aryl, or is anellated,

as well as corresponding tautomeric forms and their salts which react enzymatically to form a colored quinone diimine as a measure of the amount of the analyte.

19. The method according to claim 1, wherein said PQQ-dependent dehydrogenase is glucose dye oxidoreductase and said analyte to be determined is glucose.

14. A method for the colorimetric determination of an analyte, comprising,

enzymatically oxidizing an analyte with an oxidoreductase in the presence of a direct electron acceptor, wherein said direct electron acceptor is an electron-rich aromatic nitroso compound, wherein the electron-rich aromatic nitroso compound is reduced in the presence of a POO-dependent dehydrogenase to a colored imino compound without further enzymatic reduction to an aromatic amine with concomitant oxidation of the analyte, and

measuring any color formation of the imino compound, wherein said electron rich aromatic nitroso compound is a compound of the general formula XIII:

##STR47## wherein R¹ is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy, arylthiol, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, PO₃ H₂ or SO₃ H, an amino which is unsubstituted or substituted at least once by an alkyl which in turn is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or carboxy,

X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which are in turn unsubstituted or substituted with at least one hydroxy, carboxy, or alkoxycarbonyl residue, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

A is a saturated or unsaturated chain consisting of three members selected from the group consisting of nitrogen, sulphur and carbon, wherein the carbon atoms are unsubstituted or substituted by 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 in turn are substituted or unsubstituted with at least one hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms that are not bound via a double bond are substituted by hydrogen, aralkyl, or an alkyl which is unsubstituted or substituted by SO₃ H₂, PO₃ H₂, carboxy or dialkylphosphinyl,

as well as corresponding tautomeric forms and their salts, which react enzymatically to form a colored quinone diimine as a measure of the amount of the analyte.

15. The method according to claim 14, wherein R⁶ is an amino substituted by two alkyl residues, wherein said residues are linked to form a ring, and wherein apart from the N atom of the amino group said ring is uninterrupted or interrupted by oxygen;

sulphur;

a further nitrogen atom;

an amino which is unsubstituted or substituted by one or two groups selected from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO, alkyl, aralkyl, and arylcarbamoyl;

carboxy;

alkoxycarbonyl;

carboxamido; and

halogen.

16. The method according to claim 14, wherein A forms an imidazole, triazole, benzimidazole, thiazole or dihydroimidazole ring with an adjacent heterocycle.

17. The method according to claim 14, wherein the aromatic nitroso compound is in a concentration of more than 10-13 mol/l.

20. A reagent for the colorimetric determination of an analyte by enzymatic oxidation of said analyte, comprising PQQ-dependent dehydrogenase, an electron-rich aromatic nitroso compound, and a color-forming, non-oxidative detection reagent for imino compounds.

21. The reagent according to claim 20, wherein said nitroso compound is a compound of the general formula I, ##STR49## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂, or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, or an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H, or CO₂ H; and

an amino group NR³ R⁴, wherein said alkoxy, aryloxy, arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group, and

R³ and R⁴ are individually selected from the group consisting of hydrogen, an aryl group, an alkyl group, an alkoxy group, a hydroxyalkoxy group, a polyalkoxy group which is unsubstituted or substituted by hydroxy, PO₃ H₂, SO₃ H, COOH, a salt form of PO₃ H₂, a salt form of SO₃ H, or a salt form of COOH, and an amino group which is unsubstituted or substituted at least once by an alkyl group, wherein said aryl group and said alkyl group are unsubstituted or substituted by a hydroxy.

22. The reagent according to claim 20, wherein said nitroso compound is a compound of the general formula I, ##STR50## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂ or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H or CO₂ H, and an amino group NR³ R⁴, wherein said alkoxy, aryloxy, arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group, and

R³ and R⁴ form an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein said nitrogen is substituted by a residue selected from the group consisting of an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylylalkyl and a polyalkoxyalkyl residue each of which is unsubstituted or substituted by a hydroxy residue in the alkyl moiety.

23. The reagent according to claim 20, wherein said nitroso compound is a compound of the general formula I, ##STR51## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

an alkyl which is unsubstituted or substituted by hydroxy, COOH, PO₃ H₂ or SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

an amino which is unsubstituted or substituted at least once by an alkyl which is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or CO₂ H;

R² is selected from the group consisting of a hydroxy group, an alkoxy group, an aryloxy group, an arylthio group, an alkylthio group, an amino group which is unsubstituted or substituted at least once by alkyl, PO₃ H₂, SO₃ H, CO₂ H, or an ammonium, alkali or alkaline earth salt form of PO₃ H₂, SO₃ H or CO₂ H, and an amino group NR³ R⁴, wherein said alkoxy, aryloxy, arylthio or alkylthio groups are unsubstituted or the alkyl residue is substituted by a hydroxy group,

R¹ is in an ortho position in relation to NR³ R⁴ and

R³ or R⁴ together with R¹ form an alkylene residue.

24. The reagent according to claim 20, wherein said nitroso compound is a compound of the general formula II, ##STR52## wherein X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, dialkylphosphinyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which in turn are unsubstituted or substituted by at least one hydroxy, carboxy, or alkoxycarbonyl residues, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl groups are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

R⁷ is selected from the group consisting of alkyl, thioalkyl, aralkyl, an amino group which is unsubstituted or substituted by one or two alkyl groups which in turn are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, dialkylphosphinyl or PO₃ H₂, wherein said alkyl, thioalkyl and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H or PO₃ H₂,

wherein at least one of R⁶ and R⁷ is an amino group and

R⁸ is selected from the group consisting of an alkyl or aralkyl group which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H or PO₃ H₂,

as well as corresponding tautomeric forms and their salts.

