CS273630B2 - Method of resorufine's derivatives production - Google Patents

Abstract

A method of production of resorufin derivatives of general formulae Ia and Ib, where R<l> R<2>, R<3>, R<4> and R<5> are the same or mutually separated and mean hydrogen, halogen, the carboxyl group, carboxamide group, C1-5-alkoxy-carbonyl group, cyano group, nitro group, possibly substituted C1-5-alkyl or -alkoxyl, R<4> and R<5> can both mean annellated aromatic hydrocarbon with 6 to 10 carbon atoms, which is possibly substituted, Z means a bridging element formed from the residues X<1>, X<2>, X<3>, X<4> and M, A means a residue of hapten, an antigen, antibody, substrate or carrier and n means a whole number from 1 to 200, based on the fact that the compounds of formulae IIa and IIb, where X<1> means the reactive group, such as -COOH, -COOT, -COOCO2T<1>, -NCO, -NCS etc., are subjected to the action of diamine, dicarboxylic acid or its derivative, dialdehyde or aminocarboxylic acid of general formula IV, where X<3> and X<4> mean the reactive groups and M means the residue of the bifunctional compound with l to 5 carbon atoms, and a ligand of general formula III, where X<2> means the reactive group, such as NH2, SH, OH, COOH; the established protective groups are possibly split and the individual reactive groups X<1> to X<4> are possibly transformed to other reactive groups. The produced compounds are used as marking compounds in chemical biological processes.<IMAGE>

Description

The invention relates to a process for the production of novel resorufin derivatives which, as fluorescently labeled low molecular weight compounds, can find a wide variety of uses.

Fluorescent compounds find and because of their ability to emit certain wavelengths upon activation. Wide application as labeling compounds in chemical and biological processes, such as in clinical analytics but also in an increasing number of new areas. In biochemistry they find a fluorescent dye as a highly sensitive marker substances increasing use. Both low-molecular-weight and high-molecular-weight fluorescent dye compounds are used. Examples of such a wide range of use of such fluorescent conjugates are;

Fluorescent immunoassay using either a hapten, an ontigen or a specific antibody labeled with a fluorescent dye. By specific binding of antibody r.a hapten or antigen, the concentration of these substances or antibody can be determined by various methods.

In immunofluorescence microscopy, antigens such as whole cells or proteins become visible under the microscope by the action of fluorescently labeled antibodies (cf. Wang et al., Meth. Enzymology 85, 514 et al.).

Likewise, the distribution of hapten or antigen in the cell can be directly observed when the respective compound is fluorescently labeled and is observed under a microscope.

Fluorescently labeled latex particles find use in sorting cells in a Fluoroscent Activated Cell Sorter (FACS).

Not in the last line, the fluorescent-labeled substances are used to determine the activity of the enzymes.

Competitive immunoassays are based on competition between the ligand to be detected in the sample and the labeled and known concentration of ligands present for a known but limited number of antibody lignin binding sites that are specific for both the identified and labeled ligands. . the concentration of the ligand to be determined in the sample is critical to how many labeled molecules are bound to the antibodies. The concentration of complexes consisting of antibodies and fluorescently labeled ligands can be determined by spectroscopic methods. This concentration is inversely proportional to the concentration of ligand present in the sample. Originally, radioisotope-labeled ligands were used as labeled ligands to be added to the sample with the determined ligands at a known concentration. Because of the known disadvantages of radioisotope labeling, labeling with fluorescent compounds is becoming increasingly important. Fluorescent compounds bind to the molecules of the test substance. These conjugates can then be used in principle for a variety of fluorescent immunoassays, such as fluorescence polarization immunoassay, controlled-stop fluorescence immunoassay, or fluorescence immunoassay using auxiliary agents.

In principle, all fluorescent dyes having a high extinction coefficient and a high quantum yield as well as sufficient stability under the conditions under which the test is carried out are suitable as markers. So far, therefore, it has usually used fluoroocein or a fluorescein derivative (J. London and S. S. Kamel in slmmunooaoeyo GOa,

Univ. Park Press. Baltimore, Class, 1930, pp. 91-112). Fluoreacein or its derivatives, however, have high disadvantages or low molecular weight compounds, but have various disadvantages. The absorption and emission maxima of the fluorescein-labeled substances range between 490 and 520 nm. Since a large number of analytical methods, in particular fluorescent immunoassays, are carried out in body fluids such as serum, disturbances in the spectral range occur due to the inherent fluorescence of the biological material in the sample. At least this is due to bilirubin, which also absorbs light in the wavelength range around 500 nm and emits fluorescence.

Many measuring devices require labeling substances with as large a Stokeo shift as possible. For fluorescein derivatives this shift is at most 30 nm. This leads to light scattering problems, which affects the sensitivity of the fluorescence measurement. For such cases, compounds with a greater Stokea shift than fluorescein dorivatives would be desirable.

It was therefore an object of the present invention to provide compounds which do not have the above disadvantages. This task was solved by finding new resorufin derivatives.

Accordingly, it is an object of the present invention to provide a process for producing resorufin derivatives of formulas Ia and Ib

A (la)

in which

CS 273630 D2 and ¹ ? 3 4 5

S ', a, 0 R, which may in each case be the same or not different, represent a hydrogen atom, a halogen atom, a carboxy group, a corboxane group, an alkoxycarbonyl group having 1 to 5 carbon atoms in the alkoxy moiety, cyano, nitro or carbon atoms or alkoxy groups having from 1 to 5 carbon atoms which are optionally substituted by carboxy, carboxamido, C 1 -C 5 alkoxycarbonyl, cyano or nitro groups,

Wherein R 6 and R 8 together may represent a C 6 to C 10 aromatic hydrocarbon optionally substituted by one or more substituents selected from the group consisting of sulfo, coroxy and alkoxy. having 1-5 carbon atoms., a bridging member formed from the radicals X ~, X ^2, X ^'1, 0 M, and X ^1, X ^2, X ^, X ^, and M have the meanings indicated, is the residue of a hapten, an antigen, antibody, substrate or carrier; and

200

799 in the meaning of the substituents Β, 3 * ', R', preferably having 1 to 3 carbon atoms, in particular means an integer from 1 to

Alkyl groups, optionally alkoxy groups

R and R contain hydrocarbon chains of 1 to 5, preferably methyl and ethyl, optionally aethoxy and ethoxy.

The halogen r \ P ^2, R ^j, B-3 3 ^J is fluorine, chlorine, bromine or iodine. Particularly preferred are chlorine and bromine,

The fused C 6 -C 10 aromatic hydrocarbon consisting of substituents Ir and R is preferably benzene or naphthalene. Benzene is particularly preferred. Said aromatic hydrocarbons may be unsubstituted or may contain one or more substituents selected from the group consisting of sulfo (-SO-1H), carboxy (-COOH) or (C 1 -C 5) alkoxy.

From the bridge member is formed by conventional acid-addition or reactive substituent X \ X ^2, X ^-1, X ^ M.

Table 1 below shows examples of possible X and X substituents as well as the bridging member Z formed in this condensation reaction between the meanings of such substireactions.

