Process and electrode system for determining an analyte or an oxidoreductase as well as suitable compounds.

The invention relates to a method for the electrochemical determination of an analyte in the presence of an oxidoreductase and a reducible substance, which transfers electrons accumulating in the course of the determination reaction from the oxidoreductase to an electrode and thus leads to a signal which is a measure of the analyte to be determined. the reducible substance being enzymatically reduced and oxidized on the electrode, or a corresponding method for the electrochemical determination of an oxidoreductase in the presence of an enzyme substrate and a reducible substance characterized as above.

The invention also relates to a sensor electrode system for the electrochemical determination of an analyte in a sample containing at least two electrically conductive means, each of which is isolated from one another and which can be brought into electrical contact with the sample to be examined by means of an electrically conductive surface, at least one of which being electrically conductive surfaces an oxidoreductase and a reducible substance, is able to transmit the electrons between the oxidoreductase and the electrically conductive surface, or a corresponding sensor electrode system for determining an oxidoreductase, wherein at least one of the electrically conductive surfaces contacts an oxidoreductase substrate and a reducible substance characterized as above.

Finally, the invention relates to the use of certain compounds as electron carriers between an oxidoreductase and an electrode in an electrochemical system.

Compared to colorimetric methods for determining an analyte in a liquid, which is evaluated visually or photometrically, a corresponding electrochemical determination offers the advantage that the electrochemical reaction directly delivers current, which can be converted into a concentration. With colorimetric methods, however, the detour battery -> current -> light -> residual light (remission or transmission) -> current -> measured value is to be taken.

For electrochemical determination methods, it is necessary to oxidize the analyte to be determined or to convert it into a substance that can be oxidized using chemical or enzymatic methods. The direct electrochemical oxidation of an analyte or a substance derived therefrom on an electrode surface requires high overvoltages, ie potentials. This process is very unselective. Many other substances, which can also be in the sample to be examined, are also oxidized here. Such a method is therefore practically impossible to use analytically.

The oxidizable analyte or the oxidizable substance derived from the analyte is therefore usually reacted with a corresponding oxidoreductase and a reducible substance, the reduced form of which can be oxidized again at the electrode. Here, the oxidizable analyte or the oxidizable substance derived from the analyte is selectively oxidized by the enzyme. The enzyme reduced thereby is oxidized by the present reducible substance and the reduced reducible substance is oxidized at the electrode. The reducible substance thus serves as a carrier of the electrons from the enzyme to the electrode. The condition is therefore that the reducible substance is selected so that it is quickly and specifically implemented by the enzyme and by the electrode.

PW Carr et al. describe in "Theory and applications of enzyme electrodes in analytical and clinical chemistry", Verlag Wiley, New York (1980), pages 197-310, the reaction of glucose with oxygen as a reducible substance under enzyme catalysis by glucose oxidase and detection of the hydrogen peroxide formed on one Electrode. Side reactions of hydrogen peroxide, which itself is a strong oxidizing agent, and side reactions on the electrode surface are disadvantageous because of the high positive potential used. This method therefore requires special preliminary separations to exclude interfering components in the samples to be examined. Another disadvantage is the oxygen requirement. Especially at high glucose concentrations, the oxygen diffusion from the air into the sample and within the sample determines the speed and may falsify the results of the method.

EP-A-0 125 137 describes a sensor electrode system for determining a component of a mixture of substances, which has at least two electrically conductive means, each of which is isolated from one another and which is brought into electrical contact with the sample to be examined by means of an electrically conductive surface can be, wherein one of the electrically conductive surfaces an oxidoreductase and a so-called "mediator connection", which transfers electrons between this enzyme and the electrically conductive surface. The mediator compound used is an organometallic substance which has at least two organic rings, each of which has at least two conjugated double bonds, and a metal atom shares its electrons with each of these rings. Ferrocene or ferrocene derivatives are used as preferred mediator compounds, as in EP-A-0 078 636. It should be noted here that such compounds must first be oxidized, for example to a ferrocinium ion, before they are ready to accept electrons from the oxidoreductase. This leads to so-called "start-up currents", which already occur without the presence of an analyte, which naturally has a disruptive effect in an amperometric method in which the current occurring is a measure of the amount of the analyte to be determined. Furthermore, the poor solubility of such organometallic compounds is disadvantageous, since this leads to the preference for oxygen, for example when using oxidases, such as glucose oxidase as oxidoreductase, and thus leads to a low current and an oxygen dependency, especially at low enzyme substrate concentrations. With these electron carriers used in reduced form, poor solubility and / or the use of low concentrations are a prerequisite for acceptable starting currents.

Overall, for electrochemical determination methods the electron carriers known from the prior art are characterized in that they are reduced by an oxidoreductase in the presence of the analyte to be determined and are reoxidized at an electrode to the starting compound. If the concentration of the reducible substance acting as an electron carrier is significantly lower than the concentration of the analyte to be determined, only kinetic methods can be carried out. For end-point determinations, it is necessary that the reducible substance functioning as an electron carrier is dissolved in excess in relation to the analyte to be determined, so that the analyte to be determined is completely implemented. An amount of reducible substance proportional to the analyte to be determined is implemented. Advantages compared to the kinetic measurement are in particular the expanded linearity range of the current / concentration relationship in amperometric processes and the better competitiveness of the more concentrated reducible substance against oxygen when using oxidases as oxidoreductases. However, a disadvantage is the need to use a reducible substance for complete conversion, ie an oxidizing agent as an electron carrier with a potential significantly higher than that of the enzyme substrate, and additionally to work in the electrochemical determination in the presence of an excess of oxidizing agent, which further increases the necessary potential. However, high working potentials favor unspecific electrode reactions, especially when samples with a large number of constituents other than the analyte to be determined are to be examined.

In this respect, there are still no satisfactory solutions for the electrochemical determination of an analyte via an enzymatic redox reaction. There is a lack of universally usable reducible substances which act as electron carriers and which have both a rapid reaction with oxidoreductases and an uninhibited reaction on electrode surfaces at low potential.

The object of the present invention was to solve this problem. In particular, reducible substances should be found which can act as electron carriers between an oxidoreductase and an electrode in an electrochemical system. This object is achieved by the invention characterized in the patent claims.

The invention relates to a method for the electrochemical determination of an analyte in the presence of an oxidoreductase and a reducible substance, which transfers electrons occurring in the course of the determination reaction from the oxidoreductase to an electrode and thus leads to a signal which is a measure of the analyte to be determined , the reducible substance being enzymatically reduced and oxidized on the electrode, characterized in that that the substance formed on the electrode by oxidation is different from the reducible substance originally used.

The invention further relates to a method for the electrochemical determination of an oxidoreductase in the presence of a corresponding enzyme substrate and a reducible substance which is capable of transferring electrons from the oxidoreductase to an electrode and thus leads to a signal which is a measure of what is to be determined Is enzyme, the reducible substance being enzymatically reduced and oxidized at the electrode, characterized in that that the substance formed on the electrode by oxidation is different from the reducible substance originally used.

The invention also relates to the use of a substance which can accept electrons from an oxidoreductase to form an electron-rich aromatic amine as an electron transfer agent between an oxidoreductase and an electrode in an electrochemical system.

The invention further relates to a sensor electrode system for determining an analyte in a liquid sample containing at least two electrically conductive means which are isolated from one another and which can each be brought into electrical contact with the sample to be examined by means of an electrically conductive surface, at least one of the electrically conductive surfaces an oxidoreductase and a reducible substance, is able to transfer the electrons between the oxidoreductase and the electrically conductive surface, contacted, characterized in that a compound is used as the reducible substance which, after reduction by the oxidoreductase on the electrically conductive surface, is oxidized to a substance by the reducible substance originally used is different.

The invention also relates to a sensor electrode system for the electrochemical determination of an oxidoreductase in a liquid sample, comprising at least two electrically conductive agents which are isolated from one another and which can each be brought into electrical contact with the sample to be examined by means of an electrically conductive surface, wherein at least one of the electrically conductive surfaces contacts an oxidoreductase substrate and a reducible substance which is able to transfer electrons between the oxidoreductase and the electrically conductive surface, characterized in that a compound is used as the reducible substance, which after reduction is oxidized to a substance by the oxidoreductase on the electrically conductive surface, which is different from the reducible substance originally used. Finally, the invention relates to the use of a substance which can take up electrons from an oxidoreductase to form an electron-rich aromatic amine for producing a sensor electrode system according to the invention.