25. The reagent according to claim 24, wherein R⁶ is an amino substituted by 2 alkyl residues, and wherein these residues are linked to form a ring which 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 groups selected from the group consisting of an alkyl group, an alkoxy group, an aralkoxycarbonyl group, H₂ N--CO, an aralkyl group, an arylcarbamoyl group, hydrogen, carboxy, alkoxycarbonyl, carboxamido and halogen.

26. The reagent according to claim 20, wherein said aromatic nitroso compound is a compound of formula II:

##STR53## wherein X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl group which is unsubstituted or substituted by a residue selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, dialkylphosphinyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which in turn are unsubstituted or substituted by at least one hydroxy, carboxy, or alkoxycarbonyl residues, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl groups are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

R⁷ and R⁸ form a saturated or unsaturated chain with 3 or 4 members selected from the group consisting of nitrogen atoms, carbon atoms and sulphur atoms, wherein said carbon atoms are unsubstituted or substituted by 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 hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms which are not bound via a double bond are substituted by alkyl, aralkyl or dialkylphosphinyl;

wherein two adjacent chain substituents form an alkylene group which in turn is unsubstituted or substituted by aryl or is anellated,

as well as corresponding tautomeric forms and their salts.

27. A reagent according to claim 20, wherein said non-oxidative detection reagent for imino compounds is a chromogenic coupling reagent for imino compounds or a leuco dye.

28. A test carrier comprising a reagent according to claim 20.

29. A reagent for the colorimetric determination of an analyte by enzymatic oxidation of the analyte, comprising a PQQ-dependent dehydrogenase and an electron-rich aromatic nitroso compound, wherein the electron-rich aromatic nitroso compound is a nitrosoaniline compound of formula XIII, ##STR54## wherein R¹ is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy, arylthiol, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, PO₃ H₂ or SO₃ H, an amino which is unsubstituted or substituted at least once by an alkyl which in turn is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or carboxy,

X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which are in turn unsubstituted or substituted with at least one hydroxy, carboxy, or alkoxycarbonyl residue, wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

A is a saturated or unsaturated chain consisting of three members selected from the group consisting of nitrogen, sulphur and carbon, wherein the carbon atoms are unsubstituted or substituted by 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 in turn are substituted or unsubstituted with at least one hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms that are not bound via a double bond are substituted by hydrogen, aralkyl, or an alkyl which is unsubstituted or substituted by SO₃ H₂, PO₃ H₂, carboxy or dialkylphosphinyl,

as well as corresponding tautomeric forms and their salts.

30. The reagent according to claim 29, wherein R⁶ is an amino substituted by two alkyl residues, wherein said residues are linked to form a ring, and wherein apart from the N atom of the amino group said ring is uninterrupted or interrupted by oxygen;

sulphur;

a further nitrogen atom;

an amino which is unsubstituted or substituted by one or two groups selected from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO, alkyl, aralkyl, and arylcarbamoyl;

carboxy;

alkoxycarbonyl;

carboxamido; and

halogen.

31. The reagent according to claim 29, wherein A forms an imidazole, triazole, benzimidazole, thiazole or dihydroimidazole ring with an adjacent heterocycle.

32. A reagent for the colorimetric determination of an analyte by enzymatic oxidation of the analyte, comprising a PQQ-dependent dehydrogenase and an electron-rich aromatic nitroso compound, wherein the electron-rich aromatic nitroso compound is a nitrosoaniline compound of formula XIII:

##STR55## wherein R¹ is selected from the group consisting of hydrogen, hydroxy, halogen, alkoxy, alkylthio, aryloxy, arylthiol, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, PO₃ H₂ or SO₃ H, an amino which is unsubstituted or substituted at least once by an alkyl which in turn is unsubstituted or substituted by hydroxy, PO₃ H₂, dialkylphosphinyl, SO₃ H or carboxy,

X-Y is selected from the group consisting of NR⁵ --CO and N═

CR⁶,

R⁵ is selected from the group consisting of hydrogen, an alkyl which is unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, and dialkylphosphinyl,

R⁶ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, aralkyl, and an amino which is unsubstituted or substituted by one or two alkyl residues which are in turn unsubstituted or substituted with at least one hydroxy, carboxy, or alkoxycarbonyl residue;

wherein said alkyl, alkenyl, alkoxy, alkylthio, aryl, arylthio, and aralkyl are unsubstituted or substituted by hydroxy, carboxy, SO₃ H, PO₃ H₂, a salt of SO₃ H, a salt of PO₃ H₂, or alkoxycarbonyl,

A is a saturated or unsaturated chain consisting of three members selected from the group consisting of nitrogen, sulphur and carbon, wherein the carbon atoms are unsubstituted or substituted by 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 in turn are substituted or unsubstituted with at least one hydroxy, carboxy or alkoxycarbonyl residue and wherein any nitrogen atoms that are not bound via a double bond are substituted by hydrogen, aralkyl, or an alkyl which is unsubstituted or substituted by SO₃ H₂, PO₃ H₂, carboxy or dialkylphosphinyl;

wherein two adjacent chain substituents form an alkylene group which is unsubstituted or substituted by aryl or is anellated,

as well as corresponding tautomeric forms and their salts.