Method of analyte or oxidoreductase electrochemical determination, sensor-electrode system for this determination and application of respective suitable compounds

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Method for the electrochemical determination of an analyte or oxidoreductase, a sensor, electrode system for this determination, and use of such suitable compounds

Technical Field

The invention relates to a method of electrochemical determination of the analyte. in the presence of an oxidoreductase and a reducible substance which, during the course of the reaction, transfers the electrons from the oxidoreductase to the electrode, leading to a signal which is a measure for the analyte being determined, the reducible substance being enzymatically reduced and oxidizing on the electrode . Furthermore, the invention relates to a corresponding method of electrochemical determination of oxidoreductase in the presence of an enzyme substrate and the above-described reducible substances.

In addition, the invention relates to a sensor electrode system for the electrochemical determination of an analyte in a sample which comprises at least two electrically conductive means which are insulated with each other and can be brought into electrical contact with the sample to be subjected to the electrically conductive surface, wherein at least one of the electrically conductive surfaces contacts an oxidoreductase and a reducible substance which is capable of transferring the electrons through the oxidoxid reductase and the electrically conductive surface. The invention furthermore relates to a corresponding oxidoreductase sensor electrode system in which at least one of the electrically conductive surfaces is contacted with an oxidoreductase substrate and one of the reductable substance described above.

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

Background Art

In contrast to the colorimetric methods of analyte determination of a liquid which is evaluated visually or colorimetrically, the corresponding electrochemical determination suggests that the electrochemical reaction directly provides a current which can be converted to concentration. In the case of colorimetric procedures, it is necessary to proceed by a circle, namely the battery path --- current --- light --- residual light / remit or transmit / ---- current --- measured value.

For electrochemical determination methods, the analyte must be oxidized or transferred to a substance that can be oxidized by chemical or enzymatic methods. The direct electrochemical oxidation of the analyte, or the substance removed from it, causes a high voltage on the electrode surface, that is, the potential. This method is very ineffective. Many other substances, which may also be present in the sample under investigation, are also oxidised. This procedure is therefore practically non-analytically usable.

Typically, therefore, the oxidizable analyte or the analyte-derived oxidizable substance is reacted with a corresponding oxidoreductase and a reducible substance, the reduced form of which can be oxidized on the electrode. In this case, the oxidizable analyte or the analyte can be oxidized the substance selectively oxidizes the enzyme. The reducible enzyme is oxidized by the reducible substance and the reduced reducible substance is oxidized on the electrode. The diluent thus serves as an electron transfer agent for the enzyme on the electrode. The condition is therefore such that the reducible substance is chosen to react with the enzyme and on the electrode quickly and specifically. .PW Carr et al. describe in "Theory and applications of enzyme electrodes in analytical and clinical

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which has at least two electrically conductive means which are insulated and can be electrically contacted by the electrically conductive surface with an electrically conductive surface, wherein one of the electrically conductive surfaces is contacted with the oxidoreductase and the so-called &amp; quot; mediator compound &amp; quot; between this enzyme and the electrically conductive surface. The mediator compound is an organometallic substance having at least two organic rings each containing at least two conjugated double bonds and a metal atom dividing its electrons with each counterclock. Preferred mediator compounds are, as in EP-A-0 078 636, ferrocen or ferrocene derivatives. It should be borne in mind that such compounds must first oxidize, for example ferrocions, to be able to transfer electrons from oxidoreductase. This is the so-called "start-up currents" that occur already in the absence of an analyte, which naturally causes distraction in amperometric ways in which the current present is a measure of the amount of analyte determined. In addition, the poor solubility of such organometallic compounds is disadvantageous, which in turn leads to oxygen favoring, for example when using oxidases such as glucose oxidase as oxidoreductase, and thus, especially at low concentrations of the enzyme substrate, to detectable currents and oxygen dependence. Poor solubility and / or the use of negligible concentrations are in these reduced form of electron donors used as a prerequisite for still acceptable starting currents. In summary, electron donors for electrochemical determination methods, known in the art, characterized in that, in the presence of the analyte to be determined, they are oxidized by the oxidoreductase and oxidized back to the starting compound on the electrode. When the concentration of the reducible substance acting as an electron transfer agent is substantially less than the concentration of the analyte determined, only the kinetic methods can be performed. For the determination of the end point, it is necessary that the electron-transferable reducing substance is dissolved in excess of the analyte determined and that the analyte can be completely reacted. When the proportional amount of the reducible substance to the analyte is proportional, Benefits versus' kinetic measurements are the particularly widespread area of ​​linearity of current / concentration relationship in amperometric methods, and the better competitivity of higher concentrations of reducible substance with respect to oxygen, using oxidases as oxidoreductases. However, it is disadvantageous to add a reducible substance to the complete conversion, i.e. the oxidizing agent as an electron donor with potentially significantly higher levels than the enzyme substrate, and in addition it is necessary to work in an electrochemical assay in the presence of an oxidant residue, which potentially increases the potential . High working potentials, however, improve unsatisfactory responses on the electrodes, especially when they have to: test samples with a larger number of components than the established analyte. that is to say an oxidizing agent as an electron carrier with a potential substantially higher than that of the enzyme substrate and, moreover, it is necessary to work in an electrochemical assay in the presence of an oxidant residue, which increases the potential. High working potentials, however, improve unsatisfactory responses on the electrodes, especially when they have to: test samples with a larger number of components than the established analyte. that is to say an oxidizing agent as an electron carrier with a potential substantially higher than that of the enzyme substrate and, moreover, it is necessary to work in an electrochemical assay in the presence of an oxidant residue, which increases the potential. High working potentials, however, improve unsatisfactory responses on the electrodes, especially when they have to: test samples with a larger number of components than the established analyte.

Thus, no satisfactory solutions are yet available for the electrochemical determination of the analyte by the enzymatic oxidation reduction reaction. There are no universally applicable replicable substances acting as electron transducers capable of both rapid reaction with oxidoreductases and such uncontrolled electrode surface responses at low potentials.

SUMMARY OF THE INVENTION The object of the present invention is therefore to solve the above-mentioned problem. In particular, reducible substances which may act as electrode-transfer agents between the oxidoreduct and the electrode in the electrochemical system are to be found. In accordance with the present invention, the above object is solved by the method of electrochemical determination of the oxidant in the presence of an oxidoreductase and a reducible substance which, in the course of the reaction, transfers the electrons from the oxidoreductase to the electrode, thereby generating a signal. which is a measure of the analyte determined, wherein the reducible substance is reduced enzymatically and oxidized on the electrode, the substance consisting in the fact that the oxidation-inducing substance on the electrode differs from the originally used reducing agent. Another object of the present invention is a method of electrochemical determination of 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 which produces a signal that is a measure of the enzyme determined by which the reducible substance is enzymatically reduces and oxidizes the electrode, which is based on the fact that the zinc oxide formed by the oxidation on the electrode is different from the originally used reducible substance. The subject of the present invention is furthermore use; a substance that can accept electrons from oxidoreductase, the formation of an electron-rich aromatic amine, as electron transducer between oxidoreductase and an electrode in an electrochemical system. Further, the subject of the present invention is a sensor electrode system for analyte determination in a liquid sample,

Finally, the subject of the present invention is a sensory electrode system for electrochemical determination of oxidoreductase in a liquid sample comprising at least two electrically conductive means which are mutually isolated and each time being electrically conductive in electrically contacting the at least one electrically conductive surface being in association with an oxidoreductase substrate and a reducible substance capable of transferring the electrons between the oxidoreductase and the electrically conducting surface, the substance being characterized in that a compound which is after reduction of the oxidoreductase of the electrically conductive surface oxidizes to a substance which is different from the originally used reducible substance.

Finally, the subject of the present invention is the use of a substance which can receive electrons from oxidoreductin-7-ducase to form an electron-rich aromatic amine for the manufacture of a sensor electrode system according to the present invention .

It has been shown that the disadvantages of the methods known in the art for the electrochemical determination of the analyte in the presence of oxidoreductase and the reducible substances causing the high potency necessity, especially with the use of an excess of xxxx &amp; HKK reductable substance acting as a transducer, compared to the determined analyte , can be largely suppressed by using a non-reversible reaction. By forming an oxidant other than that originally used as a reducible substance on the electrode, the electrochemical determination can be made at an especially low potential and without the risk of disturbing reactions. The advantage of low potency may also be used when the reducible substance, acting as an electron transfer agent, can only be used in a small amount compared to the analyte determined, when the reducible substance as originally used, the tactile substance formed by oxidation on the electrode is reduced by the oxidoreductase required for the electrochemical process. When an initially used reducible substance, as well as an oxidizing substance on the electrode, reduces oxidoreductase to the same substance, the initially used reducible substance acts as a second reducible substance reservoir which is circulated between the electrode and the enzyme. the substance is different from the originally used reducible substance. The advantages of the process according to the invention result from the fact that they can be chosen as reducible substances of such compounds which are formed by the enzymatic reduction of a compound which can be oxidized at the electrode at a lower voltage. There is no oxidation on the electrode that is, the specific concentration of this new oxidized substance still desirable. Until now, the enzymatically reduced compound had to be oxidized back to the electrode to the originally used reducible substance already present in high concentration. This increased the positive potential for this.