It has been shown that the disadvantages of the methods known from the prior art for the electrochemical determination of an analyte in the presence of an oxidoreductase and a reducible substance are due to the high potential required, in particular when using an excess of the reducible substance acting as an electron carrier compared to the analyte to be determined, can largely be avoided by a non-reversible reaction. Because a different oxidized substance than that which was originally used as a reducible substance is formed on the electrode, the electrochemical determination can be carried out at a particularly low potential and thus without the risk of interfering reactions. The advantage of the low potential can also be used if the reducible substance that acts as an electron transmitter is only used in a small amount compared to the analyte to be determined, namely if both the originally used reducible substance and the one used on the electrode Oxidation arising substance can be reduced by the oxidoreductase necessary for the electrochemical process. If both the originally used reducible substance and the substance formed on the electrode by oxidation are reduced by the oxidoreductase to the same substance, the originally used reducible substance acts as a storage form for the second reducible substance which is circulated between the electrode and the enzyme, the is different from the reducible substance originally used.

The advantages of the process according to the invention are due to the fact that those which can be selected as reducible substances are those in which a connection is formed by enzymatic reduction and which can be oxidized at the electrode at low voltage. In the case of oxidation at the electrode, there is still no significant concentration of this new oxidized substance. So far, the enzymatically reduced compound had to be reoxidized at the electrode to the originally used reducible substance, which was already present in high concentration. This required an increased positive potential.

In the sense of the invention, compounds which can advantageously be used as reducible substances are those which take over the electrons resulting from the oxidation of the substrate corresponding to the oxidoreductase used and thereby form an electron-rich aromatic amine. An electron-rich aromatic amine is understood here to mean a compound which is more electron-rich than aniline and which can be oxidized at a low potential because of the electron richness at the electrode. For example, all aniline derivatives which have one or more + I or / and + M substituents, such as hydroxyl, alkyl, alkoxy, aryloxy, alkylthio, arylthio, amino, monoalkylamino and dialkylamino residues on the aromatic ring, are suitable or wear on aniline nitrogen.

Alkyl, alkoxy, alkylthio, monoalkylamino and dialkylamino radicals are radicals in which alkyl represents a hydrocarbon radical having 1 to 6 carbon atoms, which in turn is represented by a hydroxy group, an amino group which is optionally mono- or polysubstituted by alkyl having 1 to 6 carbon atoms, PO₃H₂, SO₃H or CO₂H may be substituted. The acid residues PO₃H₂, SO₃H and CO₂H can be present as such or in salt form as ammonium, alkali or alkaline earth metal salts.

Aryloxy and arylthio residues are aromatic residues with 6 to 10 carbon atoms, with phenoxy and phenylthio residues being particularly preferred.

Ammonium salts are those which contain the ammonium NH₄ + or those which contain ammonium cations substituted one or more times by alkyl, aryl or aralkyl radicals. Alkyl in alkyl and aralkyl radicals means a hydrocarbon radical with 1 to 6 carbon atoms. Aryl in aryl and aralkyl radicals is an aromatic ring system with 6 to 10 carbon atoms, with phenyl being preferred. A preferred aralkyl radical is benzyl.

Alkali salts are preferably those of lithium, sodium or potassium. Alkaline earth metal salts are preferably those of magnesium or calcium.

Aniline derivatives are also understood to mean compounds which bear an amino group which is unsubstituted or substituted by + I or / and + and M, such as, for example, alkyl, on a aromatic ring system which is fused to one or more aromatic and / or alicyclic rings . Both aromatic carbon systems and heteroaromatic compounds are suitable as aromatic rings. Examples are fused benzene or naphthalene rings or a fused pyridine ring.

Alicyclic rings are understood to mean saturated or unsaturated cycloaliphatics having 5 to 7 carbon atoms, preferably 5 or 6 carbon atoms.

Possible alkyl substituents of the amino group can be hydrocarbon radicals having 1 to 6 carbon atoms, which in turn can be substituted by a hydroxyl group, an amino group optionally substituted one or more times by alkyl having 1 to 6 carbon atoms, PO₃H₂, SO₃H and CO₂H. The acid residues PO₃H₂, SO₃H and CO₂H can be present as such or in salt form as ammonium, alkali or alkaline earth metal salts, to which the definition given above also applies here.

The examples of + I or / and + M substituents given above should not be understood as a complete list. The person skilled in the art will know in individual cases whether a given radical is a + I or / and + M substituent and in this respect all of these radicals should be possible substituents in electron-rich aromatic amines which can be used according to the present invention.

Particularly preferred as reducible substances which lead to an electron-rich aromatic amine upon electron transfer from the oxidoreductase, which can then be oxidized at an electrode at low potential, are compounds from the group of the compounds of the general formula I.         XR (I) in the<dl id="dl0001"><dt>R</dt><dd> an electron rich aromatic residue and</dd><dt>X</dt><dd> NO or NHOH represents and compounds of the general formula II         HO-N = Y (II) in the</dd><dt>Y</dt><dd> a quinoid system, which can be described as electron-rich after reduction in the aromatic state.</dd></dl>

An electron-rich aromatic radical means the possibilities given above for electron-rich aromatic amines.

Such reducible substances according to the invention are reduced to aromatic amines when electrons are transferred from oxidoreductases and are not oxidized to the original reducible substances when oxidized on an electrode. As is known to the person skilled in the art, the electrochemical oxidation of the electron-rich aromatic amines removes electrons from the aryl radical, so that radicals or quinoid systems result. However, no quinoid oximes, no hydroxylamines and no nitroso compounds are formed.

The electrochemically oxidized compounds can in turn often take over electrons from oxidoreductases and thus be reduced back to electron-rich aromatic amines. It is therefore also possible to use substances which can be reduced according to the invention in concentrations which are low in comparison to the analyte to be determined, ie in excess. They thus act as a storage form for the electron-rich aromatic amines formed when the electrons are taken over by the oxidoreductase and which can be circulated as electron carriers between the oxidoreductase and the electrode.

Have been excellent examples of electron carriers according to the invention N- (2-hydroxyethyl) -N'-p-nitrosophenyl-piperazine, N, N-bis (2-hydroxyethyl) p-nitrosoaniline, o-methoxy- [N, N-bis (2-hydroxyethyl)] - p-nitrosoaniline, p-hydroxynitrosobenzene, N-methyl-N '- (4-nitrosophenyl) piperazine, p-quinone dioxime N, N-dimethyl-p-nitrosoaniline, N, N-diethyl-p-nitrosoaniline, N- (4-nitrosophenyl) morpholine, N-benzyl-N- (5'-carboxypentyl) -p-nitrosoaniline, N, N-dimethyl-4-nitroso-1-naphthylamine, N, N, 3-trimethyl-4-nitrosoaniline, N- (2-hydroxyethyl) -5-nitrosoindoline N, N-bis (2-hydroxyethyl) -3-chloro-4-nitrosoaniline, 2,4-dimethoxy-nitrosobenzene, N, N-bis (2-methoxyethyl) -4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N- (2-hydroxyethyl) -6-nitroso-1,2,3,4-tetrahydroquinoline, N, N-dimethyl-3-chloro-4-nitrosoaniline, N, N-bis (2-hydroxyethyl) -3-fluoro-4-nitrosoaniline, N, N-bis (2-hydroxyethyl) -3-methylthio-4-nitrosoaniline, N- (2-hydroxyethyl) -N- (2- (2-methoxyethoxy) ethyl) -4-nitrosoaniline, N- (2-hydroxyethyl) -N- (3-methoxy-2-hydroxy-1-propyl) -4-nitrosoaniline, N- (2-hydroxyethyl) -N- (3- (2-hydroxyethoxy) -2-hydroxy-1-propyl) -4-nitrosoaniline, N- (2-hydroxyethyl) -N- (2- (2-hydroxyethoxy) ethyl) -4-nitrosoaniline has been proven.

A particularly preferred reducible substance according to the invention is N, N-bis (2-hydroxyethyl) p-nitrosoaniline. N- (2-Hydroxyethyl) -N- (2- (2-hydroxyethoxy) ethyl) -4-nitrosoaniline is very particularly preferred.

Many compounds of the general formula I which can be used according to the invention are known. Nitrosoaniline derivatives of the general formula III are new<chemistry id="chem0001" num="0001"><img file="EP0441222A2_D0001.tif" /></chemistry> in the<dl id="dl0002"><dt>R¹</dt><dd> Represents hydrogen, halogen, alkoxy or alkylthio,</dd><dt>R²</dt><dd> an alkyl radical and</dd><dt>R³</dt><dd> represents a hydroxyalkyl radical or</dd><dt>R² and R³</dt><dd> are identical or different and each represent a dialkylaminoalkyl radical, a hydroxyalkoxyalkyl or alkoxyalkyl radical which is optionally substituted by hydroxy in the alkyl part or a polyalkoxyalkyl radical which is optionally substituted by a hydroxy radical in the alkyl part, or</dd><dt>R² and R³</dt><dd> form an alkylene radical interrupted by sulfur or nitrogen, nitrogen being substituted by an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, dioxanylylalkyl or polyalkoxyalkyl radical which in turn is optionally substituted in the alkyl part by a hydroxy radical or</dd></dl> when R¹ is in the ortho position to NR²R³, R² together with R¹ also represents an alkylene radical, where R³ then represents a hydroxyalkyl radical or if the alkylene radical contains 3 carbon atoms optionally also represents an alkyl radical or when R¹ is not hydrogen, R² and R³, which are the same or different, each represent a hydroxyalkyl radical or a salt of this derivative.