Table 1:

Some of the meanings of the reactive groups X ¹ and X ² as well as the bridging member Z, liter;

X ²

-COOH -NH ₂ -CffllH- -CCCT ^X -kh ₂ -CONH- -C00C0 ₂ T ^lxx ~ ^KH 2 -COKH- -MGO -NHCONH- -NCS -íiHCSKH-

N-- -NH - // 'n AND -nh ₂ -KCsCH-CO- -SH -CKC -nh ₂ -SO ₂ C1 -nh ₂ -COCH ₂ - halogen -SH -CCCH8-halogen -OH - (CH ₂ ) _m -NH 4 -COOH

-S-CH-CH ₂ -CO-CH = N-SOgKH-coch ₂ -s-coch ₂ -o - (C ^H 2) _m -NH-CO-CO-N

COOI *

I® ₂

-co-n

CO-NHT means an alkyl group having 1 to 5 carbon atoms or an electronegative activated ester group such as an 11-hydroxysuccinimidoester group

T ¹ represents an alkyl group having 1 to 5 carbon atoms and r represents an integer from 0 to 3.

Instead of the primary amines, as shown in Table 1, secondary amines can also be used as reactive 12 groups X or X to form corresponding products.

Preferred bifunctional compounds of the general formula X 1 -M-X 1 are diamines, dicarboxylic acids as well as their derivatives, dialdehydes, aminocarboxylic acids and other compounds which are usually used for the production of such coupled compounds. Examples of such compounds include piperazine, 1,2'-ethylenediamine, succinic acid, glutardialdehyde glycine, sarcosine, O-alanine and piperidine-4-carboxylic acid.

Ligander as used herein means A hapten, antigen, antibody, substrate or carrier,

For the purposes of the present invention, hapten is a lower molecular weight substance that is generally not capable of generating antibodies by itself. Lower molecular weight compounds are those having a molecular weight of from about 100 to about 2000. Examples of such substances include physiologically active substances that are present in mammalian or human organisms, as well as their metabolites or drugs, which are administered to animals or humans, as well as their metabolites. However, the term hapten also refers to other low molecular weight compounds as long as they have a molecular weight only in the above range. Examples of possible haptenes include amines, steroids, hormones, carbohydrates, peptides, oligonucleotides, combinations thereof, etc.

Antigens are high molecular weight compounds. Antigens usually have the ability to produce antibodies in the organism they have been treated with. For the purposes of the present invention, high molecular weight compounds are those having a molecular weight of at least 2000, but preferably those having a molecular weight of at least about 5000. The molecular weight of such compounds cannot be limited upwards. It can be up to 20 million, but it can still be above this molecular weight. Antigens which may be present as ligands in the compounds of formula Ia / Ib are represented by proteins, nucleic acids, polysaccharides, combinations thereof or other high molecular weight substances. For the purposes of the present invention, the term antigen refers to all high molecular weight compounds having a minimum molecular weight of at least about 2000.

Antibodies are all those proteins or glycoproteins that specifically react with and form complexes with antigens or haptens, as well as with compounds derived therefrom. According to the invention, intact antibodies or fragments thereof can be used as ligands in the compounds of the formula (I) / (Ib). Also, these fragments can be referred to as antibodies according to the invention as long as they have the ability to bind antigens, hapten and compounds derived therefrom. '

Substrates are compounds which remain in the chemical reaction without a prokaryotic change. For example, these substrates may be understood to mean all those compounds or derivatives derived therefrom which are treated with enzymes, such as amino acids, peptides, proteins, glycosides, oligosaccharides or polysaccharides, nucleotides, nucleic acids, combinations thereof or other enzymatically exchangeable substances.

The carriers can be naturally occurring or synthetic, cross-linked or non-cross-linked materials with or without a particular form. They are understood to mean individual compounds or mixtures of compounds. As carriers, for example, compounds or mixtures of compounds such as polysaccharides, nucleic acids, peptides, proteins, combinations thereof or also acacia, lignin, glass, ulli, synthetic addition or condensation polymers such as polystyrene, polyacryl, vinyl compounds, polyester can be used polyethers and polyamides, or else complex formations such as latex particles, vesicles, liposomes, cell wall portions or even whole cells.

In principle, all ligands containing free amino, hydroxyl, morcapto or carboxyl groups can be used to form the compounds of formula Ia or Ib according to the invention, through which the ligands can bind to the resorufin backbone with an intermediate bridging member. Particularly preferred are free amino or carboxyl groups. Free amino groups are understood to mean both primary, flux and secondary amino groups. If the ligands have no suitable groups, then the flow groups must be introduced via a synthetic route, such as amino or carboxyl groups. It is furthermore possible to chemically activate any non-reactive functional groups of such ligands. Since the compounds thus formed are no longer exactly the same as the original compounds, ligand analogues are referred to.

The number n indicates how many resorufin molecules are bound per ligand. This number depends on the number of reactive groups in the corresponding ligand or ligand analogon. The more reactive groups the ligand has, the more resorufin molecules can bind, the number n usually being 1 to 200, particularly preferably 1 to 100.

According to the present invention, the compounds of the formulas Ia and Ib are prepared by treating nn compounds of the formulas IIa and IIb.

(Ila)

(lib) in which

S ', R ³ , j> 4 _and j> 5 have the meanings given under the general formula la}? is a bifunctional compound of formula IV

X ³ - K - X ⁴ in which

X ³ η X ⁴ represent reactive groups a

M 'is a residue of a biofunctional compound of the ligand III

X ² - A

Ib, and (IV) (III)

C3 273SJO G2 in which

O

~ V represents a reactive group and is the residue of the ligand, wherein optionally at the individual reaction steps and the protecting groups introduced .. _u may slack

2 Joto opčt cleaved with a different reactive groups X, X, X ^with the other reactive groups.

The reactive groups in the meaning of the substituents X, X ¹ , X can in principle be understood to mean all the usual reactive functional groups. Particularly preferred as functional groups are acid residues, such as carboxylic acids or sulfonic acids, as well as groups derived therefrom, such as esters, amides, onhydrides, halides, acids, or various residues such as primary or secondary amines, cyanate, icocyanate. thiocyanate, isothiocyanate, aldehyde, sulfhydryl, hydroxyl, C1-ketohalide residues, etc.

(W in which

-3, 4 and X

M o ligand (III) is a reactive group and is a residue of a bifunctional compound having from 1 to 5 carbon atoms (III) in which o

X is a reactive group such as -NH, -SH,

-CH, -COOH or a secondary omino group corresponding to the group -KH, or is as defined above for substitution X, provided that in the same reaction the substituents X and Ν 'are the same or different from each other, and

It is to have the meaning given above, where appropriate protecting groups are introduced at each reaction stage and then cleaved again to the individual reractive groups ib, in which

(IN)

The R 5, R 3, R 3 are as defined above, by reaction with resorcin derivatives of the general formula (VI) in which

X

HIM

OH

R ‘2 (VI)

are as defined above and are a reactive group.

The reaction of compounds of formula V with resorcinol derivatives of formula VI is conveniently carried out in the presence of manganese dioxide and sulfuric acid at low temperatures. The reaction initially produces resazurin derivatives which are readily convertible to resorufin derivatives of formula IIa and IIb.