In the context of the present invention, it is preferred that the compounds of the present invention are capable of converting compounds which transfer the enzyme electrons resulting from the oxidation of the substrate corresponding to the oxidoreductase enzyme to the electron enzyme, thereby forming a rich aromatic amine on the electrons. The term electron-rich electron is understood to mean a compound that carries electrons richer than aniline, and because of the richness of the electron, it can oxidize at the low potency on the electrode. Examples include, for example, aniline derivatives bearing one or more than + 1 substituents on the ring or on the aniline nitrogen. and / or + M such as hydroxyl, alkyl, alkoxy, aryloxy, alkylthio, arylthio, amino, monoalkylamino and dialkylamino.

Alkyl, alkoxy, alkylthio, monoalkylamino and dialkylamino are radicals of the alkyl moiety of a hydrocarbon radical having 1 to 6 carbon atoms which may be substituted by a hydroxyl group, an amino group which is optionally mono- or polysubstituted by a C1 to C6 alkyl group -CH2-, -CH2 -CH2 -CH2 -CH

C02H. The acid radicals R 1, R 2, R 3, R 4 and R 5 may be present as such or in the form of salts such as ammonium salts, alkali metal salts or alkaline earth metal salts.

Aryloxy and arylthio are aromatic radicals of 6 to 10 carbon atoms, especially phenoxy and phenylthio.

Ammonium salts are those containing ammonium salt W1 + or the like. which contain an ammonium cation which is mono- or polysubstituted by an alkyl group, an aryl group or an aralkyl group. The alkyl radical of the valkyl group and the aralkyl group denotes a hydrocarbon radical having 1 to 6 carbon atoms. Aryl in the aryl group and the aralkyl group is an aromatic ring system of 6 to 10 carbon atoms, phenyl being preferred. The preferred aralkyl radical is a benzyl group.

As the alkali metal salts, lithium, sodium and potassium salts are preferred. Salts with alkaline earths are preferred magnesium and calcium salts.

Aniline derivatives are also understood to mean those compounds which on the aromatic ring system are unsubstituted amino or amino groups with one or more substituted 4-1 and / or + M substituents such as, for example, an alkyl group, said ring system is annealed with one or more aromatic and / or alicyclic rings. As aromatic rings, both hydrocarbon systems as well as heteroaromatic systems are considered. Examples include, but are not limited to, the annellated benzene or naphthalene rings or the annellated pyridine ring.

The term alicyclic rings refers to saturated or unsaturated cycloaliphatic rings having 5 to 7 carbon atoms, preferably 5 or 6 carbon atoms. .

Possible alkyl substituents are straight or branched hydrocarbyl radicals which may be substituted by hydroxyl, amino substituted with one or more alkyl radicals of 1 to 6 carbon atoms, PO3H2 , SO3H and skeleton. Kyine residues and CO 2 H. may exist as such or in salt form as ammonium salts, alkali metal or alkaline earth metal salts, as defined above. The above examples for +1 and / or + M substituents can not be understood as a complete list of these substituents. It is clear to the skilled person whether the residue +1 and / or + M is a substituent and whether they have all the residues of possible substituents that can be used according to the present invention,

Particularly preferred as reducing agents which lead to the oxidation of electrons from the oxidoreductase to an electron-rich viscous-amorphous amine which can oxidize the low potentials on the electrode are compounds selected from the group of compounds of formula IX-R (I) in which R is an aromatic radical rich in electrons and X is NO or NHOH,

and compounds of formula II HO - Y = H / 11 /, · Y meaning a quinoid system which can, after re- (λ ^ ^ ζ • Τλ. Λχ · η · ··, '· ς-ίλ.ίΡ7 ·&gt; · .ί5 ι ^ \ &lt;i; i ^ -V ^ IIX / - &lt; ', Si-ÁiiSh'SS \ ·' · '- · ;,.' &lt;.- A &lt;\ v./v7 \ v, y, /, / z; YkSóv '^'; r7'VA '^ &lt;; ^ w&gt; ^ WAV duction in the aromatic state mark yacon electron rich.

The term aromatic residue-rich elektronyse meant possibilities indicated above proaromatické amines bo-Hatem to: electrons.

Such reducible substances according to the invention are transcribed from the oxidoreductase to the aromatic amine-reduced and oxidized on the electrode and are not oxidized to the original reducible substances. As is known to those skilled in the art, electrons are ablated by the electrochemical oxidation of aromatic amines, and these electrons are derived from the aryl moiety, resulting in radicals or quinoid systems. There are no quinoid oximes, no hydroxylamines and no nitroso compounds. . Electrochemically oxidized compounds can often transfer electrons from oxidoreductase and thus revert back to electron-rich aromatic amines. It is therefore possible to use reducible substances in comparison with the established analyte in a small concentration, that is, in surplus. so as a form of stock for aromatic. aiiins rich, on .electrons,

14,14-bis (2-hydroxyethyl) -3-methylthio-4-nitrosoaniline, 14,14-bis (2-hydroxyethyl) ,. 14- (2-hydroxyethyl) -4- [2- (2-ethoxyethoxy) ethyl] -4-nitroso aniline, 14- (2hydroxyethyl) 2-hydroxy-1-propyl-4-nitroaniline, N- (2-hydroxyethyl) -3- nitrosoaniline, N- [2-hydroxyethyl] -1- [2- (2-hydroxyethoxy) ethyl] -4-nitroso aniline. . A particularly preferred reducible substance of the present invention is: 1,4-bis- (2-hydroxyethyl) -p-nitrosoaniline in the form of a compound of formula I; - 13 - 14- (2-hydroxyethyl) -4- [2- (2-ethoxyethoxy) ethyl] -4-nitroso aniline, 14- (2hydroxyethyl) 2-hydroxy-1-propyl-4-nitroaniline, N- (2-hydroxyethyl) -3- nitrosoaniline, N- [2-hydroxyethyl] -1- [2- (2-hydroxyethoxy) ethyl] -4-nitroso aniline. . A particularly preferred reducible substance of the present invention is: 1,4-bis- (2-hydroxyethyl) -p-nitrosoaniline in the form of a compound of formula I; - 13 - 14- (2-hydroxyethyl) -4- [2- (2-ethoxyethoxy) ethyl] -4-nitroso aniline, 14- (2hydroxyethyl) 2-hydroxy-1-propyl-4-nitroaniline, N- (2-hydroxyethyl) -3- nitrosoaniline, N- [2-hydroxyethyl] -1- [2- (2-hydroxyethoxy) ethyl] -4-nitroso aniline. . A particularly preferred reducible substance of the present invention is: 1,4-bis- (2-hydroxyethyl) -p-nitrosoaniline in the form of a compound of formula I; - 13 -

Even more preferred is h- (2-hydroxyethyl) -? - [2- (2-hydroxyethoxy) ethyl] -4-nitrosoaniline.

Many of the useful compounds of formula (I) are known. are derivatives of the nitrosoaniline of the formula III

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in which R @ 1 and R @ 2 are; A hydrogen atom, a halogen atom, an alkoxy group or an alkylthio group, an alkyl group and a hydroxyalkyl group or are the same or different and each represent a dialkylaminoalkyl group, a hydroxyalkoxyalkyl or alkoxyalkyl group which is optionally substituted in the alkyl moiety by a hydroxyl group or a polyalkoxyalkyl optionally substituted in the alkyl moiety by a hydroxy group, or forming an alkylene radical interrupted by a sulfur atom; nitrogen atom, the nitrogen atom being substituted by an alkyl group, a hydroxyalkyl group, a hydroxyalkoxyalkyl group,

R @ 14 and R @ 14 are hydrogen, hydroxyalkyl, di-oxanyl-alkyl, or polyalkoxyalkyl groups, the radical may be optionally substituted by a hydroxyl group, or R is in the ortho position to R 1 and R 2 together with R 2 represents an alkylene group, wherein R 1 represents a hydroxyalkyl group, or when the alkylene group contains 3 carbon atoms, also means an optionally alkyl radical, or when R is not a hydrogen atom, R 1 and R, which are the same or different, denotes a hydroxyalkyl group or a salt of this derivative. In the above formula, the halogen atom is fluorine, chlorine, bromine or iodine, with fluorine and chlorine being particularly preferred. Alkyl, alkoxyl and alkylthio are radicals of 1 to 6 carbon atoms, with 1 to 3 carbon atoms being particularly preferred. The above definition for alkyl group in the hydroxyalkyl group, dialkylaminoalkyl group, hydroxyalkoxyalkyl group, alkoxy-alkyl group, polyalkoxyalkyl group, alkoxyhydroxyalkyl group and dioxanyl-alkyl group.