Halogen here means fluorine, chlorine, bromine or iodine. Fluorine and chlorine are particularly preferred. Alkyl, alkoxy or alkylthio are residues with 1-6 carbon atoms, those with 1-3 carbon atoms are particularly preferred. The definition given above for alkyl also applies to the alkyl part in hydroxyalkyl, dialkylaminoalkyl, hydroxyalkoxyalkyl, alkoxyalkyl, polyalkoxyalkyl, alkoxy-hydroxyalkyl and dioxanylyl-alkyl radicals. A dioxanylyl-alkyl radical is a radical in which a dioxane ring system is bonded to an alkyl radical. It is preferably a 1,4-dioxane ring system, ie<chemistry id="chem0002" num="0002"><img file="EP0441222A2_D0002.tif" /></chemistry>

A polyalkoxyalkyl group is a group -Alkyl- (alkoxy)_n-Alkoxy with n = 1-10. N = 1-4 is preferred. N = 1-3 is particularly preferred. An alkylene radical is a straight-chain or branched - preferably straight-chain -, saturated or unsaturated - preferably saturated -, hydrocarbon chain of 2-5, preferably 2-4 C atoms, with two free binding sites. The meaning of an alkylene radical interrupted by R² and R³ by sulfur or nitrogen is the thiomorpholine or. Piperazine residue preferred. The piperazine residue is particularly preferred.

In the meaning of an alkylene radical formed by R¹ and R², the indoline or 1,2,3,4-tetrahydroquinoline radical formed by inclusion of the aromatic ring of the general formula III is preferred. Preferred salts of a nitrosoaniline derivative according to the invention of the general formula III are, in particular, those of strong acids, in particular mineral acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid. Hydrochlorides, which are salts of hydrochloric acid, are very particularly preferred.

Of the new nitrosoaniline derivatives, preferred according to the invention are above all:<ul id="ul0001" list-style="none"><li>a) 2,2 '- [(3-fluoro-4-nitrosophenyl) imino] bis-ethanol,</li><li>b) 2,2 '- [(3-chloro-4-nitrosophenyl) imino] bis-ethanol,</li><li>c) 2,2 '- [(3-methoxy-4-nitrosophenyl) imino] bis-ethanol,</li><li>d) 2,2 '- [(3-methylmercapto-4-nitrosophenyl) imino] bis-ethanol,</li><li>e) 2 - [(2-hydroxyethoxy) ethyl- (4-nitrosophenyl) amino] ethanol,</li><li>f) 2 - [(2-methoxyethoxy) ethyl- (4-nitrosophenyl) amino] ethanol,</li><li>g) 1- [N- (2-hydroxyethyl) - (4-nitrosoanilino)] - 3-methoxy-2-propanol,</li><li>h) 1- [N- (2-Hydroxyethyl) - (4-nitrosoanilino)] - 3- (2-hydroxyethoxy) -2-propanol</li><li>i) 1-methyl-4- (4-nitrosophenyl) piperazine,</li><li>j) 4- (4-nitrosophenyl) -1-piperazinoethanol,</li><li>k) 5-nitroso-1-indolineethanol,</li><li>l) 1-methyl-6-nitroso-1,2,3,4-tetrahydroquinoline,</li><li>m) 6-nitroso-3,4-dihydro-1 (2H) quinolineethanol and their salts.</li></ul>

Of these, the compounds a), d), e), f), g), and h) and their salts are particularly preferred. Compound e) or its salts, in particular the hydrochloride, is very particularly preferred.

The compounds of the general formula III can be prepared by reacting a compound of the general formula IV<chemistry id="chem0003" num="0003"><img file="EP0441222A2_D0003.tif" /></chemistry> in which R¹, R² and R³ have the same meaning as in compounds of the general formula III, with nitrite. An analogous method is described in JJ D'Amico et al., J. Amer. Chem. Soc.<u style="single">81</u>, 5957 (1959).

Alkali nitrite is primarily used as the nitrite, lithium, sodium, potassium, rubidium or cesium being suitable as alkali metal; Sodium and potassium nitrite are preferred. Sodium nitrite is especially preferred. The reaction preferably takes place in an acidic medium at low temperature. The temperature should advantageously be below 10 ° C, preferably between -10 and + 5 ° C. The reaction of a compound of general formula IV with nitrite advantageously takes place in an aqueous medium. The pH should preferably be less than 3, particularly preferably less than 2.

In a preferred embodiment, a compound of the general formula IV or a salt thereof, preferably a salt of a mineral acid, such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, is initially introduced and cooled in an aqueous acid medium for the reaction. For this purpose, nitrite, preferably in dissolved form, is added to the reaction mixture while maintaining a low temperature. An aqueous medium is advantageously also used as the solvent for the nitrite. After the addition of the nitrite, the reaction mixture is kept at a low temperature until the reaction has ended. To work up the reaction mixture, it is preferably extracted with an organic solvent and the product is isolated from the extract.

The compounds which can be used according to the invention as electron carriers can be stored and used in oxidized form. In this way, starting currents are avoided and end point determinations with an excess of electron carriers can be carried out. The compounds which can be used according to the invention as electron carriers are stable on storage and can react rapidly with oxidoreductases. They are particularly competitive with oxygen when using oxidases and can also be used in excess over the highest analyte concentration to be determined. The latter property in particular is made possible by the good solubility of the electron carriers according to the invention in an aqueous medium. A particular advantage of the compounds which can be used according to the invention as electron carriers is their property of not undergoing enzymatic reduction in body fluids, or only to a minor extent, of electrochemical determination of analytes in body fluids. The electron transmitters according to the invention are quickly oxidized on the electrode surface and, in a reduced form, are not sensitive to oxygen. These compounds can be used at the electrode for low oxidation potential.

In the present invention, an analyte is a substance to be determined. This is usually a component of a mixture of substances. Above all, the method according to the invention offers advantages in this regard when determining an analyte in a body fluid, such as blood, plasma, serum, urine, because a specific reaction of only one component of the biological multi-component system is very important.

The method according to the invention for the electrochemical determination of an analyte is based on the fact that the analyte itself is oxidized by an oxidoreductase, that is to say that it represents a corresponding enzyme substrate, or the analyte is converted in one or more preceding reactions, preferably enzymatic reactions, into a compound which is oxidized by an oxidoreductase can be. The electrons produced by such an oxidation are proportional to the amount of the analyte to be determined. If these electrons are transferred from a substance which can be reduced according to the invention to an electrode, this leads to a signal which is a measure of the analyte to be determined. Amperometric methods are possible, whereby a current is measured or potentiometry, ie measurement of a voltage.

Preferred oxidoreductases for the process according to the invention are oxidases, non-NAD (P) -dependent dehydrogenases or diaphorase. For example, according to the invention, glucose with glucose oxidase, lactate with lactate oxidase, glycerol phosphate using glycerophosphate oxidase or cholesterol using cholesterol oxidase can be determined. As a non-NAD (P) -dependent dehydrogenase, for example, glucose-dye oxidoreductase can be used to determine glucose. Diaphorase, which can also be referred to as NADH: dye oxidoreductase, can advantageously be used for the detection of NADH.

In cases in which an analyte is to be electrochemically determined that does not itself serve as a substrate for an oxidoreductase, this analyte can be converted into a compound by one or more upstream reactions, in particular enzymatic reactions, which is accepted as a substrate by an oxidoreductase. For example, triglycerides can be determined in such a way that they are cleaved into glycerol and acid residues by means of an esterase, glycerin is converted with glycerol kinase and ATP into glycerol phosphate and this is finally oxidized by means of glyerophosphate oxidase and the resulting electrons are released from an electron carrier according to the invention to an electrode, generating a current proportional to the amount of triglycerides in the sample to be determined.

Analogously, total cholesterol can also be determined, for example, by cholesterol esters being split by cholesterol esterase and the cholesterol thus formed being determined by means of cholesterol oxidase. Here, too, the amount of cholesterol formed in this way and the electrons released upon oxidation by cholesterol oxidase, which are transferred to an electrode by means of a reducible substance according to the invention and thus generate a current, are proportional to the amount of total cholesterol to be determined.

The enzyme diaphorase can be used to determine NADH. Using the reducible substances according to the invention, electrons can also be transferred from diaphorase to an electrode. Since a large number of biological substances can be converted enzymatically with the formation of NADH, it is possible in this way to convert many analytes into NADH by means of enzymatic reaction sequences and then to finally determine this on an electrode via diaphorase and a reducible substance used according to the invention.