The reaction of compounds of formula V with compounds of formula VI is generally carried out at temperatures between -10 and 50 ° C, preferably at temperatures between 0 and 30 ° C. The reaction is particularly gentle when the compounds of formula V and formula VI are mixed at a temperature of about 0 ° C and the reaction mixture is then allowed to warm to room temperature. The concentration of manganese dioxide should preferably be 0.5 to 5, preferably 1 to 2 mol / liter. The sulfuric acid concentration should be 0.5 to 5, preferably 1 to 3 mol / liter.

The reduction of the initially formed resazurin derivatives to resorphins of the formulas IIa and IIb is preferably carried out in an ammoniacal solution of zinc dust / cf. Nietzki et al., Ber. Dtsch. Chem Ges. 22. 3020 (1889) / or sodium borohydride. Suitably, the solvent used is a mixture of water and an alcohol, preferably a mixture consisting of 1 part water and 0 to 4 parts methanol. N 1 mol of the reduced compound is added with 1 to 20, preferably 1 to 5 mol of zinc dust or sodium borohydride. The temperature of the reaction solution is maintained at -10 to +35 ° C, preferably at +5 to +10 ° C. Accurate adherence to the temperature range has proven to be a necessary measure for a clear reaction. Without cooling, the exothermic reaction leads to by-products that are difficult to separate.

Under selected mild conditions, the reaction between the compounds of formula (V) and formula (VI) proceeds unambiguously and in good yield. However, the synthesis method chosen is capable of modification. This opens up numerous possibilities for synthesis, in particular with respect to the preparation of unsymmetrically substituted resorufin derivatives. As a result of this extremely variable production process, a number of resorfinine-labeled compounds are available which open up a wide range of colors by their different substituents at different positions of the chromophore.

Before the reaction of the resorufin derivatives of the formulas IIa and IIb with the compounds of the formulas IV and III, the compounds of the formulas IIa and IIb are expediently converted with n8 triocyldihydroresorufin derivatives of the formula VII ο

CS 272630 Ε2

(VID in which τ 2 * 3 / c: τ

H ^A , B \ S. 2 \ ?? X ¹ P. ^{6 has the} meaning given in formulas IIIa and IIb as a modified alkyl group, an aryl group or an aralkyl group.

The alkyl group in the above substituent B is an alkyl group of 1 to 5 carbon atoms, preferably of 1 to 3 carbon atoms. A methyl group and an ethyl group are particularly preferred. Phenyl is particularly preferred as an aryl group. The aralkyl group preferably contains a phenyl group as the aryl moiety, while the alkyl moiety contains 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms. Benzyl 3 is particularly preferred as the aralkyl group.

In order to prepare triacyl derivatives of the general formula VII, the corresponding resorufin derivatives are prepared. of formulas IIIa and IIb are first reduced electrochemically with a strong reducing agent such as stannous chloride or chronic acetate. For reduction, the resorufin derivative is heated for 10 minutes to one hour with 2 to 10, preferably with 2 to 6 equivalents of a reducing agent in a suitable solvent, preferably tin (II) chloride in 5 to 35% aqueous hydrochloric acid. Upon cooling, the di-derivative is eliminated. The acylation is carried out in a conventional manner by treatment with a suitable acylating agent, for example acetic anhydride. benzoyl chloride, etc. Preferably, the compounds of formula VII are prepared by a single-pot process by reductive acylation of resorufin derivatives of formula IIa and IIb. The corresponding resorufin derivative. For this purpose, it is heated to 2 to 6 equivalents of the reducing agent for 5 minutes to 3 hours in the presence of the acylating agent in a suitable solvent under reflux or at room temperature for 4 to 16 hours.

In the triacyldihydrorescrufine derivatives thus obtained, they can be converted to a reactive group X před before the next reaction optionally to another reactive group. In particular, when X 'represents a carboxyl group, it is expedient to convert this group to a carboxylic acid chloride, a carboxylic anhydride or a reactive ester function. It can alcohol alcohols position numerous procedures to perform a variety of

K-hydroxyaukcinim known from the literature in what ways, for example by the action of carbodiimides idi, etc. They are known to the skilled person in this regard. It is particularly advantageous to convert the function of corbexylic acid with carboxylic acid chloride, for example by treatment with a mixture of hionyl chloride and dimethylformamide or a mixture of oxalyl chloride and dimethylformamide, as well as to an activated ester, for example the N-hydroxysuccinimide ester.

If the ligand or ligand analog contains a free amino group, a hydroxyl group or a mercapto group, then these ligands can react as reactive groups X ² . The ligands or ligand analogues of the formula X ² -A may then react directly with compounds of formula IIa and IIb, if appropriate after prior conversion to derivatives triacyldihydroresorufinu and / or after activation of the group X ³ to form amides, esters or thioesters. On account of its stability, the formation of at least one amide bond is particularly advantageous, starting with compounds of the formulas IIa / IIb or VII, in which X1 is a carboxylic acid halide. Its reaction with an amino-containing ligand and optionally an amino-containing ligand analogue is carried out according to conventional methods, for example in an organic solvent such as dichloromethane with the addition of a tertiary amine such as triethylamine as the base. Depending on the size of the ligand or the analog of the ligand and the associated number of free amino groups thereof and the amount of compounds of the formulas IIIa / IIb used, several chromophores can be bound to one ligand molecule or one ligand analogone molecule.

When triacyl derivatives of formula VII are used to produce the compounds of formulas Ia and Ib according to the invention, the acyl residues of the dihydroresorphin may be selectively cleaved after reaction and the resulting leuco dye residue is oxidized to the chromophore of the compound of formula Ia / Ib.

The O-acyl residues of a portion of the dihydroresorphin are cleaved particularly preferably by reaction with 2 to 10 mol, preferably 2 to 4 mol of sodium sulfite, in a mixture of water and a water-soluble solvent such as 1,4-dioxane, methanol or ethanol, preferably in a 1: 1 mixture of water and 1,4-dioxane. The reaction temperature is 20 to 100 ° C, preferably 80 to 100 ° C. Under these reaction conditions, N-acyldihydroresorfinine derivatives can be produced in high yields.

Oxidation of a portion of dihydroresorufin to compounds of formulas Ia and Ib can be accomplished by treatment with mild oxidizing agents. Preferably potassium hexacyanoferrate is used which is present in a 2 to 6 fold molar excess, particularly preferably a 2 to 4 fold molar excess relative to the leuco dye in a water-miscible solvent-water mixture such as 1,4-dioxane, methanol or ethanol, preferably in a 3: 1 mixture of water and methanol. The reaction is preferably carried out in the presence of an auxiliary base such as sodium bicarbonate or sodium carbonate. The reaction temperature is 10 to 40 ° C, preferably at room temperature.

It is particularly advantageous to carry out the selective cleavage of the N-acyl residue which has been used as a protecting group and to oxidize a portion of the dihydroresorphin in a single reaction vessel. To this end, 2 to 4 moles of sodium bicarbonate and an equimolar amount of 1N sodium hydroxide solution and then a 2- to 4-fold molar excess of potassium hexacyanoferrate are first added to the N-acylated dihydroresorphin derivative in a 3: 1 mixture of water and methanol. After about 10 minutes to 2 hours, preferably 0.5 hours at room temperature, the reaction is complete.

It is expedient to combine the compounds of formulas IIIa / IIb with the ligand and optionally with the anaT ligand III logo to interleave the X -MX grouping. For this purpose, all compounds usually used for this reaction can be used, preferably with at least two reactive groups. . Particularly preferred are diamines or aminocarboxylic acids. Selecting the same time depends on the type of functional groups X ¹ and X ² which are to be coupled with a compound forming a bridge.