The dioxanyl-alkyl moiety is a group in which the monooxane ring system is attached to the alkyl moiety. Preferably, this is a 1,4-dioxane ring system, i.e., O-alkyl-15-

The polyalkoxyalkyl group is a radical of the formula -alkyl- (alkoxy) n-alkoxy wherein n is 1 to 10, preferably 1 to 4, particularly preferably 1 to 3. An alkylene group may. to be straight-chain, but preferably straight-chain, saturated or unsaturated, but preferably saturated, hydrocarbon chain contains from 2 to 5 carbon atoms, preferably from 2 to 4 carbon atoms, with two salt binding sites.

The meaning of the substituents R1 and R2 denoting an alkylene radical interrupted by a sulfur or nitrogen atom is a preferred thiomorpholine or piperazine residue formed with the nitrogen atom of formula III. Particularly preferred is the piperazine residue.

As an alkyl radical of the radical formed by the substituents R1 and R2, preferred is indoline or 1,2,3,4-tetrahydroquinoline residue formed with the aromatic ring of formula III. As salts of the nitroso-aniline derivative of the formula (III) according to the invention, salts with strong acids, especially mineral acids, such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, are particularly advantageous. More preferred are hydrochlorides, i.e. salts of the acid salt.

4 &amp; quot; dihydro-1 (2H) quinolineethyl alcohol, and salts thereof. Of these compounds, the compounds (a), (d), (e), (f), (g) and (h) are particularly preferred. Particularly preferred compound (e) and optionally salts thereof, in particular hydrochloride.

Compounds of formula (III) are obtainable by reacting a compound of formula (IV)

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/ IV /, -17 -

Wherein R, R and R are as defined for compound of formula (III), with a sulfite. The analogous method is known from J.J. D'Amico et al., J. Amer. Chem. Soc. 81, 5957 (1959).

Nitrite is preferably used with nitrite of alkali metals, with alkali metal, such as lithium, sodium, potassium, rubidium and cesium, but preferably sodium nitrite and potassium nitrite are used. Especially preferred is sodium nitrite. React. is preferably carried out in acidic medium at low temperatures. The suitable temperature should be below 10 ° C, preferably in the range of -10 to +5 ° C.

The reaction of the compound of formula IV with the nitrite is preferably carried out in an aqueous medium. Suitably, the pH is less than 3, particularly preferably less than 2.

For the reaction, a compound of formula (IV) or a salt thereof, preferably a mineral acid salt such as, for example, hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, to an aqueous, acidic medium and cooled. In order to maintain a low temperature, the reaction mixture is preferably nitrite, preferably in dissolved form. Preferably, an aqueous medium is used as the nitrite solvent. After the addition of nitrite, the reaction mixture is kept at a low temperature until the reaction is complete. Treatment of the reaction mixture is preferably carried out by extraction with an organic solvent and the product is isolated from the extract.

The compounds of the present invention, useful as electron donors, can be stored and used in a form of oxidized form. This eliminates the starting currents and the end point of the determination is feasible with an excess of the electron transfer device. The compounds of the present invention, useful as electron donors, are stable in storage and can enter the fast reaction with oxidoreductases. In particular, the use of oxidases is oxygen-friendly and may be used in excess also against high concentrations of the analyte to be determined. Particularly the latter property is made possible by the good solubility of the electron transfer device according to the invention in the feed medium.

A particular advantage of the compounds of the present invention useful as electron donors is their ability to perform non-enzymatic reduction with reducing agents in body fluids when electrochemically determining analytes in body fluids. The electron donors according to the invention are rapidly oxidized on the surface of the electrodes and are not oxygen-sensitive in reduced form. These compounds can work at low potential for oxidation on the electrode. In the present invention, as the analyte, it denotes the constituent substance. This is normally a compound component. In particular, the method according to the invention offers the advantages of analyzing in the body fluids, such as blood, plasma, serum, urine,

The method of electrochemical determination of the analyte of the present invention consists in that the analyte itself is an oxidoreductase, i.e. the corresponding enzyme substrate, or the analyte is converted into a compound which can be oxidized by oxidoreductase in one or more precursor reactions, especially enzymatic reactions. In such reactions, the released electrons are proportional to the amount of analyte set up. When these electrons are converted by the reducible substance of the invention to. electrode, causes a signal that is a measure for the analyte to be determined. weighted amperometric methods in which the current is measured, or post-tenometric methods where voltage is measured. ¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿" . '', '', '', ''. ' - - - - - - - - - - - -

For the process of the present invention, such oxidase reductases are preferably oxidases, NAD / P / -containing dehydrogenases or diaphorase. For example, according to the invention, it is possible to stabilize glucose with glucose oxidase, lactate with lactatoxidase, glycerophosphate with glycerophosphate oxidase or cholesterol by cholesterol oxidase. As. NAD / P / - independent dehydrogenase may be used, for example, glucose-dye oxidoreductase for glucosing. Diaphorase, which may also be referred to as NADH: dye-oxidoreductase, may be used advantageously. NADH. In the case of an electrochemically determined analyte which does not itself serve as an oxidoreductase substrate, the analyte can be converted by one or more precursor reactions, in particular enzymatic reactions, to a compound which is acceptable for the oxidoreductase as a sucrose. For example, triglycerides can be determined by cleaving them with glycerol residues and acid residues, glycerol by glycerol kinase and ATP being converted into glycerophosphate and finally oxidized with glycerophosphate oxidase and released electrons. are fed by one of the electron donors according to the invention, generating a stream that is proportional to the triglycerides in the sample being examined.

Analogously, for example, the total cholesterol can be determined by cholesterol cleavage by cholesterol ester synthesis and the cholesterol thus produced is determined by cholesterol oxidase. There are also many cholesterol produced by cholesterol oxidase and electrons liberated by cholesterol oxidase oxidation. and converted with the reductable substance of the present invention to the electrode of the current, proportional to the amount of total cholesterol determined.

For the determination of NADH, the enzyme is diaphorase.

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20 -

By using the reductable substance of the present invention, the electrons can also be transferred from the diaphorase to the electrode. Since many biological agents can enzymatically react to form NADH, many analytes are converted by enzymatic reaction sequences to MDH and then finally determine through the diaphorase and the reducible substance of the invention on one electrode. From the above interpretation, it is understood that the oxidoreductase can be determined naturally by the use of the corresponding compound which is acceptable as the enzyme substrate and the reducible substance of the invention, for example the lacquer can be electrochemically determined by the glucose oxidase that the glucose and electron transfer device according to the invention are contacted in the presence of a corresponding sensor electrode system.

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IN-

Zn 'au

The process according to the invention is characterized in that the reducible substance used for the transfer electrode from the oxidoreductase to the electrode is stable in its oxidation form and is also readily soluble in water. This is especially important for the determination of analytes in body fluids, such as blood, plasma, serum and urine. In accordance with the present invention, useful reductants exhibit a rapid reaction with oxidoreductases and are particularly well-reactive with oxygen in reaction with oxidases. Due to their solubility, they can be very well-suited for amperometric methods where excess over the highest concentration of analytes is required. Since the reducible substances usable in the present invention admit in the body of fatigue, it is negligible. wound reduction. here come to · ·; with the same reducing agents on the surface of the electrodes are rapidly oxidized and in a reduced form are not sensitive to oxygen, and these substances are very well suited for specific, trouble-free, electrochemical determinations. Reliable and specific electrochemical analyte determinations are possible when, in addition, it is above all caused that the reducible substance of the invention requires only minimal potential on the electrons.

The method of the present invention for the electrochemical determination of the analyte is not limited to certain electrochemical devices. For example, it is possible to use sensor-electrode systems according to the state of the art. In principle, suitable sensor electrode systems for determining the analyte in a sample are provided which comprise at least two electrically conductive means such as electrodes which are isolated from one another and capable of being electrically contacted with the investigated sample by means of an electrically conductive surface. It is to be assumed that only two electrodes, namely the working electrode and the reference electrodes, are used. It is also possible to arrange measurements without a reference electrode, that is, only the processing electrode and counter electrode. Only a constant external voltage is maintained. However, it is also possible to use a second electrode, namely reference electrodes, working electrodes and counter-electrodes. Corresponding sensor electrode systems are known in the art, for example, from G. Kenz and R. lleeb, "Blektrochemische Analytik", Springer-Verlag (1986).