It goes without saying from the preceding statements that, according to the invention, oxidoreductases can of course also be determined if a corresponding compound which is accepted as an enzyme substrate and a substance which can be reduced according to the invention are presented. For example, glucose oxidase can be determined electrochemically if glucose and an electron carrier according to the invention are contacted with the sample to be determined in the presence of a corresponding sensor electrode system.

The method according to the invention is characterized in particular by the fact that the reducible substance used for electron transfer from an oxidoreductase to an electrode is stable in storage in its oxidized form and is also readily water-soluble, which is particularly important for the determination of analytes in body fluids such as blood and plasma , Serum, urine is very important. The reducible substances which can be used according to the invention have a rapid reaction with oxidoreductases and are very well competitive with oxygen, in particular when reacting with oxidases. Because of their solubility, they can be used very well for amperometric endpoint methods where an excess over the highest analyte concentration to be determined is required. Since the reducible substances which can be used according to the invention are negligible in non-enzymatic reduction by reducing agents present there, are quickly oxidized on the electrode surface and are hardly sensitive to oxygen in the reduced form, these substances are very suitable for specific, interference-free electrochemical analyte determinations. The fact that interference-free and specific electrochemical analyte determinations are possible is furthermore primarily due to the fact that the reducible substances which can be used according to the invention require only a low electrode potential.

The method according to the invention for the electrochemical determination of an analyte is not restricted to certain electrochemical devices. For this purpose, sensor electrode systems from the prior art can be used, for example. In principle, sensor electrode systems for determining an analyte in a liquid sample are suitable which contain at least two electrically conductive means as electrodes, which are isolated from one another and which can each be brought into electrical contact with the sample to be examined by means of an electrically conductive surface. It is conceivable here that only two electrodes, namely a working and a reference electrode, are used. A measuring arrangement without a reference electrode, ie only with a working and counter electrode, is also possible. The voltage is only kept constant externally. However, it is also possible to use three electrodes, namely a reference, a working and a counter electrode. Corresponding sensor electrode systems are known from the prior art, for example from G. Henze and R. Neeb, "Electrochemical Analysis", Springer-Verlag (1986).

It is important that for the electrochemical determination of an analyte (at least) one electrode, ie an electrically conductive surface, contacts an oxidoreductase and a reducible substance which is able to transfer electrons between the oxidoreductase and the electrically conductive surface. It is conceivable that all the necessary reagents are in solution together with the sample to be examined, or that some of the reagents, preferably the oxidoreductase and / or the electron-transferable reducible substance, are immobilized on an electrode and the rest is in solution, or that all reagents necessary for the determination are immobilized on one electrode. In principle, it is not critical for the function of a sensor electrode system whether the working electrode contacts the oxidoreductase and the reducible substance that acts as an electron transmitter as dissolved substances or whether these substances are applied to the electrode as solid substances and, if necessary, come into contact with the liquid to be determined Loosen the sample or remain immobilized on the electrode even after contact with the liquid sample to be determined.

It goes without saying that the above description applies analogously to the determination of an oxidoreductase. It must then be taken into account that the sensor electrode system contacts an oxidoreductase substrate and a reducible substance according to the invention. For the rest, the statements made for the determination of an analyte apply accordingly.

The accompanying figures explain the invention in more detail. Show it

1 in part a) shows a diagram of the function of the reducible substances that can be used according to the invention in methods and sensor electrode systems according to the invention if the concentration of the electron carrier is greater than or equal to the analyte concentration to be determined.

Fig. 1 in part b) a scheme for the function of electron-transferring substances in methods and sensor electrode systems of the prior art.

2 in part a) shows a diagram of the function of the reducible substances which can be used according to the invention in methods and sensor electrode systems according to the invention if the concentration of the electron-transferring substance is very much lower than the concentration of the analyte to be determined.

Fig. 2 in part b) a scheme for the function of electron-transferring substances in methods and sensor electrode systems of the prior art.

3 shows a sensor electrode system for carrying out the method according to the invention in which the required substances are in solution.

Fig. 4 shows a sensor electrode system for performing the method according to the invention, which is designed as a disposable or disposable sensor.

5: Diagram with the values ​​for anodic current density maxima obtained from cyclic voltammograms at various glucose concentrations with N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline as the electrode-transmitting substance in an electrochemical glucose test according to the invention.

6: Diagram of the relationship between current density and NADH concentration in an NADH test according to the invention.

Fig. 7: Cyclic voltammograms for N- (2-hydroxyethyl) -N'-p-nitrosophenyl-piperazine and N, N-bis (2-hydroxyethyl) -p-nitrosoaniline.

Fig. 8: Diagram of the dependence of the current density on the glucose concentration according to the inventive method with N-methyl-N '- (4-nitrosophenyl) piperazine as an electron-transferring substance in the presence and in the absence of atmospheric oxygen.

Fig. 9: Diagram of the dependence of the current density on the glucose concentration according to methods of the prior art with tetrathiafulvalene as an electron transfer substance in the presence and in the absence of atmospheric oxygen.

10: Diagram of the dependence of the current density on the LDH concentration by the method according to the invention with N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline as an electron-transferring substance at various times after the start of the determination reaction by lactate dehydrogenase.

11: Current-time curves for the method according to the invention with a single-use electrode according to FIG. 4 for glucose detection.

FIG. 12: diagram of the dependence of the current on the glucose concentration according to the inventive method with a single-use electrode according to FIG. 4 after a reaction time of 10 seconds.

1 and 2 show the differences between the method (a) according to the invention and the method of the prior art (b) when using an excess of the electron-transmitting substance over the analyte to be determined (FIG. 1) and when using an analyte concentration very small amount of electron-transmitting substance (Fig. 2) shown. According to the prior art method according to FIG. 1 b), the electron transfer substance (E_{ox 1}) in the presence of the analyte to be determined or the substance derived from the analyte (p_red), which are enzymatic to (p_ox) is oxidized, in the reduced form (E_red) transferred. The reduced electron carrier (E_red) by electron donation into the originally used reducible substance (E_{ox 1}) oxidized back.

In contrast, the reducible substance (E_{ox 1}) in the enzymatic oxidation of the analyte to be determined or the substance derived from the analyte (p_red) to (p_ox) in the reduced form (E_red) transferred. In the case of anodic oxidation on an electrode, an oxidized form of the electron carrier (E_{ox 2}) formed by the reducible substance originally used (E_{ox 1}) is different. Because of the complete absence of E at the beginning of the electrochemical oxidation_ox2 can E_red are oxidized at a particularly low potential. The electron-transmitting reducible substance according to the invention (E_{ox 1}) can be chosen so that for the anodic oxidation of the enzymatically formed reduced form (E_red) a relatively low potential is sufficient. In this way, disruptive accompanying reactions can be avoided, which occur when accompanying substances are oxidized in the samples to be examined when high potentials are applied to the electrodes, thus leading to a current flow and thus to a false positive result. In the method of the prior art according to FIG. 1 b), for the reoxidation of the enzymatically formed reduced form of the electron carrier (E_red) because of the excess of E_ox1 a higher potential than that of the reducible substance originally used (E_{ox 1}) required.

If the reducible substance that acts as an electron carrier (E_{ox 1}) in deficit compared to the analyte to be determined or the substance derived from the analyte to be determined (p_red), the reducible substance between the electrode and the enzyme can be circulated according to the method of the prior art (FIG. 2 b), since the reduced form (E_red) anodically into the reducible substance originally used (E_{ox 1}) is oxidized back.

According to the method according to the invention (FIG. 2a), if the oxidized form of the electron carrier (E_{ox 2}) is reduced by the reduced enzyme as well as the reducible substance originally used (E_{ox 1}), then E_{ox 1} for example as a storage-stable form of storage for the electron transfer system E._{ox 2}/ E_red serve.

In principle, all sensor electrode systems which are also suitable for carrying out the methods of the prior art can be used for the method according to the invention. A sensor electrode system according to FIG. 3 can be used, as is known, for example, from G. Henze and R. Neeb, "Electrochemical Analysis", Springer-Verlag (1986).

A working electrode (1), a counter electrode (2) and a reference electrode (3) are immersed in the liquid sample (4) to be determined. Conventional materials can be used for the electrodes. Working and counter electrodes (1, 2) can, for example, advantageously consist of noble metals or be produced using such metals. Preferred materials for working and counter electrodes (1, 2) are, for example, gold and platinum. The reference electrode (3) can also be constructed from systems which are customary for this purpose. The silver / silver chloride system is preferred, for example. The reference electrode (3) is advantageously connected to the rest of the electrode system (1, 2) in the liquid sample (4) to be determined via a salt bridge, for example a potassium chloride solution.

The oxidoreductase or the oxidoreductase system required for the method according to the invention (depending on whether an analyte or an oxidoreductase is to be determined) and the reducible substance functioning as an electron carrier can be dissolved in the sample (4) to be determined or they can also be all or some of them are on the working electrode (1). How the electrodes must be electrically connected to one another and regulated depending on the electrical signal to be measured is a matter of course for the person skilled in the art.