Compounds which are obtained by reacting resorphin derivatives of formulas IIIa / IIb with bifunctional compounds of formula IV in one or more stages, for example via compounds of formula VII, can be represented by formulas VIIIa and VHIb.

(Vlila)

(9IIIb) wherein r 1, R ² , r 3, r ⁴ and have the meanings given in formulas 1a and 1b and

M and X ⁴ have the meanings given in formula IVa

X ^ is a functional group formed by the reaction of X ^ and X ^.

The functional groups X 1 may be all possible groups which are formed by the reaction of the reactive groups mentioned as X ¹ and X ¹ together. Preferred groups X @ ¹ include amides, thioethers, ethers, secondary or tertiary amine, ureido and thioureido etc.

This means, if the substituent X ¹ in the formulas IIa / IIb, optionally in the formula VII, the carboxylic acid function, or derived thereof reactive group and rep resent when reactive substituents X ² ligand or Analogon ligand amino, hydroxyl or mercapto, then Suitably, X @ 1 -MX @ ⁴ is selected, wherein X @ 4 is amino and X ^{@ 4} is a carboxylic acid function or a reactive derivative thereof. The terminal amino group can bind to the carboxylic acid function of the compounds of the formulas IIa / IIb, or of formula VII, and terminal carboxyl may bind to the ligand or ligand analogue of X ² -A. However, if both X and X each represent the function of the carboxylic acid or the derivative derived therefrom, the diamine is chosen as the intermediate.

The reaction of the resorufin derivatives of formula IIa and IIb with the bifunctional intermediate IV and the ligand or the anologone of the ligand is preferably carried out in two steps. First, the compounds of the formulas IIIa / IIb may be combined, optionally after prior conversion to the active compounds VII, with the intermediate compound of formula IV. The derivatives formed from this reaction can then be reacted in a second step with a ligand or an analog of a ligand of the formula III. It is, of course, also possible to select the reverse order, i.e. to bind the intermediate IV in the first step to the ligand or the analog of the ligand III and then to react the resulting product in the second step with a resorufin derivative of formula IIIa / IIb or an activated compound. VII.

Preferred aminocarboxylic acids are aminocarboxylic acids. Glycine, alanine, sarcosine and piperidine-4-carboxylic acid have proven to be particularly preferred. Coupling of resorphin derivatives of formulas IIa / IIb and VII, respectively, with aminocarboxylic acids to form an amide linkage is carried out according to methods common to one skilled in the art. It is particularly preferred for these purposes to use methyl esters or tert-butyl esters of the corresponding amino acids. After formation of the amide bond, the previously introduced protecting groups are optionally cleaved selectively according to conventional methods. For example, an activated derivative of formula (VII) and an aminocarboxylic acid having a protected carboxy group are reacted, then selectively hydrolyze the O and N-acyl groups of the dihydroresorfin skeleton, re-oxidize the leuco dye to the resorufin system and then cleave the carboxy carboxy acid, preferably with trifluoroacetic acid. The free carboxylic acid can then be activated to conjugate the ligand or analog of the ligand of formula III in an appropriate manner as described for resorufin derivatives of general formulas IIIa and IIb. This conjugation itself is performed analogously to that described for direct conjugation of ligands or analog ligand to resorufin derivatives of formulas IIa and IIb.

In the case of carboxyl group-containing ligands or carboxyl group-containing ligands analogs, it is expedient to convert the resorphin derivatives of the formulas IIa / IIb and VII respectively to compounds containing amino groups which can then be reacted with the carboxyl-group-containing ligands optionally ligand analogs containing carboxyl groups to form an amide bond. For this purpose, the reaction of the compounds of formulas IIIa / IIb and VII, respectively, with diamines (formula IV with the amino groups X1 and X1) has proved to be particularly simple and advantageous. Preferred diamines in this regard are, for example, piperazine, 1,2-diaminoethane or 1,3-diaminopropane.

The conversion of the resorphin derivatives of the formulas IIa / IIb or the formula VII with diamines to the free amino group derivatives is carried out according to methods which are common to one skilled in the art. In order to achieve particularly high yields of monosubstituted diamines, diamines having only one reactive amino group and whose other functional group is blocked by a protecting group are reacted. In principle, all conventional amino protecting groups which can be cleaved again without adversely affecting the amide bond are usable. butoxycarbonyl protecting groups or benzoyloxycarbonyl protecting groups.

After reaction of the diamines with one amino-protected group with resorufin derivatives of formulas IIa / IIb and VII, respectively, the optionally introduced protecting groups are cleaved again and optionally the intermediate leuco dye is oxidized to the resorufin system. The cleavage of the amino protecting groups from the bound diamines is carried out under conventional conditions, preferably by treatment with trifluoroacetic acid.

The amino group-containing resorufin derivatives thus obtained may be conjugated in a conventional manner to ligands or ligands analogs of formula III which contain:

to activate them. This activation can be carried out as described above. Preference is given to the use of ligands or ligand analogs of the general formula (III) in which X @ ² represents activated ester groups.

In particular, K-hydroxysuccinimide esters have proven to be particularly advantageous. The conjugation itself is essentially carried out analogously to the method described for direct conjugation of resorufin derivatives with ligands or with analogs of ligands, taking into account the roles of the functional groups exchanged.

Further reactions of the products obtained after coupling of resorphin derivatives of formulas IIa / IIb and VII, respectively, with an intermediate of formula IV, i.e. from ^3- i-X ⁴ , with low molecular weight ligands X 1 -A, for example α haptens, is preferably carried out in a mixture of water or a buffer and a water-soluble solvent such as 1,4-dioxane, methanol or ethanol. Preferably, a mixture of 0.1 M potassium phosphate buffer solution at pH 8.5 and 1,4-dioxane in a 1: 1 ratio is used. It is also possible to monitor the progress of the reaction by thin layer chromatography. The reaction may take between 1 and 24 hours. after 1 to 18 hours.

K-hydroxysuccinimide esters have proven to be particularly advantageous for the reaction of resorphin derivatives of formulas IIIa / IIb, optionally VII or VIHa / VIIIb with high molecular weight ligands of formula III. Thus, for example, when coupling to rabbit IgG, 6 moles of resorufin derivative per 1 mole of IgG are sufficient to achieve a level of labeling of 3 resorufin molecules per 1 mole of IgG. Fluorescent dyes which have been conventional for this purpose and have a maximum absorption wavelength% max / · 470 nm containing a reactive group containing an isothiocyanate or sulfonic acid chloride residue, such as fluoreaceinisothiocyanate or Texas red must be used in a substantially higher excess of dye to be coupled to rabbit IgG in order to achieve the same degree of labeling.

The conjugation of high molecular weight cli compounds of formula III, such as proteins, is carried out in a buffer, particularly preferably in a 0.1 M potassium phosphate buffer solution at pH

The reaction temperature should always be 10 to 35 ° C. Preferably, the reaction is carried out at room temperature.

In view of the particularly favorable spectral properties, the present invention also relates to the use of the compounds of the formulas Ia and Ib in analytical procedures in which the fluorescence property of the compounds of the formulas Ia / 1b or the conversion products of these compounds is determined or measured.