It is important that for the electrochemical determination of the anolyte (at least) one electrode, i.e. one electrically conductive surface, contacts the oxidoreductase and a reducible substance capable of transferring the electrons between the oxidoreductase and the electrically conductive surface. It is clear that all of the reagents required, together with the sample to be examined, are in solution or that some of the reagents, in particular oxydipine and / or reductable electron transferring substances, are immobilized on the electrode and the rest of the reagents are dissolved in the solution or the whole of the reagents required for the determination immobilized on the electrode. The principle for the function of the sensor electrode system to decide whether the working electrode contacts the oxidoreductase and a reducible substance acting as an electron transfer agent of the dissolved substance,

Of course, the same applies to the determination of oxidoreductase as described above. It should be noted here that the sensor electrode system must contact;

oxidoreductase substrate and a reducible substance according to the invention. Otherwise, the same requirements apply to this case; and the form of execution as in the determination of the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS The enclosed drawings illustrate the subject matter in greater detail. These show:

Fig. 1, part a) Scheme of the useable reducible substances according to the invention in the process of the invention and in the sensor electrode system when the concentration of the electron donor is greater than or equal to the concentration of the stabilized analyte.

Fig. 1st part. b) Scheme of the function of the substance transferring the electrons of the process and of the sensor electrode system of the prior art. - 23 -

Fig. 2, part a and /

Fig. 2, part b /

Scheme of the function of the reducible substance usable. according to the invention in the process of the invention in a sensor system when the electron transferring concentrates are substantially lower than the concentration of the analyte to be determined.

Scheme for the function of the electron-transferring substance in the process and the sensor electrode system, as before.

Fig. A sensor electrode system for carrying out the method of the invention in which the required substances are dissolved in solution.

Fig. 4 Sensor electrode system for carrying out the process. according to the invention, designed as a single-use sensor, or a disposable sensor.

Fig. 5 Diagram with the value obtained from cyclo-talmograms for the anodic current peak at different glucose concentrations .s. D-bis- (2-hydroxyethyl) -p-nitrosoaniline as the electron-transferring agent for the glucose electrochemical assay of the invention.

Fig. .6 Diagram for Relationship Between Current Density and Concentration M) H in the NADH Teflon of the Invention 9

Fig. D- (2-hydroxyethyl) -D-p-nitrosophenylpiperazine and D, D-bis- [2-hydroxyphenoxy] p-nitrosoaniline cyclicotamogram.

Fig. 8 Diagram of current density versus glucose concentration according to the method of the present invention with n-methyl-N, N (4-nitrosophenyl) -piperazine as 24-transferring electrons in the presence and absence of air oxygen.

Fig. 9 Diagram of current density dependence on glucose concentration according to the prior art with tetrathia fulvale as an electron donating agent, in the presence and absence of air oxygen.

Fig. 10 Diagram of current density dependence of kjjjSsntraxE on LDH-concentration according to the method of the invention with N, N-bis- (2-hydroxyethyl) -p-nitrosaniline as a substance transferring the electrons at different time intervals following the start of the lactate dehydrogenase assay.

Fig. The current-time curve for the method of the invention with the disposable electrode of Fig. 4 for glucose addition.

Fig. FIG. 12 is a graph of the current-dependent glucose concentration according to the invention with the disposable electrode of FIG. 4 after 10 seconds of the reaction time.

Figures 1 and 2 illustrate the differences between the intermediate process according to the invention (a) and the method of the prior art (b) using an excess of the electron-transferring agent relative to the analyte (Fig. 1) and the admission of a very small amount of the electrode transfer agent relative to the concentration of the analyte determined (Fig. In the prior art method of Figure 1b), the electron donating agent (E), in the presence of the analyte to be determined, or the analyte of the derivative (4, 4), which is enzymatically oxidized to (3), is converted into reduced ox form (Epsilon) and a 3 g of electrophoresis reduced carrier

I V). ? ý 7:; .ipí; / 2β - electrons / oxidized / oxidized by electron donation of the naturally occurring reducible substance (Et 3);

On the other hand, in the process according to the invention, lb / reductant / E, acting as an electron-transferring enzyme in the enzymatic oxidation of the analyte to be determined, or, if appropriate, from the analyte of the derived substance to be converted into a reduced form (E-Fe) oxidation on the electrode then forms an oxidized electron beam form (E &amp; quot;), which differs from the originally used reducible substance (E). Since the woman of the origin of electrochemical oxidation is completely absent, the substance E can be oxidized at a particularly low potential. The transferable electron transferring agent according to the invention may be selected such that a relatively low potential is required for the anodic oxidation of the enzymatically formed reduced form / Epsilon. This can eliminate the interfering accompanying reactions that occur, when the high potentials of the accompanying electrode inserts in the sample under investigation oxidize and thus cause the production of a stream, resulting in a false positive result. In the prior art process of FIG. lb) the redundant form of the transducer means for the redox oxidation of the reduced electron transfer vector (E) is needed for the excess reductable substance (E), a potential higher than that of the originally used reducible substance (Eqx-j)

When the reducible substance (E), acting as an electron transfer agent, is used in a smaller amount of the analyte determined relative to the analyte, or with respect to the substance from the stabilized analyte, it can then be derived from the prior art FIG. 2b) reducible or reduced as originally used in the circulation between the electrode and the enzyme, the form / Ere (i) anodically oxidized reducible substance (EQX). In the process according to the invention (Fig. 2a) when the oxidized form of the electron transfer / Eqx formed on the electrode is reduced by the reduced enzyme as well as the initially used reducible substance (E), it can be reduced by reducing matter (BQX), for example when stored stable form supplies for electron transferring system / Eox 2 / Ere &lt;i /.

For the process of the present invention, all sensor electrode systems which are also suitable for carrying out the prior art processes can be used. The sensor electrode system of Figure 3, for example known from G. Henze and R. Reeb, "Electro Springer-Verlag / 1986. The working electrode 1, the counter electrode 2 and the reference electrode J are immersed in the determined liquid drop, and the electrodes can be used for the electrodes The working electrode 1 and the counter electrode 2 may, for example, be formed preferably of precious metals or the use of such metals.The preferred materials for the working electrode 1 and the counter electrode 2 are, for example, gold and platinum.The reference electrode 3 can also be formed from the systems used here.The silver / chloride system silver.The reference electrode J 'is preferably over a salt bridge, for example a potassium chloride solution, in conjunction with the other electrode system 1, 2 for the determined liquid sample.

The oxidoreductase or oxidoreductase system required for the process according to the invention (depending on whether an analyte or oxidoreductase is to be determined) and an electron-transferring reducing agent can be dissolved in the test sample, or they can also all or to be located on the working electrode _1. As the electrodes must always be electrically connected and regulated according to the measured electrical signal, it is obvious to a person skilled in the art.

Figure 4 illustrates the construction of the electrode for prolonged use, which may be used, for example, for the demonstration of glucose. On the insulated carrier material 8, for example a polycarbonate film, suitable electrodes and associated feed lines can be used by suitable methods. Suited methods include screen printing methods, injection methods, steaming techniques or thin film techniques. Figure 4 illustrates a working electrode with a suitable electrically conductive supply 55, a reference electrode 6 with an inlet 66 and a counter electrode 1 with a corresponding supply line 77. Electrodes and their conduits can be used with conventional electrically conductive materials. For example, for the production of electrically conductive conduits, it is possible to use commercially available graphite pastes on the market.

The electrodes contain mostly precious metals, such as hawthorn, gold or platinum. In the sensor electrode system according to the invention shown in FIG. 4, the electrode has the necessary reagents required to perform the electrochemical determination of the analyte or oxidoreductase.

For glucose determination, for example, the glucose oxidase, the electron transferable reducing agent according to the present invention, contains a buffer which optimizes the pH of the sample to be used for the enzymatic reaction, as well as, for example. a detergent and a swelling agent to obtain a mixture which has the consistency needed to produce the electrode and the mixture is processable as a paste by means of a conductive material. As a material for improving conductivity, for example, a graphite powder can be added.

Reference electrode 6. a protielektroda 7, * * * * * * * * * * * * * * And the corresponding feed lines 66 and 77 can, for example, be made from a marketable silver-conductive paste which contains powdered silver chloride.