4 shows the construction of a disposable electrode, as can be used for example for the detection of glucose. The necessary electrodes and associated leads can be applied to an insulating carrier material (8), for example a polycarbonate film, using suitable methods. Suitable methods can be, for example, screen printing, inkjet, vapor deposition or thin film technology. In Fig. 4th mean (5) the working electrode, (55) the associated electrically conductive feed lines, (6) a reference electrode with feed line (66) and (7) a counter electrode with a corresponding conductor track (77). Known electrically conductive materials can be used for electrodes and conductor tracks. For example, commercially available graphite printing pastes can be used to produce the electrically conductive leads to the electrodes. The electrodes usually contain precious metals such as silver, gold or platinum. In the sensor electrode system according to the invention according to FIG. 4, the working electrode contains the reagents necessary for carrying out the electrochemical determination of an analyte or an oxidoreductase. For the determination of glucose, for example, glucose oxidase, an electron-transferable reducible substance according to the invention, a buffer substance that optimizes the pH of the sample to be examined for the enzymatic reaction, and optionally a detergent and swelling agent in order to use a material that makes the mixture conductive Production of an electrode to achieve the necessary consistency and make the mixture processable as a paste. For example, graphite powder can be added as the conductive material. Reference (6) and counterelectrode (7) as well as the corresponding feed lines (66) and (77) can be produced, for example, from commercially available silver conductive pastes which contain powdered silver chloride. A sensor electrode system according to FIG. 4 can be produced in a size of approximately 10 × 30 mm. The solution to be examined can be applied to the electrode surfaces or the test carrier can be immersed in the solution to be examined so that the electrode surfaces are covered with liquid. In the case of amperometric measurement, a potential can then be applied to the electrodes and a current measured that is proportional to the analyte to be determined.

For this purpose, the current is measured and regulated between the counter electrode (7) and the working electrode (5) in such a way that a predetermined voltage is maintained between the reference (6) and working electrode (5). The voltage measurement between the working (5) and reference electrode (6) takes place without current, so that resistances of the conductor track do not play a role. If there is less requirement for the accuracy of the electrode potentials, the currentless voltage measurement can also be dispensed with or the reference electrode (6) can be operated simultaneously as a counter electrode (7).

The invention is explained in more detail below by examples.

example 1

Glucose test

A sensor electrode system according to FIG. 3 is used. The working electrode (1) consists of a gold wire with an area of ​​0.1 cm². The counter electrode (2) is a platinum wire with a surface area of ​​0.1 cm 2 and the reference electrode (3) is a silver / silver chloride system from Orion Research Inc. (Boston, Massachusetts, USA).

There is a solution in the reaction vessel 0.1 mol / l potassium phosphate buffer and 0.1 mol / l potassium chloride, pH 7.0; 10 mmol / l N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline and Glucose in a concentration between 0 and 100 mmol / l.

The determination reaction is triggered by adding glucose oxidase (EC 1.1.3.4) to the reaction mixture and subsequent mixing. Sufficient glucose oxidase is added so that the concentration in the reaction mixture is 0.5 mg / ml (125 U / ml). One minute after the addition of glucose oxidase, a cyclic voltammogram is measured at a scan rate of 100 mV / s using a potentiometer (Mod. 273 EG &amp; G, Princeton Applied Research, Princeton, New Jersey, USA). The currents of the first oxidation maximum at 150 mV are evaluated. The results obtained are shown in FIG. 5. Corresponding measurements after 5 minutes after the addition of glucose oxidase or with exclusion of oxygen (under argon) do not result in any significant change.

As can be seen from the diagram according to FIG. 5, there is a linear dependence of the anodic current density maximum on the glucose concentration up to a glucose concentration of approximately 30 mmol / l. If the glucose concentration is higher than 30 mmol / l, the N, N-bis (2-hydroxyethyl) -p-nitrosoaniline used as the electron-transfer substance is completely converted to the corresponding phenylenediamine. Concentrations higher than 30 mmol / l glucose therefore do not lead to a further increase in current. Since two glucose molecules are necessary for the production of a molecule phenylenediamine and only about two thirds of the total glucose is in β-form and are therefore available for the conversion by means of glucose oxidase, the complete conversion of 10 mmol / l of electron-transferring substance by 30 mmol / l found Glucose exactly the theoretical stoichiometry.

When using glucose dye oxidoreductase (EC 1.1.99.17) instead of glucose oxidase (EC 1.1.3.4) in 0.1 mol / l Tris buffer, 0.1 mol / l potassium chloride, pH 7.0 with the addition of 1% bovine serum albumin , comparable results are obtained.

Example 2

NADH proof

Setup and measuring arrangement are as described in Example 1. The reaction vessel contains 0.1 mol / l potassium phosphate buffer, 0.1 mol / l potassium chloride, pH 7.0, 10 mmol / l N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline and NADH in concentrations between 0 and 10 mmol / l.

The measurement is started by adding and mixing diaphorase (NADH: dye oxidoreductase) from microorganisms and mixing the enzyme with the reaction mixture. Enough enzyme is added so that the enzyme concentration in the reaction mixture is 0.2 mg / ml (3 U / ml). Measurement of the current density after a reaction time of 1 minute gives the linear current density-concentration relationship shown in FIG. 6.

Example 3

Determination of lactate

Lactate can also be determined with the same experimental setup and the same electron carrier as in Example 1. Lactate oxidase (EC 1.1.3.2) is used as the enzyme and 0.1 mol / l citrate buffer, 0.1 mol / l potassium chloride, pH 5.5 is used as the buffer.

Example 4

Determination of glycerine phosphate

In example 1, the enzyme glucose oxidase is replaced by glycerophosphate oxidase (EC 1.1.3.21) and the buffer 0.1 mol / l Tris buffer, 0.1 mol / l potassium chloride, pH 8.0 replaced, can be determined analogously to glycerol phosphate.

Example 5

Determination of cholesterol

In example 1, glucose oxidase is replaced by cholesterol oxidase from Streptomyces (EC 1.1.3.6), the electron acceptor by 10 mmol / l of N-methyl-N '- (4-nitrosophenyl) piperazine and the buffer 0.1 mol / l potassium phosphate buffer, 0.1 mol / 1 potassium chloride, pH 5.5 with 2% Triton X 100^R replaced, cholesterol can be determined analogously to Example 1.

Example 6

Electron-transferable reducible substances according to the invention

The compounds mentioned in Table 1 below are mixed in a concentration of 10 mmol / l in 0.1 mol / 1 potassium phosphate buffer, 0.1 mol / l potassium chloride, pH 7.0 50 mmol / l glucose and 0.5 mg / ml glucose oxidase (125 U / ml) reacted. A measuring arrangement as described in Example 1 is used here. Corresponding cyclic voltammograms give the peak potentials of the electron carrier reduced with glucose oxidase and glucose, which are given in mV against a normal hydrogen electrode.

Table 1 shows the ratio of the oxidation currents at the potential of the highest oxidation peak after one and after ten minutes as a measure of the conversion rate.<tables id="tabl0001" num="0001"><img file="EP0441222A2_D0004.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0441222A2_D0005.tif" /></tables>

7 shows the cyclic voltammograms for N- (2-h-hydroxyethyl) -N'-p-nitrosophenyl-piperazine and N, N-bis (2-hydroxyethyl) -p-nitrosoaniline. The cyclic voltammograms were made with 10 mmol / l glucose measured in order to avoid interference by reactions of residual glucose during the recording of the cyclic voltammogram.

Example 7

Comparison of an electron carrier according to the invention with one of the prior art

<ul id="ul0002" list-style="none"><li>a) In an experimental set-up, as described in Example 1, N-methyl-N '- (4-nitrosophenyl) piperazine is used in a concentration of 10⁻⁴ mol / l in a phosphate buffer pH 7.0. The measurement of cyclic voltammograms at glucose concentrations between 0 and 3 mmol / l reveals a dependence of the current density on the glucose concentration as shown in FIG. 8. At low concentrations, an influence by atmospheric oxygen is determined, which can be avoided by measurement under argon. The same result as when using argon as protective gas is achieved by using the electron carrier in a higher concentration (10⁻² mol / l). The influence of the measurement by oxygen can also be avoided by using glucose dehydrogenase instead of glucose oxidase.</li><li>b) If, instead of N-methyl-N '- (4-nitrosophenyl) piperazine, tetrathiafulvalene is used as the electron carrier of the prior art as the electron carrier according to the invention, the dependence of the current density on the glucose concentration is shown as shown in FIG. 9. Tetrathiafulvalene shows a significantly higher oxygen disturbance than is the case with the electron transmitter according to the invention. In addition, much lower current densities are measured.</li></ul>

Tetrathiafulvalen is quite difficult to dissolve. In order to achieve a concentration of 10⁻⁴ mol / l in a phosphate buffer pH 7.0, 2.5% Tween 20^R can be used as a detergent. The setting of significantly higher concentrations of tetrathiafulvalene to reduce the oxygen disturbance, as is possible in the case of the electron carrier according to the invention, is ruled out here because of the poor solubility.