In heterogeneous immunoassays, separation of antibody-bound ligands and free ligands by precipitation with suitable substances or the use of antibodies bound to solid supports is required before the cell determines the concentration of free ligands or bound ligand concentrations. In homogeneous immunoassays, the determination of complex antibody-ligand binding in a sample is performed without such separation. Methods of homogeneous immunoassay include, for example, fluorescence immunoassays with controlled stopping of the reaction, fluorescence immunoassay and the use of adjuvants, as well as fluorescence polarization methods using fluorescent substances as a marker. In particular, the latter method still lacks suitable marking means because of the disadvantages of the useful fluorescent marking means which have been used. Since the compounds of formulas Ia or Ib have absorption maxima and emission maxima that are well above the maxima of the biological material that acts disruptive in body fluids due to their own fluorescence, they are particularly suitable for use in fluorescence polarization immunoassays. A further advantage of the compounds produced by the process of the invention is that they have a particularly high Stokes shift of up to about 70 nm with suitable substitution.

Fluorescence polarization immunoassay is fundamentally based on the principle of conventional fluorescence immunoassay. _of

If suitable fluorescently labeled ligands are activated for fluorescence by linearly polarized light, then the molecule may rotate before it emits radiation due to the slight time delay between activation and emission. This also rotates the plane of the linearly polarized light by a certain angle. For many molecules, some fluorescent emission depolarization may occur by rotational diffusion during this short period of time. The polarization of the emitted fluorescence is that the larger the molecule is, and consequently the slower the rotation. This correlation can be used to measure the binding of ligands to antibodies since free, labeled ligands have a smaller molecule volume than complexes of labeled ligands bound to the antibodies. Conversely, the polarization is proportional to the concentration of ligands being determined and present in the sample.

The concentration of labeled ligands or ligand analogs and antibody required for such immunological procedures depends on the measurement equipment used, as well as the respective characteristics of the labeled ligand or labeled ligand analogue used and the antibody itself. In principle, these concentrations naturally also depend on the concentration of the ligands to be determined and must therefore be determined empirically. These findings can be made by any expert in a simple optimization.

The concentration of ligand to be determined, varies generally from about 10 to about 10 ^ M. It is advantageous if the measurement adjust the concentration of ligand in the sample determination from about 10 "^ to 10 ^ ² M, preferably from about 10 "to about 10 ^ ^- ^ M. Higher concentrations of ligand may be measured after diluting the original sample.

The measurement is carried out at certain pH values, which may range from about 3 to about 12. They typically range from about 5 to about 10, preferably in the pH range and from about 6 to about 9, to reach and maintain the pH throughout. various buffers, such as borate buffer, phosphate buffer, carbonate buffer or Tris buffer, may be used in the measurement. It is not critical to the present invention which buffer is used. The choice depends primarily on the antibody used, according to the ligands to be determined as well as the fluorescent labeling agent used.

Fluorescent polarization immunoassays are preferably performed at a constant temperature. Typically, a temperature in the range of about 0 to 50 ° C, preferably in the range of about 10 to about 40 ° C can be selected.

The exact relationship between polarization and the concentration of ligands or ligand analogs to be determined can always be read from the calibration curve. Such curves are obtained by measuring the polarization values of solutions of substances and different but known concentrations. The unknown concentrations of ligands in the sample under investigation can then be obtained from such calibration curves, knowing the polarization.

The wide field of application of the compounds of the formulas Ia and 1b produced by the process of the invention is to be seen in their general utility as a fluorescent labeling agent. For example, for immunofluorescence microscopy, they can be made visible by fluorescently labeled antibodies as protein antigens or whole cells · Correspondingly, the distribution of hapten or antigen within the cell can also be directly observed when the corresponding fluorescently labeled compound is introduced into the cell and monitored, for example . In contrast to the fluorescence immunoassays described above, the fluorescence properties of resorufin derivatives are not altered.

Claims (2)