The sensor electrode system according to Figure 4 can be made in a size of about 10 x 30 mm. The sample can be applied to the surface of the electrodes or the test carrier can be immersed in the test solution so that the surface of the electrodes is covered by the amperometric the measurement can then be placed on the electrodes of the potential and measure the current which is proportional to the analyte determined. For this purpose, between the working electrode 5 and the counter electrode 7, the current is measured and controlled so as to maintain the pre-tension between the reference electrode 6 and the working electrode 5. Measurement of the voltage between the working electrode and the electrophoresis electrode. 6 is carried out without a current so that the council does not play a role here. With slight requirements, the precision of potential on the electrodes can also be dropped from the faultless measurement of the voltage,

The invention is illustrated by the following examples. EXAMPLES Example 1

Glucose test

A sensor, electrode system, 3. The working electrode 1 consists of a gold wire with a surface area of ​​0.1 cm. Counter-Electrode 2 is made of platinum-30-? a 0.1 cm wire and a reference electrode 3 formed with silver / silver chloride system (Orion Researchlne., Boston, Massachusetts, USA). The reaction vessel contains a solution of 0.1 mol / l potassium phosphate buffer and 0.1 mol / l chloride, pH 7.0; 10 mmol / l of N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline agglomerate at a concentration ranging from 0 to 100 mmol /

The reaction is determined by addition of glucose oxidase (EC 1.1.3.4) to the reaction mixture, followed by stirring. Add as much glucose oxidase as the concentration in the reaction mixture is 0.5 mg / ml (125 U / ml) . One minute of the addition of glucose oxidase using a potentiestate (Mod. 273EG &amp; G, Princeton Applied Research, Princeton, New Jersey, USA) measures the cycloltammogram at a scanning rate of 100 mV / s. The currents of the first oxidation peak and 150 mV are evaluated. The results obtained are shown in FIG. Corresponding measurements after five minutes after the addition of glucose oxidase did not show any significant changes in oxygen access (under argon).

As can be seen from the diagram in FIG. 5, the glucose concentration is about 30 mmol / l linear dependence of the maximum anode current density on the glucose concentration. At higher concentrations of glulisine than 30 mmol / l, N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline, used as an electron transfer agent, is completely converted to the corresponding phenylenediamine. Higher concentrations than 30 mmol / l do not lead to further 'upward, current. Since only one-third of the total glucose is present in the β-form and therefore are usable for conjugation with glucose oxidase, the complete conversion is found to be suitable for the production of one phenylenediamine molecule, of; 10 mmol / l of electron donating 30 mmol / l of glucose to the exact theoretical stoichiometry. Using glucose-dye oxidoreductase (EG 1.1.99. 17) instead of glucose oxidase (EC 1.1.3.4) in 0.1 mol / l of tris-buffer, 0.1 mol / l of potassium chloride, pH 7.0 and with the addition of 1% bovine serum albumin yields comparable results. Example 2

Proof NADH

The construction and layout of the measurement is the same as that described in Example 1. The reaction vessel contains 0.1 mol / l of potassium phosphate buffer, 1.0 mol / l of potassium chloride, pH 7.0, 10 mmol / l of N, N-bis (2-hydroxyethyl) p -nitrosoaniline and NADH in the range of 0 to 10 mmol / l. Measurement is started by addition and mixing of diazepase (NADH: dye-oxidoreductase) from the reaction mixture microorganisms. Ensure that the enzyme concentration of the reaction mixture is 0.2 mg / ml (3 U / ml). Measuring the flow density after one minute of the reaction time gives a linear relationship of the current density and concentration shown in FIG. Example 3

Determination of lactate

With the help of the same experimental arrangement as well, di. - / -. - 32 - with the same electron donor as described in Example 1se can also be determined by lactate. Lactate oxidase (EG 1.1.3.2) is used as the enzyme and as a buffer of 0.1 mol / l citrate buffer, 0.1 mol / l potassium chloride and pH 5.5. Example 4

Determination of glycerophosphate

When glycerophosphate oxidase (EG 1.1.3021) and buffer 0.1 mol / l buffer, 0.1 mol / l potassium chloride and pH 8.0 are replaced in Example 1 with glycosoxoxidase enzyme, glycerophosphate can be determined analogously. Example 5

Cholesterol determination

When replacing in Example 1 glucose oxidase by cholesterol oxidase from streptomycetes (EC 1.1.3.6), acceptor nucleotides 10 mmol / l N-methyl-N- (4-nitrosophenyl) -piperazine and 0.1 mM potassium phosphate buffer, 0.1 mol / l of potassium chloride and pH 5.5 with 2% Triton X 100, analogous to that described in Example 1, the cholesterol. Example 6

Reductible substances transferring the electrons according to the invention

The compounds listed in Table 1 are then combined with 10 mmol / l in 0.1 M potassium phosphate buffer, 0.1 mol / l potassium chloride and pH 7.0 are treated with 50 mmol / l of glucose and 33 0.5 mg / ml glucose oxidase (125 U / ml). For measurements, the arrangement described in Example 1 is used. Corresponding cyclo-talgrams provide the potential potentials, reported in mV against the normal hydrogen electrode, of the electron donor reduced by glucose oxides. dase and glucose. tt

As a rate for the conversion rate is in Table 1? the ratio of oxidation currents to the highest oxides potential is given; of the dawn peak after one and ten minutes. and J, 5b

Table 1 s? a) the electron transfer vector &amp; apos; s peak potential. / conversion of N- [2-hydroxyethyl-N-p-nitrosophenyl) piperazine 340 97 N, N-bis (2-hydroxyethyl) p- ) -aniline 210 94% of o-methoxy- [N, N-bis- [2 ', 1'-hydroxyethyl] -p-n-L-trosoaniline 170 35.3; Iň; p-nitrosophenol 220 62-8 C-p-quinondioxime 2 50 35 N, N-dimethyl-4-nitroso-1-naphthylamine 175 25 - $

Table 1 / continued / - 34 - electron transfer vector peak potential and / conversion rate H, U-3-trimethyl-4-nitrosoaniline 220 K- (2-hydroxyethyl) -5- nitrosoindoline 80 liters - (2-hydroxyethyl) -3-chloro-4-nitrosoaniline 315 2,4-Dimethoxynitrobenzene 130 H, 1 -bis (2 -methoxyethyl) -4- aniline 245 3-Methoxy-4-nitrophenol 140 N- (2-hydroxyethyl) -6-nitroso-1,2,3,4-tetrahydroquinoline 95 K, - 4-nitrosoaniline 275 li, 11 bis- (2-hydroxyethyl) -3-fluoro-4-nitrosoaniline 2b0 56 86 72 95 68 30 82 27 74 35 -

Table 1 / Continuation / Electron Transmitter. peak potential α // conversion rate h, 1β-bis- (2-hydroxyethyl) -3-methylthio-4-nitrosoaniline 195 21- N- [2- (2-methoxyethoxy) ethyl] -4-nitroaniline 210 mg; N- (2-Hydroxyethyl) -3,4- (3-methoxy-2-hydroxy-1-propyl) -4-nitroso aniline 225 N- (2- h - [3- (2-Hydroxyethoxy) -2-hydroxy-1-propyl] -4-nitrosoaniline 210 ii) The first peak potential of the electron transferring cellulose-reduced glucose oxidase and glucose against Ag / AgCl; b / First peak current in the cycloltogram at one minute reaction time: flow at 10 min reaction time in% c / Concentration 5-10 mol /

FIG. 7 shows cyclotolmograms for N- (2-hydroxyethyl) -α-p-nitrosophenyl-piperazine and N, N-bis- (2-hydroxyethyl) -p-nitrosoaniline. These cyclo-lactams were measured with 10 mmol / l of glucose to avoid residual glucose reactions during cyclic-lambda sensing. · ?. Example 7

Comparison of the xykixxxxxxxag electron donor according to the invention by the prior art carrier a) In the experimental setup as described in Example 1, R-methyl-N- (4-nitrosophenyl) -piperazine in a concentration of 10-4 mol / l phosphate buffer at pH 7.0. Measurement of cycloltammograms at glucose concentrations of 0 and 3 mmol / l gives the current density dependence on glucose concentration as shown in Figure 8. At low concentrations, atmospheric oxygen is detected, which can be eliminated by measuring under argon. Same results as using argon as the protective gas are achieved by using electron transfer at higher concentrations (10 μmol / l). It is also possible to exclude the influence oxygen measurement using glucose dehydrogenase instead of glucose oxidase. bb) When instead of N-methyl-N- (4-nitrosophenyl) -piperazine as an electron donor according to the invention, an electron transfer electrode of tetrathiable is used, the current density depends on the concentration of glucose as shown in Fig. 9 . Tetrathia-valen exhibits considerably higher levels of oxygen-induced damage. than the electron donor according to the invention.