Example 8

Enzyme determination

<ul id="ul0003" list-style="none"><li>a) <u style="single">Lactate dehydrogenase test</u>Analogous to the test setup according to Example 1, the following solutions are presented: 0.1 mol / l sodium phosphate buffer, 0.1 mol / l potassium chloride, pH 9.0 10 mmol / l N, N-bis (2-hydroxyethyl) p-nitrosoaniline 0.1 mol / l D, L-lactate (sodium salt) 1 U / ml diaphorase from microorganisms 10 mmol / l NAD⁺. With strong stirring (magnetic stirrer, 1000 revolutions per minute) current is measured against silver / silver chloride at a constant potential of 75 mV. You start by adding lactate dehydrogenase (EG 1.1.1.27). Various amounts of lactate dehydrogenase are added and measured after every 100, 200, 300, 400, 500 and 600 seconds. The current / time curves obtained are shown in FIG. 10. The LDH activities indicated on the ordinate were determined using the usual pyruvate reduction test.</li><li>b) <u style="single">Glucose dehydrogenase test</u>Analogous to that described under a), 0.1 mol / l potassium phosphate buffer, 0.1 mol / l potassium chloride, pH 7.0 10 mmol / l NAD⁺, 10 mmol / l electron carrier according to the invention, 1 U / ml diaphorase and 0.1 mol / l glucose, a test for NAD-dependent glucose dehydrogenase can be carried out. Oxidases, diaphorase or non-NAD-dependent dehydrogenases can also be determined accordingly.</li></ul>

Example 9

Disposable electrode system for glucose detection

A sensor electrode system according to FIG. 4 is produced in such a way that the working electrode (5), reference electrode (6), counter electrode (7) and conductor tracks (55, 66, 77) are applied to a polycarbonate film (8) by screen printing with suitable printing pastes. The conductor tracks consist of commercially available graphite printing paste (Acheson 421 SS, Deutsche Acheson Colloids, Ulm, Federal Republic of Germany). The reference electrode (6) and the counter electrode (7) consist of commercially available silver conductive paste, which is mixed with 20% by weight of powdered silver chloride (Acheson SS 24566, Deutsche Acheson Colloids, Ulm, Federal Republic of Germany).

For the working electrode (5) in a swelling of 2% by weight of hydroxyethyl cellulose (Natrosol 250 G, Hercules BV, Rijswijk, the Netherlands) in 0.05 mol / l sodium phosphate buffer (pH 7.0) 3 mmol / l N, N -Bis-hydroxyethyl-p-nitrosoaniline, 500 KU glucose oxidase (glucose oxidase, purity grade II, Boehringer Mannheim GmbH, Mannheim Federal Republic of Germany) per 100 g mixture, 30% by weight graphite powder (UF 296/97, Graphitwerke Kropfmühl, Federal Republic of Germany) and 4% by weight of ethylene glycol. The electrode surfaces are for the working electrode (5): 4x6 mm² = 24 mm², for the reference electrode (6): 1x1.5 mm² = 1.5 mm² and for the counter electrode (7): 1x1.5 mm² = 1.5 mm² large.

The sensor electrode system produced by means of screen printing is immersed in a measuring solution which contains 0.05 mol / l sodium phosphate buffer (pH 7.0), 0.1 mol / l sodium chloride and 0-45 mmol / l glucose in such a way that the electrode surfaces of of the liquid to be examined are covered. At 200 mV potential against the integrated silver / silver chloride reference electrode (6) current / time curves are recorded, which are shown in Fig. 11. The application of the current values ​​after 10 seconds of measurement results in the calibration curve shown in FIG. 12, which indicates the dependence of the current flow on the glucose concentration.

Example 10

Preparation of 2,2 '- [(4-nitrosoaryl) imino] bis-ethanols

In a 4 l three-necked flask with stirrer, thermometer and dropping funnel, 2 mols of N, N-bis (β-hydroxyethylaniline) (or its aryl-substituted analogs) are added in portions with vigorous stirring in a mixture of 200 ml of water and 400 ml of conc. Hydrochloric acid. The resulting solution is cooled to 0 ° C. in a cold bath and a solution of 148 g (2.1 mol) of sodium nitrite in 200 ml of water is added dropwise at 0 to 2 ° C. with stirring within 20 minutes. The mixture is subsequently stirred at 0 ° C. for 30 minutes, the mostly crystalline yellowish to greenish colored nitroso compound is filtered off and the filter cake is washed with 2 × 200 ml of ice-cold, half-concentrated hydrochloric acid. For cleaning, the crude product is dissolved in 900 ml of water, 400 ml of concentrated hydrochloric acid are added with vigorous stirring, the mixture is stirred at room temperature for 30 minutes, then with ice cooling for 30 minutes. The crystals obtained are then dissolved in 580 ml of water, with 265 ml of conc. Hydrochloric acid added, stirred for 30 minutes at room temperature and 30 minutes with ice cooling. The crystals formed are filtered off, washed three times with 150 ml of ice-cold acetone, twice with 200 ml of diethyl ether and dried in vacuo at room temperature. It is obtained as follows:<ul id="ul0004" list-style="none"><li>a) <u style="single">2,2 '- [(4-nitrosophenyl) imino] bis-ethanol hydrochloride</u>Yield 32.8% of theory, green crystals; Mp 160 ° C (dec.)</li></ul>

The following are obtained analogously with corresponding aryl-substituted analogs:<ul id="ul0005" list-style="none"><li>b) <u style="single">2,2 '- [(3-fluoro-4-nitrosophenyl) imino] bis-ethanol hydrochloride</u>Yield: 26.5% of theory, yellow crystals; Mp 140 ° C (dec.). TLC: Kieselgel 60 (Merck) - mobile phase: ethyl acetate / methanol = 5: 1, R_F = 0.59 from 3-fluoro-N, N-bis- [2-hydroxyethyl] aniline (Chem. Abstr. <u style="single">57</u>, 13922 [1962])</li><li>c) <u style="single">2,2 '- [(3-chloro-4-nitrosophenyl) imino] bis-ethanol hydrochloride</u>Yield 21% of theory, yellow crystals; Mp 154 ​​° C (dec.). TLC: Kieselgel 60 (Merck) - eluent: methylene chloride / methanol = 5: 1, R_F = 0.72 from 3-chloro-N, N-bis- [2-hydroxyethyl] aniline (M. Freifelder, GR Stone, J. Org. Chem. <u style="single">26</u>, 1499 (1961))</li><li>d) <u style="single">2,2 '- [(3-methoxy-4-nitrosophenyl) imino] bis-ethanol hydrochloride</u>Yield 32% of theory, ocher crystals; Mp 145-146 ° C (dec.). TLC: Kieselgel 60 (Merck) - eluent: methylene chloride / methanol = 5: 1, R_F = 0.4 from 3-methoxy-N, N-bis [2-hydroxyethyl] aniline (M. Freifelder et al., J. Org. Chem. <u style="single">26</u>, 1499 (1961))</li><li>e) <u style="single">2.2 '- [(3-Methylmercapto-4-nitrosophenyl) imino] bis-ethanol hydrochloride</u>Yield 59.3% of theory, red-brown crystals; Mp 148 ° C (dec.). TLC: Kieselgel 60 (Merck) - mobile phase: ethyl acetate / methanol = 5: 1, R_F = 0.53 from 3-methylmercapto-N, N-bis- [2-hydroxyethyl] aniline (obtainable from: 0.1 mol of 3-methylmercaptoaniline dissolved in 50 ml of 4N acetic acid and 0.35 mol of ethylene oxide and stirring at room temperature for 12 hours. Add Excess NaHCO₃ solution, extraction with methylene chloride and column chromatography purification on silica gel 60 (Merck) - mobile phase: toluene / acetone = 5: 2, R_F = 0.18, yield 25%, colorless oil).</li><li>f) <u style="single">2- [methyl (3-chloro-4-nitrosophenyl) amino] ethanol hydrochloride</u>Yield 15% of theory, yellow crystals; Mp 147 ° C (dec.), TLC: silica gel 60 (Merck) - mobile phase: methylene chloride / methanol = 19: 1, R._F = 0.34 from 2- [methyl (3-chlorophenyl) aminoethanol (obtained from 2 - [(3-chlorophenyl) amino] ethanol by boiling for three hours with methyl iodide in the presence of 10% NaOH; column chromatography purification on silica gel 60 (Merck) - mobile phase: toluene / Acetone = 5: 2, R_F = 0.39, yield 25%, colorless oil).</li></ul>