Known dyes that have hitherto been used as fluorescent labeling agents are, for example, fluoresceins, such as fluorescein isothiocyanate and noble rhodamine dyes, such as Texas Red. However, as previously mentioned, fluoresceins have the disadvantage that they fluoresce at relatively low wavelengths. In addition, the yield of the coupling reaction is generally low according to experience on the respective support and, moreover, the color stability of the coupling products is poor. The same applies to rhodamine dyes. It is with respect to these products that the compounds of the formulas Ia and Ib show distinct advantages over prior art fluorescent labeling agents. They fluoresce in the long wave range with good color fastness and can be produced in good yield from the compounds of formulas IIa or IIb and the corresponding coupling components. The compounds of formula (IIa) or (Hb) prepared by the process of the invention can naturally also be used for labeling substances in other or mentioned fluorescent immunological procedures. Thus, it is also possible with these substances to label the components of another complex-forming system with the use of these reactive resorufin derivatives. Complex-forming systems can be understood to mean all those combinations of substances which, by virtue of their specific exchange activity, are able to form complexes. Known combinations are, for example, a combination of a hormone and a specific receptor, a combination of biotin and avidin, a combination of a carbohydrate derivative and a lectin, etc. Examples include biotin-labeled proteins which can be determined using avidin and the reactive compound of formula IIa or IIb. Another advantageous utility of the compounds of formula (Ia) and (Ib) produced by the process of the present invention lies in the assay of the lectin / carbohydrate derivative component. Fluorescently labeled latex particles are used to screen cells in a Fluorescent Activated Cell Sorter. Such particles can also be very fluorescently labeled, for example by reacting latex particles containing hydroxyl groups, mercapto groups, aminoacids, or also carboxyl groups or sulfo groups, with reactive resorphin derivatives of the formulas IIa or Hb. The compounds of the formulas Ia and Ib can also be advantageously used for the determination of enzymes. For this purpose, a resorufin derivative of the formula IIa or IIb can be bound to a substrate which can be cleaved by the enzyme to be determined. This coupling product is also a compound of formula (Ia) and (Ib) prepared by the process of the invention. The enzyme activity can be determined by reacting the substrate, which is labeled with resorufin, with the enzyme, and separating the cleavage products from the unreacted substrate. For example, a reactive derivative of resorufin of formula IIa or Hb can be coupled to a glycopeptide, and the thus obtained coupling product can then be used as a substrate to demonstrate the efficacy of endoglucosidase. For the determination of endoglucosidases, dansyl isomer labeling is known. Iwase et al., Anal, Biochem. 113, 93-95 (1981)). Resorufin derivatives have a particularly high sensitivity compared to these compounds. The following examples serve to illustrate the invention. These examples, which are not intended to limit the scope of the invention in any way, are intended to demonstrate the essential possibility of labeling low-molecular and high-molecular compounds and their uses. Example 1 / 3- (1-diphenylhydantoinyl) ethylcarbonyl / piperazide resorufin-4-carboxylic acid a) Resorufin-4-carboxylic acid 16 g nitrosoresoroin, 15.5 g 2,6-dihydroxybenzoic acid and 8.6 g manganese dioxide (burel) The suspension was suspended in 200 ml of methanol and then cooled to 0 ° C. To the cooled suspension was added dropwise 10.6 ml of concentrated sulfuric acid and the reaction mixture was stirred at room temperature for 2 hours. The resulting red resazurine-4-carboxylic acid is filtered off, washed with methanol and dried. The resazurin derivative is dissolved in 200 ml of water and 50 ml of a 25% ammonia solution and filtered. 2 blue filtrate was added portionwise with 50 g of zinc powder under ice-cooling, and the mixture was allowed to warm to room temperature. The progress of the reduction can be easily followed by thin layer chromatography (solvent system: 1: 1 methanol / ethyl acetate} thin layer chromatography silica gel plate). The reaction solution was filtered, and the filtrate was then acidified with glacial acetic acid and a small amount of concentrated hydrochloric acid. The precipitated resorufin-4-carboxylic acid is filtered off and dried under vacuum with phosphorus pentoxide. .. ’. ’. '·. . Yield: 16.33 g of Rf (silica gel) solvent system: n-butanol / glacial acetic acid / water (4: 1: 1) = 0.4. b) N, O, O-triacetyldihydroresorufin-4-carboxylic acid 12.9 g of resorufin-4-carboxylic acid are dissolved in 30 ml of glacial acetic acid and 30 ml of acetic anhydride, 27.6 g of stannous chloride are added and the mixture is stirred for 1 day. hour at 80 ° C. The mixture was poured into 600 ml of ice water, then stirred for 1 hour and the precipitate was filtered off. After drying, the solid is taken up in 500 ml of acetone. The solution was filtered and the filtrate was evaporated. After drying, 11.3 g of product are obtained. @ 1 H-NMR (CD2): .delta. = 2.24, 2.26 and 2.29 (each s, 9H); 6.94 (double d, J = 8.5 and 2.2 Hz, 1H)} 6.98 (d, J = 2.2 Hz, 1H)} 7.04 (d, J = 9 Hz, 1H)} 7.61 (d, 2 = 8.5 Hz, 1H)} 7.67 ppm (α, J = 9 Hz, 1 H). Rf value = 0.46 (silica gel, solvent system: 9: 1: 0.1 chloroform / methanol / glacial acetic acid). c) N, O, O-Triacetyldihydroresorufin-4-carboxylic acid chloride 38.5 g of the triacetate described in Example 1b) are mixed with 54 ml of oxalyl chloride and the mixture is cooled to 0 ° C. A few drops of dimethylformamide were added to this mixture and allowed to warm to room temperature. The starting material dissolves during the evolution of gas. The mixture is evaporated to dryness under reduced pressure, the residue is taken up three times with 200 ml of absolute methylene chloride and the methylene chloride solution is again evaporated to dryness. Yield: 41 g. D) K-tert-butoxycarbonylpiperazine 12.61 g of N-benhydrylpiperazine 100 ml of a 1: 1 mixture of 1,4-dioxane and water. To this solution was added dropwise a solution of 12.0 g of di-tert-butyl dicarbonate dissolved in 50 ml of 1,4-dioxane. After stirring for 1/2 hour, 50 ml of water was added dropwise, the mixture was filtered and the precipitate was dried. Yield: 16.2 g of N-tert-butoxycarbonyl-N-benzhydrylpiperazine Value = 0.92 (silica gel, solvent system: 9: 1: 0.1 chloroform / methanol / glacial acetic acid). 7 g of N-tert-butoxycarbonyl-N-benzhydrylpiperazine are dissolved in 100 ml of ethyl acetate and 5 ml of glacial acetic acid. 0.3 g of palladium on active ul is added and hydrogenation is carried out. Then the catalyst is filtered off and the filtrate is evaporated. 