In addition, a significantly lower current density is measured,

Tetrathiafulvalen is rather difficult to dissolve. To reach a concentration of 10 μmol / l in phosphate buffer pH 7.0, 2.5% Tween 20 as a detergent must be used. Setting a substantially higher concentration of tetrathia fulvalen to avoid oxygen-induced disturbances, which is possible in the case of the elkktrone transducer of the present invention, is poor due to poor solubility. Example 8

Enzyme Determination and / Lactate Dehydrogenase Assay The following solutions are used in the assay procedure as described in Example 1: 0.1 M sodium phosphate buffer, 0.1 M potassium chloride, pH 9.0 10 mmol / 1 N, N-bis (2-hydroxyethyl) -p-nitrosoaniline 0.1 M D-Lactate (sodium salt), 1 U / ml diaphorase / 1 NAH +. 2a of vigorous stirring / magnetic stirrer, 1000 rpm, at a constant potential of 75 mV silver / silver chloride counter current. Lactation starts with the addition of lactate dehydrogenase / EC 1.1.1.27/. Different amounts of lactate dehydrogenase are added and each time is measured. 100 200, 300, 400, 500, and 600 seconds. The current / time curves obtained are shown in Figure 10. The LDH activities applied on the x-axis were detected using i '

<img img-format="tif" img-content="drawing" file="CS9100261A2D00381.tif" id="idf0008" />

- 38 - pyruvate reduction test. b) Glucose dehydrogenase assay

Analogously as described in Example 8b), 0.1 mol / l of potassium phosphate buffer, 0.1 mol / l, can be used. chloride, pH. 7.0, performed with 10 mmol / l NAD +, 10 mmol / l of the electron donor according to the invention, 1 U / ml of diaphorase and 0.1 mol / l of glucose, a NAD-dependent glAcoso-dehydrogenase assay. . .

Oxidases, diazease or NAD-independent dehydrogenases can be determined in the same manner. Example

X

Disposable electrode system for glucose proof

The sensor electrode system according to Figure 4 is made by applying a screen-printing to the polycarbonate film. suitable leads are the working electrode J, the reference electrode 6, counter electrode 7 and the corresponding devices (55, 66 and 77). The inlets are made available on the available graphite printing paste (Acheson 421 SS, Deutsche Acheson Colloids, Ulm, BRD). The reference electrode 6 and the counter electrode 7 are composed of a silver conductive paste which is mixed with 20 wt.% Silver powder (Acheson SS 24566, Deutsche Acheson Colloids, Ulm, BRD). Y '%

For the working electrode 5, 2% by weight of hydroxyethylcellulose (Natrosol 250G, Hercules) is homogenized upon swelling. B7, 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 / - 39 -

Reinheitsgrad II, Boehringer Mannheim GmbH, Mannheim, BRD / per 100 g of mixture, 30% by weight of graphite powder (UF296 / 97, Graphitwerke Kropfmtlhl, BRD) and 4% by weight of ethylene glycol. The electrodes are 2 at the working electrode 5 x 6 mm = .24 mm, 2 at the reference electrode 6: 1 x 1, 5 mm = 1.5 mm and 2 at the counter electrode 7: 1 x 1.5 mm = 1.5 mm

The screened electrode system is immersed in a measured solution containing 0.05 mol / l sodium phosphate buffer (pH 7.0), 0.1 mol / l sodium chloride and 0 to 45 mmol / l glucose so that the areas electrically-tested liquid. At the 200 mV potential of the integrated reference electrode 6 (Silver / Silver Silver), the current / time curve shown in Figure 11 is measured. application of the current values ​​after ten seconds of the measuring time, the calibration curve shown in FIG. 12, which shows the current flow dependence on glucose concentration. Example 10 9 Production of 2,2 - (4-nitrosoaryl) imino / bis-ethyl alcohols

To a 4-liter three-necked flask with a stirrer, heat-meter, and dropping funnel, two moles of N, B-bis (β-hydroxyethylaniline) . The resulting solution is cooled to 0 ° C by means of a cooling bath and a solution of 148 g (2.1 mol) of sodium nitrite in 200 ml of water is added dropwise over a period of 20 minutes to a temperature ranging from 0 to 2 ° C. The reaction mixture is stirred for a further 30 minutes at 40 DEG C. at 0 DEG. most of the crystalline yellowish to greenish-colored nitroso compound is aspirated and the filter cake is washed twice with 200 ml of ice-cold, semi-concentrated hydrochloric acid. For purification, the crude product is dissolved in 900 ml of water, with vigorous stirring, 400 ml of concentrated hydrochloric acid, the solution is stirred for 30 minutes at room temperature and then for an additional 30 minutes with ice bath cooling. The crystallizate was then dissolved \ in 580 ml of water, mixed with 265 ml of concentrated hydrochloric acid i- and again stirred for 30 minutes at Heat-, * that room for 30 minutes with ice bath cooling. You? The crystals are filtered off with suction, washed three times with 150 ml each

acetone and two times with 200 ml of diethyl ether and finally dried under vacuum at room temperature. It is obtained as follows:. A) 2,2 - [(4-nitrosophenyl) imino] bis-ethyl alcohol hydrochloride. Yield: 32.8% of theory, green crystals, melting point 160 ° C (decomposition).

Analogously, the corresponding arylsubstituted analogs of b) 2,2 - [(3-fluoro-4-nitrosorinyl) imino] bis-ethyl alcohol hydrochloride are obtained. Yield: 26.5% theory, yellow crystals, melting point 140 ° C / /, DC: silica gel (Kieselgel 60, Merck), mobile phase ethyl acetate / ethyl alcohol = 5: 1, = 0.59.

Manufactured from 3-fluoro-1, N-bis- (2-hydroxyethyl) aniline (Chem. Ab.str. (C) 2,2 - [(3-Chloro-4-nitrosophenyl) imino] bis-ethyl alcohol hydrochloride 41 - Yield: 21% of theory, yellow crystals, melting point 154 DEG C. (decomposition), DC: silica gel (Kieselgel 60, Merck), phases of methylene chloride / methyl alcohol = 5 : 1, Rf = 0.72.

Manufactured from 3-chloro-N, N-bis- (2-hydroxyethyl) aniline / M. Rreifelder, GR Stone, J. Org. Chem. 26, 1499 (1961). 9d / 2,2 - [(3-methoxy-4-nitrosophenyl) imino] bis-ethyl alcohol hydrochloride Yield: 32% of theory, ocher crystals, mp 145-146 ° C. / decomposition /. DC: silica gel / Kieselgel 60, Merck /, pooled phase methylene chloride / methanol = 5: 1,

Rf = 0.4;

Made from 3-methoxy-K, K-bis (2-hydroxyethyl) aniline / M. Rreifelder et al., J. Org. Chem. 26, 1499 (1961). e] 2,2 '- [(3-methylmercapto-4-nitrosophenyl) imino] bis-ethyl alcohol hydrochloride Yield: 59.3% of theory, reddish brown crystals, melting point 148 DEG C. (decomposition) Kieselgel 60, Merck), mobile phase ethyl acetate / methanol = 5: 1, Rf = 0.53;

Manufactured from 3-methylmercapto-L1, bis- (2-hydroxyethyl) -aniline. This can be obtained by dissolving 0.1 mole of 3-methylmercaptoaniline in 50 ml of 4N acid, sulfuric acid and 0.35 moles of ethylene oxide, and the reaction mixture is stirred for 12 hours at room temperature. Then, excess sodium bicarbonate solution is added, methylene chloride and purified by column chromatography over silica gel (Kieselgel 60, Merck, luene / acetone = 5: 2, 1L. = 0.18), yield 25% of the product as a colorless oil. f) 2- (methyl) -3-chloro-4-nitrosophenyl] amino] ethyl alcohol hydrochloride. Yield: 15%. yellow crystals, melting point 147 DEG C. (decomposition), i. DC: silica gel (Kieselgel 60, Merck), mobile phase methylene chloride / methanol = 19: 1, = 0.34 g

Prepared from 2- (methyl) 3-chlorophenyl] -amino] -ethanol. [

This can be obtained from 2 - [(3-chlorophenyl) amino] ethylalcohol with a 3 hour boiling point with methyl iodide in the presence; 10% sodium hydroxide. . After purification by column chromatography on silica gel. / Kieselgel 60, Merck, eluted with toluene / acetone = 0732 5: 2, = 0.39. gives &lt;25% of product as colorless oily cathode. . . ; . ; Example 11 2- [7-Hydroxyethoxy) ethyl] -4-nitrophenyl] amino] ethyl alcohol hydrochloride A) 2- (2-Hydroxyethoxy) ethylphenyl] SíffíS ^^

<img img-format="tif" img-content="drawing" file="CS9100261A2D00431.tif" id="idf0009" />

CH2CH2OCH2CH2OHH.