Example 11

2 - [(2-Hydroxyethoxy) ethyl (4-nitrosophenyl) amino] ethanol hydrochloride

<ul id="ul0006" list-style="none"><li>A) <u style="single">2 - [(2-Hydroxyethoxy) ethyl- (phenyl) amino] ethanol</u><chemistry id="chem0004" num="0004"><img file="EP0441222A2_D0006.tif" /></chemistry> 146 g (0.8 mol) of 2- (2-anilinoethoxy) ethanol (obtained by reacting aniline with 2- (2-chloroethoxy) ethanol, yield 54%, colorless oil, Kp₁ 131 - 133 ° C) are in 500 ml of 4N acetic acid dissolved, cooled to 0 ° C. with stirring in a cold bath and 70.5 g, ie approximately 79 ml (1.6 mol) of ethylene oxide were added dropwise at 0-10 ° C. in the course of five minutes. After standing at room temperature for 12 hours, 500 ml of water are added, neutralized with stirring and careful addition of a total of 200 g of NaHCO 3 in portions. The extracted base is then extracted with 500 ml of methylene chloride, shaken three times with 250 ml of methylene chloride each time, the organic phases are combined, dried over sodium sulfate, suction filtered and concentrated in vacuo. 178.2 g of product are obtained. TLC silica gel 60 (Merck) solvent: toluene / acetone = 5: 2, R_F = 0,18</li><li>B) <u style="single">2- [2-hydroxyethoxy) ethyl (4-nitrosophenyl) amino] ethanol hydrochloride</u><chemistry id="chem0005" num="0005"><img file="EP0441222A2_D0007.tif" /></chemistry> A mixture of 280 ml of concentrated hydrochloric acid and 140 ml of water is added to a 2 1 three-necked flask with stirrer, dropping funnel and thermometer, and the mixture is cooled to -5 ° C. with a carbon dioxide bath, and 178 g (0.79 mol) drips within 10 minutes at a constant temperature. Substance obtained under A) and stirred for 15 minutes. A solution of 60 g (0.87 mol) of sodium nitrite in 120 ml of water is then added at 0 ° C., which turns from blood red to brown, and the mixture is stirred at 0 ° C. for 30 minutes. Then it is diluted by adding 500 ml of water (pH of the reaction mixture 1.4) and 218 ml of concentrated aqueous ammonia solution to pH 9 are added dropwise with ice cooling at a maximum of 15 ° C. The released nitroso base is shaken five times with 400 ml of n-butanol and the solvent is distilled off on a rotary evaporator. 212.8 g of dark green oil are obtained. To remove inorganic products, this is stirred with a mixture of 250 ml of toluene / acetone = 1: 1, the insoluble portion is filtered off with suction and washed with 50 ml of toluene / acetone = 1: 1. 18.4 g of inorganic material remain as a residue. The filtrate is purified by chromatography on a silica gel 60 column (7.5 cm diameter, filling height 90 cm - separating liquid: toluene / acetone = 1: 1). 155 g of nitroso base, dark green oil are obtained. This is dissolved in 600 ml of acetone and 250 ml of saturated ethereal hydrochloric acid are added dropwise with ice cooling. After stirring for 30 minutes while cooling with ice, the crystals formed are filtered off with suction, washed three times with 100 ml of acetone and dried in vacuo at room temperature over diphosphorus pentoxide. 159.9 g (= 69.6% of theory) of the title compound are obtained; Mp 118 ° C, TLC: silica gel 60 (Merck) - mobile phase: toluene / acetone = 1: 1, R._F = 0,24</li></ul>

Example 12

The following compounds are prepared in an analogous manner to that in Example 11:

<ul id="ul0007" list-style="none"><li>a) <u style="single">1- [N, N- (2-Hydroxyethyl) - (4-nitrosoanilino) 1-3- (2-hydroxyethoxy) -2-propanol hydrochloride</u><chemistry id="chem0006" num="0006"><img file="EP0441222A2_D0008.tif" /></chemistry> Yield 10.5% of theory, orange crystals, mp. 104 ° C (dec.); TLC - silica gel 60 (Merck) - mobile phase: toluene / methanol = 5: 1, R_F = 0.13 from 1- [N, N- (2-hydroxyethyl) (anilino)] - 3- (2-hydroxyethoxy) -2-propanol<chemistry id="chem0007" num="0007"><img file="EP0441222A2_D0009.tif" /></chemistry> (this from 1- [N- (anilino)] - 3- (2-hydroxyethoxy) -2-propanol<chemistry id="chem0008" num="0008"><img file="EP0441222A2_D0010.tif" /></chemistry> obtained from aniline with 1-chloro-3- (2-hydroxy-ethoxy) -2-propanol - yield: 21.5% colorless oil, TLC: silica gel 60 (Merck) - eluent: toluene / acetone = 5: 2, R_F = 0.6) by reaction with ethylene oxide in the presence of 4 N acetic acid. 71% colorless oil, TLC: silica gel 60 (Merck) - mobile phase: toluene / acetone 5: 2, R_F = 0,43)</li><li>b) <u style="single">1- [N- (2-Hydroxyethyl) - (4-nitrosoanilino)] - 3-methoxy-2-propanol hydrochloride</u><chemistry id="chem0009" num="0009"><img file="EP0441222A2_D0011.tif" /></chemistry> Yield 44.5%, light yellow crystals, mp. 122 ° C (dec.). TLC: Kieselgel 60 (Merck) - mobile phase: methylene chloride / methanol = 49: 1, R_F = 0.55 from (±) -3- [N- (2-Hydroxyethyl) anilino] -1-methoxy-2-propanol (German Reichspatent 603808 (1933) - Friedländer <u style="single">21</u>, 295), (Kp ₁ 212 - 214 ° C)</li><li>c) <u style="single">2 - [(2-methoxyethoxy) ethyl- (4-nitrosophenyl) amino] ethanol</u><chemistry id="chem0010" num="0010"><img file="EP0441222A2_D0012.tif" /></chemistry> Yield 25% of theory, dark brown resin. TLC: Kieselgel 60 (Merck) - eluent: methylene chloride / methanol = 19: 1, R_F = 0.49; Methylene chloride / methanol = 5: 1, R_F = 0.77 (via the amorphous hygroscopic hydrochloride with NH₃); from 2 - [(2-methoxyethoxy) ethyl- (phenyl) aminoethanol (A)<chemistry id="chem0011" num="0011"><img file="EP0441222A2_D0013.tif" /></chemistry> which is obtained from aniline and 2-methoxy-ethoxy-chloroethane (heating for 1 hour at 90 ° C. and column chromatography on silica gel 60 (Merck) with toluene / ethyl acetate = 5: 1. N- (2-methoxyethoxy-ethyl) thus formed aniline (R_F = 0.69, colorless oil)<chemistry id="chem0012" num="0012"><img file="EP0441222A2_D0014.tif" /></chemistry> gives with ethylene oxide and 4 N acetic acid (A) as a colorless oil, TLC: silica gel 60 (Merck) - mobile phase: toluene / acetone = 5: 2, R_F = 0,31.</li><li>d) <u style="single">2 - [(2- (2- (2- (2-methoxy) ethoxy) ethoxy) ethyl) -4- (nitrosophenyl) amino] ethanol</u><chemistry id="chem0013" num="0013"><img file="EP0441222A2_D0015.tif" /></chemistry> Yield: 63% of theory, green oil, TLC silica gel 60 (Merck) - mobile phase: toluene / acetone = 1: 5, R_F = 0.64 from 2 - [(2- (2- (2- (2-methoxy) ethoxy) ethoxy) ethyl) -4- (phenyl) amino] ethanol.</li></ul>

The starting compound was prepared as follows:

From aniline and diethylglycol bis (2-chloroethyl ether) (Perry, Hibbert Can. J. Res. <u style="single">14</u>, 81 (1936)) is obtained by heating to 140 ° C. for four hours and then separating by column chromatography on silica gel 60 (Merck) with toluene / ethyl acetate = 2: 1, 20.5% of theory yellowish oil, R_F = 0,5 <chemistry id="chem0014" num="0014"><img file="EP0441222A2_D0016.tif" /></chemistry> Its reaction with ethylene oxide in 4 N acetic acid is practically quantitative<chemistry id="chem0015" num="0015"><img file="EP0441222A2_D0017.tif" /></chemistry> as a beige oil, TLC: Kieselgel 60 (Merck) solvent: methylene chloride / methanol = 19: 1, R_F = 0,61.

With NaOCH₃ in methanol (reflux for 24 hours, concentration, addition of water, taking up in ethyl acetate and subsequent column chromatography purification of the crude product on silica gel 60 (Merck) with toluene / acetone = 5: 2 gives 51.3% of product theory as a colorless oil, R_F = 0,21.