100 ml of water and 20 ml of 1N hydrochloric acid solution were added to the residue, and the mixture was filtered. The filtrate was extracted twice with ethyl acetate and then the aqueous phase was made alkaline with sodium hydroxide. The product, which had precipitated in oily form, was extracted with dichloromethane. After drying over sodium sulfate and evaporating to give 3.2 g of K-tert-butoxycarbonyl-piperazine as an oil, which crystallizes completely after a few days. Values = 0.05 (silica gel, solvent system: 9: 1: 0.1 chloroform / methanol / glacial acetic acid). The use of ninhydrin produces a blue color. e) N, O-N-tert-butoxycarbonylpiperazide N, O, O-triacetyl-dihydroresorufin-4-carboxylic acid 25 g of the acid chloride described in Example 1 c) and 17.3 g of triethylamine in 450 ml of dichloromethane are stirred at room temperature. A solution of 13.0 g of K-tert-butoxycarbonylpiperazine in 50 ml of dichloromethane is added dropwise at 0 ° C. The reaction mixture was stirred without cooling for 1 hour, extracted three times with water and the organic phase was evaporated. Yield: 36.0 g. Rf value = 0.64 (silica gel, solvent system: chloroform / methanol / glacial acetic acid = 9: 1: 0.1). f) N * -tert-butoxycarbonylpiperazine K -acetyldihydroresorufin-4-carboxylic acid 34.3 g of triaoetate as described in Example 1e) and 17.1 g of sodium sulfite were stirred for 1 hour at 60 ° C in 500 ml of mixture 1. , 4-dioxane and water 1: 1. Then the reaction mixture is evaporated, the residue is taken up in ethyl acetate, the ethyl acetate solution is filtered to remove insoluble salts and chromatographed on 2 liters of silica gel (eluent; ethyl acetate / dichloromethane 4: 1 as the product starts to elute to pure product). ethyl acetate). Yield; 14 g. Rf value = 0.28 (silica gel, solvent system: ethyl acetate / dichloromethane 4: 1). g) Resorufin-4-carboxylic acid N-tert-butoxycarbonylpiperazide 5 g of the N-acetyl derivative described in Example 1f) are dissolved in 200 l of methanol and 600 ml of water. 1.8 g of sodium hydrogen carbonate and 10.7 ml of 1N sodium hydroxide solution are added, followed by 14 g of potassium ferrocyanide. After stirring at room temperature for 1/2 hour, the pH is adjusted to 5. The product precipitates and is filtered off. Yield: 2.72 g. Value = 0.28 (silica gel, solvent system: 9: 1: 0.1 chloroform / methanol / glacial acetic acid). h) Resorufin-4-carboxylic acid trifluoroacetate (1) Sample: human serum from a donor with known amounts of diphenylhydantoin. Human donor serum containing diphenylhydantoin at a concentration is used to generate the calibration curve (a) 2,5 µg / ml (b) 5 µg / ml (c) 10 µg / ml (d) 20 µg / ml e) 40 µg / ml. antibody solution: 450 µg antibody / ml 0.1 M sodium phosphate buffer solution (pH 7.8) Antibodies are obtained by immunizing sheep with diphenylhydantoin, which is bound to bovine serum albumin, according to conventional methods. The antiserum is purified by ammonium sulfate precipitation and DEAE cellulose chromatography, (3) diphenylhydantoinresorufin solution (10 ~ ⁶ M): the diphenylhydantoin-resorufin (b) conjugate of Example 1i) in 0.1 M sodium phosphate buffer solution (pH 7.8). The measured results obtained with diphenylhydantoin solutions 1a), 1b), 1c), 1d) and 1e) are shown in the attached drawing. In this drawing, the concentrations of diphenylhydantoin of the formulas (µg / ml) are plotted against the measured polarization values (mP). The concentration of diphenylhydantoin in samples with unknown diphenylhydantoin content can also be determined using such a calibration curve. A comparable calibration curve is also obtained when the diphenylhydantoin conjugate of example 1) is used instead of the diphenylhydantoin conjugate of example 2, 7 or 9f). Example 16 Determination of endoglycosidase activity using mannose-based resorufin- (higher) glycopeptide Note: The term (higher) mannosaglycopeptide will be used in the rest of the text to simplify the expression of the meaning of the word, which is the higher glycopeptide in which mannose is part of the sugar. a) Labeling of (higher) mannosaglycopeptide with K'-hydroxysukoinimide ester of H- (4-resorufinylcarbonyl) sarcosine 50 mg (higher) of mannosaglycopeptide (manufactured by Huang et al., Carbohydrate Ses. 1, 127-137 (1970)) is mixed with 10 ml of a 0.1 M potassium phosphate buffer solution at pH 8.0. Then, 25 mg of N (4-resorufinylcarbonyl) sarcosine dissolved in 3 ml of dioxane was added, after one hour the same amount of N-hydroxysuccinimide ester of dye in 3 ml of dioxane was added, and the mixture was stirred at room temperature for 14 hours. , then dioxane is evaporated in vacuo and the residue diluted with water to 70 ml and then with buffer A (= 0.02 M TEIS-HCl solution, 2 mM magnesium chloride, 2 mM manganese chloride, 2 mM calcium chloride, pH 7.2) The pH is adjusted to 7.2 with aqueous ammonia and the precipitate formed ^is centrifuged and the supernatant is transferred to a Con A-Sepharose column (1.times.15 cm) with Buffer A eluting free dye. not red, wash out A 1. fraction of resorufin (higher) mannosaglycopeptide was eluted with 2% methyl mannoside in buffer A as eluent (about 100 mL). The 2nd fraction was then eluted with 2% aqueous methyl mannoside. Both fractions were dialyzed against water and lyophilized. Both fracoes are suitable for the determination of endoglycosidase activity as described in b) below. b) Determination of endoglycosidase activity Eesorufin- (higher) mannosaglycopeptide is incubated in a suitable buffer with endoglycosidase, for example endoglycosidase H and citrate buffer pH 5.5 (= sample 1). In parallel to this experiment, an experiment is conducted simultaneously with a sample that does not contain indoglycosidase but is otherwise identical (= sample 2). After incubation, Con A-Sepharose is added to both samples and the mixture is shaken to bind resorufin- (higher) mannosaglycopeptide. A resorfin-labeled peptide in which part of the sugar was cleaved due to enzyme activity does not bind. After 15 minutes, Con A-Sepharose is centrifuged, the supernatant is adjusted to pH 7.5 and the fluorescence measured (excitation, for example, 550 nm, emission λmax 95 95 nm). The difference between Sample 1 and Zero (= Sample 2) gives the amount of Cleaved Resorufin- (higher) mannosaglycopeptide and is therefore a measure of enzyme activity. THE INVENTION in which E ^1, E ^2, Ε · \ p4 θ _K 5 _j] n _ERJ 4 _mo hou always be the same or different, are hydrogen, halogen, carboxy, carboxamido, alkoxycarbonyl having 1-5 carbon atoms in the alkoxy part, cyano, nitro, C 1 -C 5 alkyl or C 1 -C 5 alkoxy optionally substituted by carboxy, carboxamido, C 1 -C 5 alkoxycarbonyl, cyano or nitro, Wherein E and E may together represent a fused aromatic hydrocarbon having from 6 to 10 carbon atoms optionally substituted by one or more substituents selected from the group consisting of sulfo, carboxy and alkoxy and 1 to 5 carbon atoms, Z is a bridging member formed from the radicals X ^"1 · X ^2, X ^, ^{Χ? Ί} and M and X ^1, X ^2, X \ χ4 θ _M have the meanings indicated, A is a residue of a hapten, antigen, antibody, substrate or carrier, and n is an integer from 1 to 200, characterized in that the compounds of the formulas are R ¹ , R ² , R ⁵ , and R ⁵ have the meanings given in formula Ia and Ib, and X ¹ represents a reactive group such as -COOH, -COOT, -GOOCOgT · ¹ ·, -NCO, -NCS, -HC = CH-CO- -CCH3 -NH3, -CHO, -SO ₂ Cl, or -CG-N100T, wherein T represents a C 1 -C 5 alkyl group or an electronegated ester moiety such as an N-hydroxysuccinimide ester moiety, Znamená * 'represents an alkyl group having 1 to 5 carbon atoms and m represents an integer from 0 to 3, and Hal represents a halogen atom and, in the case of groups containing a primary amino group, is also a group having a corresponding secondary amino group, or has the meaning of X below, provided that X and X are the same or different from each other, act with diamine, dicarboxylic acid or a derivative thereof, a cialdehyde or an aminocarboxylic acid of formula IV X ³ - M - X ⁴ (IV) in which X ³ and X ⁴ represent reactive groups α M represents a residue of a bifunctional compound having 1 to 5 carbon atoms and a ligand of the formula III X ² - A (III) in which o X represents a reactive group, such as -NHg, -SH, -OH, -COOH or a secondary amino group corresponding to -NH ₀ , or has the meaning of the aforementioned substituent X, provided that in the same reaction the meaning of χΐ and X ^{2 is the} same or different from each other, and A is as defined above, whereby protecting groups are optionally introduced in the individual reaction steps and then cleaved again and the individual reactive groups χ ⁵ , X ² , X ³ and X ⁴ are optionally converted to other reactive groups. 