CbLCrLCH LMMM

<img img-format="tif" img-content="drawing" file="CS9100261A2D00432.tif" id="idf0010" />

146 g (0.8 mol) of 2- (2-anilinoethoxy) ethanol obtained by reacting aniline with 2- (2-chloroethoxy) ethyl alcohol in a yield of 54% and a colorless oil of boiling in the range of 131 133 DEG C.) was dissolved in 500 ml of 4N hydrochloric acid, cooled to 0 DEG C. with stirring, and dropped to a temperature within the range of 5 minutes0 to 10 UC 70.5 g, which is about 79 ml / 1.6 mol / ethylene oxide. After 12 hours of standing the reaction mixture at room temperature, 500 ml of water are added and 200 g of sodium bicarbonate are neutralized with cautious succession with stirring. The free base is extracted with 500 ml of methylene chloride, the mixture is then shaken three times with 250 ml of methylene chloride each time, the organic phases are combined, dried over anhydrous sodium sulfate and concentrated by evaporation in a vacuum. 178.2 g of product are thus obtained. DC: silica gel (Eiesegel, Merck), toluene / acetone = 5: 2, = 0.18. B) 2 - [(2-hydroxyethoxy) ethyl] -4-nitrosophenyl] amino]

<img img-format="tif" img-content="drawing" file="CS9100261A2D00441.tif" id="idf0011" />

CH 2 CH 2 CH 2 CHO OHS dddd

In gh &amp; quot; 2 d HCl

To a 3-necked, 2-liter flask equipped with a stirrer, dropping funnel and a thermometer is added a mixture of 280 ml of concentrated hydrochloric acid and 140 ml of water, cooled to -5 DEG C. in the cooling bath, maintaining the temperature within 10 minutes 178 g (0.79 mol) of the compound obtained according to A) are added dropwise and the reaction mixture is stirred for another 15 minutes. Then a solution of sodium nitrite (60 g, 0.87 mol) in water (120 ml) is added at 0 DEG C. and the reaction mixture is stirred at 0 DEG C. for 30 minutes. It is then diluted with addition. 500 ml of water (a reaction mixture of 1.4), and 218 ml of concentrated aqueous ammonia solution are added dropwise to a pH of 9 at maximum 15 ° C while cooling with an ice bath. The liberated nitrosobases are shaken with 400 ml of n-butyl alcohol five times, and the solvent is evaporated on a rotary evaporator. 212.8 g of the product is obtained as a dark green oily liquid.

The product was stirred with toluene / acetone (1: 1) (250 ml), the insoluble matter was filtered off with suction and washed with 50 ml of the solvent mixture. The remainder remains 13.4 g of inorganic material. The filtrate is chromatographically purified on a silica gel column (7.5 cm diameter, 90 cm height, 1: 1 toluene / acetone splitting point). 155 g of n-45-trosobase are obtained as a dark green oily liquid.

This product is dissolved in 600 ml of acetone and the solution is treated dropwise with 250 ml of saturated ethereal hydrochloric acid while cooling with an ice bath. After thirty minutes of stirring under ice-bath cooling, the crystals formed are filtered off with suction, washed with three 100 ml portions of acetone and dried over a diphosphor pentoxide. 159 g (63.6%) of the title compound are obtained,

Melting point: 118 DEG C., DC: silica gel (Kieselgel 60. Merck), toluene / acetone 1: 1, Rf = 0.24. EXAMPLE 12

In a manner analogous to that described in Example 11, the following compounds were prepared: a) 1- [K, N - (2-hydroxyethyl) - [4- nitrosoanilino] -3- (2- hydroxyethoxy) -2- propyl- HCl hydrochloride

<img img-format="tif" img-content="drawing" file="CS9100261A2D00451.tif" id="idf0012" />

X CH 2 CH 2 OCH 2 CH 2 OH

Yield: 10.5% of theory, orange crystals, m.p. 104 ° C (dec), DC: silica gel / Kieselgel 60, Merck /, 5: 1 = 0.13 .

It will be made from .... 1 - [[(1,1-difluorophenyl) amino] -3-

<img img-format="tif" img-content="drawing" file="CS9100261A2D00461.tif" id="idf0013" />

and this zirconiumanilino] -3- (2-hydroxyethoxy) -2-propyl alcohol

<img img-format="tif" img-content="drawing" file="CS9100261A2D00462.tif" id="idf0014" />

which was obtained from aniline and 1-chloro-3- (2-hydroxyethoxy) -2-propanol, yielding 21.5% of product 47 as a colorless oil, DC: silica gel / Kieselgel 60, Merck / , mobile phase acetone / acetone 5: 2, Rf = 0.6. 1

The above compound is obtained by reacting said 1- [N- [amino] -3- [2-hydroxyethoxy] -2-hydroxyethoxy) -2-propanol by reaction with ethylene oxide in the presence of 4 U of acetic acid to give 71% of product as a colorless oil, DC: silica gel (Kieselgel 60, Merck), mobile phase acetone / acetone 5: 2, = 0.43. B) 1- [1- (2-hydroxyethyl) -4-nitrosoanilino] -3-methoxypropyl alcohol

<img img-format="tif" img-content="drawing" file="CS9100261A2D00471.tif" id="idf0015" />

Yield: 44.5%, pale yellow crystals, melting point 122 ° C, (decomposition), DC: silica gel (Kieselgel 60, Merck), 49: 1 Rf = 0.55;

This compound was obtained from (±) -3- [1- (2-hydroxyethyl) amino] -1-methoxy-2-propanol (Mehmet Reich patent) - - - - - - - - -. L; ~ 48 - No. 603 808 (1933) - Pridl &amp; amp; amp; 21, 295), (temperature 212-214 ° C). c) 2 - [(2-methoxyethoxy) ethyl] - (4-nitrosophenyl) amino] ethyl] alcohol.

<img img-format="tif" img-content="drawing" file="CS9100261A2D00481.tif" id="idf0016" />

CH 2 CH 2 CH 2 CH 2 OCH 3 Yield 25%.

Dark brown resin, DC: silica gel (Kieselgel 60, Merck), 19: 1, phase methylene chloride / methanol, Rf = 49, methylene chloride / methylalcohol 5: 1, Rf. = 0.77 (via amorphous hygroscopic hydrochloride with KH 4).

This compound is obtained from 2- (2-methoxyethoxy) ethyl] phenyl] amino] ethyl alcohol (A)

<img img-format="tif" img-content="drawing" file="CS9100261A2D00482.tif" id="idf0017" />

(which is prepared from aniline and 2-methoxyethoxychloethanol), heated at 90 ° C for one hour and separated by column chromatography on silica gel with toluene / ethyl acetate 5: 1, where the E- (2-methoxyethoxymethan-ethyl) aniline is obtained as a beryllium oil, Rf = 0.69, and the product

<img img-format="tif" img-content="drawing" file="CS9100261A2D00491.tif" id="idf0018" />

with ethylene oxide and 4N acetic acid (A) as a colorless oil, DC: silica gel (Kieselgel 60, Merck), mobile phase acetone 5: 2, Rf 0.31 .... d / 2- [2- [2- [2- (2-methoxy) ethoxy] ethoxy] ethyl] -4- (nitrosophenyl) ethyl alcohol 0 = w

<img img-format="tif" img-content="drawing" file="CS9100261A2D00492.tif" id="idf0019" />

Yield: 63% of theory, green oil, DC: silica gel (Kieselgel 60, Merck), mobile phase acetone / acetone 1: 5 Rf = 0.64

The title compound is obtained from 2- [2- [2- [2- (2-methoxy) ethoxy] ethoxy] ethyl] -4- (phenyl) amino] ethanol as follows. (From aniline and diethylglycol-bis- (2-chloroethyl ether) (Perry, Hibbert Can, J. Res., 14, 81, 1936), is obtained by heating for four hours at 140 DEG C. and subsequent separation on a column chromatography on silica gel (Kieselgel 60, Merck) with a 2: 1 mixture of toluene / ethyl acetate

<img img-format="tif" img-content="drawing" file="CS9100261A2D00501.tif" id="idf0020" />

CH 2 CH 2 OCH 2 CH OCH 2 CH 2 OCH 2 CH 2 Cl in 20.5% yield as a yellowish yellow oil; Rf = 0.5. Reaction of this compound with ethylene oxide produces 4-acetic acid in virtually quantitative form 51-

<img img-format="tif" img-content="drawing" file="CS9100261A2D00511.tif" id="idf0021" />