Example 13

N- (4-nitrosophenyl) -N - [(2-diethylamino) ethyl] -N, N'-diethyl-1,2-ethanediamine tris hydrochloride

<chemistry id="chem0016" num="0016"><img file="EP0441222A2_D0018.tif" /></chemistry> Mp 125 ° C (dec.), TLC: silica gel 60 (Merck) - mobile phase: Isopropanol / n-butyl acetate / water / concentrated aqueous NH₃ = 50: 30: 15: 5, R_F = 0.56 from N- [di- (2-diethylamino) ethyl] aniline.

Example 14

Preparation of 1-N-substituted 4- (4-nitrosophenyl) piperazines

<chemistry id="chem0017" num="0017"><img file="EP0441222A2_D0019.tif" /></chemistry><ul id="ul0008" list-style="none"><li>a) <u style="single">1-methyl-4- (4-nitrosophenyl) pinerazine dihydrochloride</u><chemistry id="chem0018" num="0018"><img file="EP0441222A2_D0020.tif" /></chemistry> 17.62 g (0.1 mol) of 1-methyl-4-phenyl-piperazine (from 0.3 mol of 1-phenylpiperazine by heating for four hours with 0.2 mol of trimethylphosphate to 150 ° C., isolation by adding NaOH and extraction with Diethyl ether, purification by column chromatography on silica gel 60 (Merck) with methylene chloride / methanol = 5: 1 gives 40.1% of theory of a colorless liquid, b.p._0,05 82-84 ° C, R_F = 0.31 (according to Stewart et al., J. Org. Chem. <u style="single">13</u>, 134 (1948)) are dissolved in a mixture of 20 ml of concentrated hydrochloric acid and 10 ml of water, then a solution of 8 g (0.12 mol) of sodium nitrite in 16 ml of water is added dropwise at 0-2 ° C. in the course of 15 minutes and 30 Stirred minutes at 10 ° C. With further cooling, 60 ml of concentrated aqueous ammonia are added at the same temperature, diluted by adding 100 ml of water and the red-brown solution (pH 9) is shaken three times with 100 ml of methylene chloride each time, the organic phase is dried over Na₂SO₄, suction filtered and constricts. The residue (20.6 g of moss-green crystals) is taken up in 40 ml of methanol and 20 ml of saturated ethereal hydrochloric acid are added with cooling. After suction and washing with twice 20 ml of ether, 15.8 g = 56.8% of the theory of the title compound moss green crystals. Mp 187-189 ° C (decomp.), TLC: silica gel 60 (Merck) - mobile phase: methylene chloride / methanol = 5: 1, R._F = 0,72</li></ul>

The following are produced analogously:

<ul id="ul0009" list-style="none"><li>b) <u style="single">4- (4-nitrosophenyl) -1-piperazinoethanol dihydrochloride</u><chemistry id="chem0019" num="0019"><img file="EP0441222A2_D0021.tif" /></chemistry> from 2- (4-phenyl-piperazino) ethanol (Kremer, J. Amer. Chem. Soc. <u style="single">58</u>, 379 (1936)) as light gray crystals; purely by recrystallization from methanol / water = 7: 1, mp 170-173 ° C. (dec.), TLC: silica gel 60 (Merck) - mobile phase: methylene chloride / methanol = 5: 1, R_F = 0,67</li><li>c) <u style="single">3- [4- (4-nitrosophenyl) -1-piperazinyl] -1,2-propanediol dihydrochloride</u><chemistry id="chem0020" num="0020"><img file="EP0441222A2_D0022.tif" /></chemistry> from 1-phenyl-4- (2,3-dihydroxypropyl) piperazine (H. Howell et al., J. Org. Chem. <u style="single">27</u>, 1711 (1962)) as green crystals, mp. 163 ° C (dec.) - TLC: Kieselgel 60 (Merck), eluent: ethyl acetate / methanol = 2: 1, R_F = 0,41.</li><li>d) <u style="single">4- (4-nitrosophenyl) -α- (methoxymethyl) piperazino-1-ethanol dihydrochloride</u><chemistry id="chem0021" num="0021"><img file="EP0441222A2_D0023.tif" /></chemistry> from 1-phenyl-4- (2-hydroxy-3-methoxypropyl) piperazine (H. Howell et al., J. Org. Chem. <u style="single">27</u>, 1711 (1962)) as yellow crystals, mp. 162 ° C (dec.) - TLC: silica gel 60 (Merck), eluent: methylene chloride / methanol = 19: 1, R_F = 0.51 e) <u style="single">2- [2- [4- (4-nitrosophenyl) -1-piperazinyl] ethoxy] ethanol dihydrochloride</u><chemistry id="chem0022" num="0022"><img file="EP0441222A2_D0024.tif" /></chemistry> from 2- [2- [4- (phenyl) -1-piperazinyl] ethoxyethanol (obtained from 2 moles of 1-phenylpiperazine and 1- [2-chloroethoxy] -2-methoxyethane (the latter according to US Pat. No. 2,837,574) as green Kirstalle, mp. 134 ° C (dec.) - TLC: silica gel 60 (Merck) - mobile phase: ethyl acetate / methanol = 5: 1, R_F = 0,31.</li><li>f) <u style="single">1- (1,4-dioxanylyl) methyl-4- (4-nitrosophenyl) piperazine dihydrochloride</u><chemistry id="chem0023" num="0023"><img file="EP0441222A2_D0025.tif" /></chemistry> from 1- (1,4-dioxanylyl) methyl-4- (phenyl) -piperazine (obtained by heating 1-chloro-3- (β-hydroxyethoxy) -2-propanol for five hours (MS Kharash, W. Nudenberg, J. Org. Chem. <u style="single">8</u>, 189 (1943)) with 1-phenylpiperazine at 130 ° C., extraction with ethyl acetate and concentration. Column chromatographic purification on silica gel 60 (Merck) - mobile phase: toluene / acetone = 5: 2) as green-yellow crystals, mp. 166 ° C (dec.), TLC: silica gel 60 (Merck) - mobile phase: toluene / methanol = 5: 1 , R_F = 0,69</li></ul>

Example 15

Nitrosoheterocycles

<ul id="ul0010" list-style="none"><li>a) <u style="single">5-nitroso-1-indolineethanol hydrochloride</u><chemistry id="chem0024" num="0024"><img file="EP0441222A2_D0026.tif" /></chemistry> From 1-indolinoethanol (obtained by heating 1 mol of indoline with 1 mol of 2-chloroethanol in the presence of 1 mol of finely powdered K₂CO₃ under reflux for one hour) gives 63.8% of theory colorless oil, bp. _0,1 128 - 130 ° C, TLC: Kieselgel 60 (Merck) solvent: toluene / acetone = 5: 2, R_F= 0.42) the nitroso compound is obtained, which is isolated by adding ammonia with methylene chloride as the base. It is converted into the hydrochloride using ethereal hydrochloric acid. Light brown crystals are obtained, mp. 180 ° C., TLC on silica gel 60 (Merck) - mobile phase: methylene chloride / methanol = 5: 1, R._F = 0,51</li><li>b) <u style="single">1-methyl-6-nitroso-1,2,3,4-tetrahydro-quinoline hydrochloride</u><chemistry id="chem0025" num="0025"><img file="EP0441222A2_D0027.tif" /></chemistry> The title compound is prepared from 1-methyl-1,2,3,4-tetrahydroquinoline (obtained from 1,2,3,4-tetrahydroquinoline by heating with trimethyl phosphate (according to Huisgen et al., Chem. Ber. <u style="single">92</u>, 203 (1959)). The crude product is prepared in a conventional manner as in Examples 10 and 11 and purified on silica gel 60 (Merck) with isopropanol / n-butyl acetate / water = 5: 3: 2. By dissolving in acetone after adding ethereal hydrochloric acid, the title compound is obtained, mp 123-124 ° C. (dec.), TLC: silica gel 60, mobile phase: isopropanol / n-butyl acetate / water = 5: 3: 2, R_F = 0,7</li><li>c) <u style="single">6-nitroso-3,4-dihydro-1 (2H) -quinoline-ethanol hydrochloride</u><chemistry id="chem0026" num="0026"><img file="EP0441222A2_D0028.tif" /></chemistry> The title compound is obtained from 2- (3,4-dihydro-2H-quinolin-1-yl) ethanol (Zaheer et al., Indian J. Chem. <u style="single">1</u>, 479 (1963), bp₅ 140-144 ° C). The crude product is purified by column chromatography on silica gel 60 (Merck), eluent: methylene chloride / methanol = 19: 1. Precipitation of the hydrochloride from isopropanol with ethereal hydrochloric acid and recrystallization from ethanol gives 10.5% of theory of ocher-colored crystals of the title compound, mp. 193-195 ° C (dec.), TLC: silica gel 60 (Merck) - mobile phase: methylene chloride / methanol 19: 1, R_F = 0,36</li></ul>