XCH2CH2OH. CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 Cl as a colored oily liquid, DC: silica gel (Kieselgel 60, Merck), 19: 1, Rf = 0.61. On heating the compound with KaOCH3 in methanol for 24 hours under reflux, concentration, addition of water, removal into ethyl acetate and subsequent purification by column chromatography on silica gel (Kieselgel 60, Merck / with toluene and acetone 5: 2 gave 51.3% of theory of the product as a colorless oil, Rf = 0.21. EXAMPLE 13 N - (4-Nitrosophenyl) -N- (2-diethylamino) ethyl] -N, N -diethyl-1,2- ethanediamine tris

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xch2ch2k / c2h5 / 2 2X15 / 2. 3 HG1 52 -

125 DEG C. (decomposition), DC: silica gel (Kieselgel 60, Merck), mobile phase isopropanol / n-butyl acetate / water / concentrated aqueous ammonia 50: 30: 15: 5, Rf =

The title compound was prepared from N- [di- (2-diethylamino) ethyl] aniline. Example 14 Production of 1-substituted 4- (4-nitrosophenyl) -piperazines

0 = N

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/ - \

(R) - (R) -1-methyl-4- (4-nitrosophenyl) -piperazine dihydrochloride

0 = U

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/ ~ N N h - CUL. HCl 17.62 g (0.1 mol) of 1-methyl-4-phenylpiperazine (prepared from 0.3 mol of 1-phenylpiperazine by heating for four hours with 0.2 mol of trinethylphosphine 53g at 150 ° C, isolation by addition of sodium hydroxide and extraction with diethyl ether, purification by column chromatography on silica gel (Kieselgel 60, Merck) using methylene chloride / methanol 5: 1 to give 40.1% of the product as a colorless oil with a boiling point of 82 DEG-84 DEG C. and a. 0.31 according to Stewart et al., J. Org. Chem. 13, 134 (19W); is dissolved in a mixture of 20 ml of concentrated hydrochloric acid and 10 ml of water, then a solution of sodium nitrite (8 g, 0.12 mol) in water (16 ml) is added dropwise at a temperature in the range of 0 DEG to 2 DEG C. the reaction mixture is stirred for 30 minutes at 10 ° C. With constant cooling, 60 ml of concentrated aqueous ammonia is added at the same temperature, the reaction mixture is diluted with 100 ml and the red-brown solution (pH 9) is shaken three times with 100 ml of methylene chloride. The organic phase is then dried over sodium sulphate and then suctioned off and concentrated. The residue (20.6 g of green crystals) is taken up in 40 ml of methanol and mixed with 20 ml of saturated ethereal hydrochloric acid under cooling with cooling. Purification and washing with two 20 mL portions of diethyl ether gave 15.8 g (5.8%, theory) of the title compound as a greenish crystalline solid. is taken up in 40 ml of methanol and mixed with 20 ml of saturated ethereal hydrochloric acid under cooling. Purification and washing with two 20 mL portions of diethyl ether gave 15.8 g (5.8%, theory) of the title compound as a greenish crystalline solid. is taken up in 40 ml of methanol and mixed with 20 ml of saturated ethereal hydrochloric acid under cooling. Purification and washing with two 20 mL portions of diethyl ether gave 15.8 g (5.8%, theory) of the title compound as a greenish crystalline solid.

Melting point: 187 DEG-189 DEG C. (decomposition), DC: silica gel / Kieselgel 60, a phenol / methylene chloride / methanol 5: 1,

Rf = 0.72

Analogously: b) 4- (4-nitrophenyl) -1-piperazino-ethyl alcohol-dihydrochloride-

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~ ^ Ύ

N H. --CH2CH2OH V / 2 HCl from 2- (4-phenylpiperazino) -ethyl alcohol (Kremer, J.

Amer. Chem. Soc. 58, 379 (1936) as light gray rats; the pure material is recrystallized from methanol / water 7: 1, m.p. 170-173 ° C (decomposition), DC: silica gel (Kieselgel 60, Merck), mobile phase: methylene chloride / methanol 5: 1 Rf = 0.67; c) 3- [4- (4-nitrosophenyl) -1-piperazinyl] -1,2-propanediol di-

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of 1-phenyl-4- (2,3-dihydroxypropyl) -piperazine (H). -Kowella et al., J. Org. Chem. 27, 1711 (1962) as a green crystalline solid, m.p. 1 DEG C. and 3 DEG C. (decomposition), DC: silica gel (Kieselgel 60, Merck), 2: 1 mobile phase ethyl acetate /

Rf = 0.41; D] [4- (4-nitrophenoxy) phenyl] -piperazine-1-ethyl alcohol dihydrochloride

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CH2CHCH2OCH3

OH 2 HCl from 1-phenyl-4- (2-hydroxy-3-methoxypropyl) piperazine / H. Howell et al., J. Org. Chem. Melting point: 162 DEG C. (decomposition), DC: silica gel (Kieselgel 60, Merck), mobile phase methylene chloride / methanol 19: 1 = 0.51 g 2- 4- (4-nitrosophenyl) -1-piperazinyl] ethoxy] hexylalcohol dihydrochloride

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/ RTI &amp; gt; NN-CHOCHOOCHOCHOOC1-L. 2 HCl from 2- [2- [4- (phenyl) -1-piperazinyl] ethoxyethyl alcohol (obtainable from 2 mol. 1-phenylpiperazine. .alpha .- [1- (2-chloro-ethoxy) -2-methoxyethoxy] carbonate according to U.S. Pat. 2,837,974) as green crystals, m.p. 134 DEG C./dissolve 56 M; (silica gel / Kieselgel 60, Merck), ethyl acetate / methanol 5: 1, Rf = 0.31; f /. 1- (1,4-dioxanyl) methyl-4- (4-nitrosophenyl) -piperazine dihydrochloride

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. 2 HCl from 1- (1,4-dioxanyl) methyl-4- (phenyl) -piperazine (obtained from a five hour warming of 1-chloro-3-beta-hydroxyethoxy) -2- propyl alcohol MS Kharash, W Nudenberg, J. Org. Chem. 8, 189 (1943)] and 1-phenylpiperazine in vacuo at 30 ° C, extraction, ethyl acetate and concentration. Silica gel / Kieselgel 60 column chromatography (Merck), toluene / acetone mobile phase 5: 2 as a yellow-green crystals, melting point 166 DEG C. (decomposition), DC: silica gel (Kieselgel 60, Merck) / methanol 5: 1,

Rf = 0.69. 15

Nitro with heterologous cycles and / 5-nitroso-1-indolinethyl alcohol hydrochloride 57 -

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HCl from 1-indolinoethyl alcohol (obtained by heating for 1 hour with 1 mole of inulin with 1 mole of 2-chloroethyl alcohol) in the presence of finely powdered potassium carbonate under reflux to give 63% of theory as a colorless oil b.p. 128 DEG-130 DEG C., DG silica gel (Kieselgel 60, Merck), toluene / acetone, 5: 2, Rf = 0.42,

The nitroso compound is obtained which is isolated as the base by the addition of ammonia with methylene chloride. With etheric hydrochloric acid, the base is converted to the hydrochloride. This gives a light brown crystalline solid melting point 180 DEG C., DG: silica gel (Kieselgel 60, Merck), methylene chloride / methanol 5: 1 Rf = 0.51; b) 1-methyl-6-nitroso-1,2,3,4-tetrahydro-quinoline hydrochloride

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The title compound is prepared from 1-methyl-1,2,3,4-tetrahydroquinoline from the 1,2,3,4-tetrahydroquinoline by heating with trimethyl phosphate according to Huisgen et al. , Chern. Ber. The crude product is prepared in a conventional manner analogous to Examples 10 and 11 and purified on silica gel (Kieselgel 60, Merck) with isopropyl alcohol, n-butyl acetate and 5: 3: 2. Dissolving in acetone and adding ether. hydrochloric acid there was obtained the title compound, m.p. 123-124 ° C (dec.): silica gel (Kieselgel 60, Merck), mobile phase isopropyl alcohol / n-butyl acetate / water 5: 3; , Cis-6-nitroso-3,4-dihydro-1 (2H) -quinoline-ethyl alcohol -hydrochloride

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59 -

The title compound is obtained from 2- (3,4-dihydro-2H-quinolin-1-yl) ethanol (Zaheer et al., Indian J. Cit. 1, 479 (1963). The crude product was purified by column chromatography on silica gel (Kieselgel 60, Merck) using a 19: 1 mixture of methylene chloride and methanol. Recrystallization of the hydrochloride from isopropanol with ethereal acidic hydrochloric acid and recrystallization from ethyl alcohol gave 10.5% (theory) of ocher crystals in the title compound, m.p. 193-135 ° C (dec.): Silica gel (Kieselgel 60, Merck) mobile phase methylene chloride / methanol 19: 1, Rf. = 0.36.