1.6 g of the triacetyl derivative described in Example 9b) is reacted in an analogous manner to that described in Example 8d) with oxalyl chloride and N-butoxycarbonylpiperazine. Yield: 1.2 g. @ 1 H-NMR spectrum (CDCl3): .delta. cf = 1.49 (s, 9H), 2.12, 2.27, 2.46 (each s, 12H), 3.0-3.9 (m, 8H), 7.03 (d, J = 9 Hz, 1H), 7.16-7.94 ppm (m, 6H). d) 9'-hydroxy-5-benzo [i] - phenoxazone-8-carboxylic acid N'-butoxycarbonylpiperazide In analogy to Example 1f), 0.51 g of the product was obtained from 0.93 g of the triacetyl derivative of Example 9c). The value of E _f = 0.69 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). e) 9-Hydroxy-6-benzo [i] - phenoxazone-8-carboxylic acid piperazide trifluoroacetate Starting from 0.5 g of the butoxycarbonyl protected compound described in Example 9d), 0.5 g of the product was obtained in an analogous manner to that described in Example Ih). Value E ^. = 0.02 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). £) Coupling of 9-hydroxy-5-benzo [a] phenoxazone-8-carboxylic acid piperazide with 2- (1-diphenylhydantoinyl) acetic acid H-hydroxysuccinimide ester From 50 mg of the piperazide prepared according to Example 9e) and 150 mg of 2- (1-diphenylhydantoinyl) -acetic acid N-hydroxysuccinimide ester, 70 mg of the product was obtained in an analogous manner to that described in Example 1e. E ^ - value (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). UV / VIS (0.1 molar solution of potassium phosphate buffer, pH 8.0): - 560 nm γG max Fluorescence emission: λ = 615 nm. JC max @ 1 H-NME spectrum (DMSO): 3.0-4.5 (m, 10H), 6.37 (s, 1H), 6.80 (d, J = 8 Hz, 1H), 7.2-7.35 (m, 10H), 7.35-8.0 (m, 3H), 8.10 (double d, J = 8 and 2 Hz, IH), 8.56 (double d, J = 8 and 2 Hz, IH), 9.60 ppm (s, 1H). Example 10 8-Ethylresorufin-4-carboxylic acid 1-diphenylhydantoinylmethylcarbonyl-piperazide (a) 6-ethyl-4-nitrosoresorcin In analogy to Example 8a), 6-ethyl-4-nitrosoresorcin is obtained as a yellow solid from 7.5 g of 4-ethylresorein, 4.5 g of potassium hydroxide and 5 ml of isopentyl nitrite. Yield: 7.5 g. Rf value = 0.37 (silica gel, solvent system; mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). b) 8-ethylresorufin-4-carboxylic acid In analogy to Example 1a), from 7.4 g of 6-ethyl-4-nitrosoresorcin, 6.8 g of 2,6-dihydroxybenzoic acid, 3.9 g of manganese dioxide and 5 ml of concentrated sulfuric acid after reduction of 8 g zinc powder yielded 9.5 g of product. B-value = 0.05 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). c) H, O -Butoxycarbonylpiperazide K, O, O-triacetyl-8-ethyldihydroresorufin-4-carboxylic acid In an analogous manner to that described in Example 1d), 7.7 g of 8-ethylresorfinine were obtained -4-carboxylic acid, 15.4 g of stannous chloride, 30 ml of glacial acetic acid and 15.3 ml of acetic anhydride yields N, O, O-triacetyl-8-ethylresorfinine-e-carboxylic acid, which is further processed as the crude product in an analogous manner to that described in Example 1c) into the acid chloride and in an analogous manner as described in of Example 1e) is directly further processed to butoxycarbonylpiperazide. Yield: 4 g. Value = 0.86 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). d) 8-ethylresorufin-4-carboxylic acid H-butoxycarbonylpiperazide 4 g of N, O, O-triacetyl-8-ethyldihydroresorphin-4-carboxylic acid N-butoxycarbonylpiperazide is obtained in an analogous manner to that described in Example 1f) and 1g) of the corresponding N'-butoxycarbonylpiperazide carboxylic acid. Yield: 0.5 g. Value = 0.67 (silica gel, solvent system: 4: 1 chloroform / methanol). 8-Ethylresorfin-4-carboxylic acid piperazide trifluoroacetate 330 mg of the corresponding butoxycarbonylpiperazide are left to stand for 1.5 hours in 35 ml of a 6: 1 mixture of dichloromethane and trifluoroacetic acid. After evaporation, the residue was digested with ether, the product was filtered off and dried. Yield: 350 mg. Value = 0.33 (silica gel, solvent system: mixture of butanol, glacial acetic acid and water in a ratio of 4: 1: 1). f) Reaction with 2- (1-diphenylhydantoinyl) acetic acid K-hydroxysuccinimide ester GS 273630 B2 In analogy to Example 1i), 120 mg of the product are obtained from 325 mg of 8-ethylresorfin-4-carboxylic acid piperazide trifluoroacetate and 435 mg of 2- (1-diphenylhydantoinyl) -acetic acid N-hydroxysuccinimide ester. Value = 0.43 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). 1 H-NMR (perdeuterated dimethylsulfoxide); (f = 1.15 (t, J = 7.2Hz, 3H), 2.52 (q, J = 7.2Hz, 2H), 3.1-4.0 (m, 8H), 4, 25-4.45 (m, 2H), 6.42 (s, broad, 1H), 6.94 (d, J = 9.0 Hz, 1H), 7.3-7.5 (m, 11H) 7.68 (d, J = 9.0 Hz, 1H), 9.54 (s, 1H), 11.2 ppm (s, broad, 1H). UV / ViS (0.1 molar solution of potassium phosphate buffer, pH = 8,0): = 575 nm. fo max Fluorescence emission: = 598 nm. λ ^max Example 11 8-Chloro-resorufin-4-carboxylic acid (1-diphenylhydantoinylmethylcarbonyl) piperazide (a) 8-Chloresazurine-4-carboxylic acid From 17.3 g of 4-chloro-6-nitrosoresorcin (prepared according to Plampin and Gain, J. Med. Chem. , 247 (1963)), 15.4 g of 2,6-dihydroxybenzoic acid, 8.6 g of manganese dioxide and 10.7 ml of concentrated sulfuric acid are obtained in an analogous manner to that described in Example 1a) 8-chlorresazurin-4. -carboxylic acid. Yield: 17.1 g. Rf value = 0.58 (silica gel, solvent system; mixture of butanol, glacial acetic acid and water in a ratio of 4: 1: 1). b) N, O, O-triacetyl-8-chlorodihydroresorufinecarboxylic acid 16.3 g of 8-chlorosazurine-4-carboxylic acid and 18.9 g of tin (II) chloride are heated for 1 1/2 hours in 100 ml of 1: 1 glacial acetic acid / acetic anhydride to 80 ° C and then poured into 500 ml of ice-cooled water. The reaction mixture is stirred for 2 hours, then filtered to remove the precipitate and dried with Sicapent. The solid is taken up in 500 ml of acetone and the insoluble residue is filtered off. The filtrate was evaporated to give 12.3 g of product after drying. Rf value. = 0.39 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). @ 1 H NMR Spectrum: (DMSO): (Τ '= 2.25 (s, 3H), 2.33 (s, 6H), 7.16 Cd, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.76 ( d, J = 8.8 Hz, 1H), 7.90 ppm (s, 1H). c) 8-Chlororesorfin-4-carboxylic acid N'-butoxycarbonylpiperazide In analogy to Example 1b), c) and e) to g), 0.8 g of the product is obtained from 5 g of Ν, Ο, Ο-triacetyl-8-chlorodihydroresorufin-4-carboxylic acid. Rp value = 0.7 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). d) 8-Chloro-resorfin-4-carboxylic acid piperazide trifluoroacetate Starting from 0.8 g of the butoxycarbonyl protected compound of Example 11e), 0.81 g of the product was obtained in an analogous manner to that described in Example 1h). Value = 0.07 (silica gel, solvent system; mixture of chloroform, methanol and glacial acetic acid (9: 1: 0,1) e) Coupling of 8-chlorosorfinine-4-carboxylic acid piperazide with 2- (1-diphenylhydantoinyl) acetic acid N-hydroxysuccinimide ester In analogy to Example 1i), the desired product is obtained from 400 mg of 8-chloro-resorfin-4-carboxylic acid piperazide trifluoroacetate and 410 mg of 2- (1-diphenylhydantoinyl) -acetic acid N-hydroxysuccinimide ester. Yield: 150 mg. Value = 0.38 (silica gel, solvent system; mixture of chloroform, methanol and glacial acetic acid (9: 1: 0,1) WAIS (0.1 M potassium phosphate buffer pH 8.0): = 581 nm .nu.max = 597 nm. Fluorescent emission: J max Example 12 Coupling of 8-chloro-resorfin-1-carboxylic acid piperazide with theophylline-7-propionic acid (2-aminoethyl) amide (a) 8-Chlororesorfinine-1-carboxylic acid 8.7 g of 4-chloro-6-nitrosoresorcin and 7.71 g of 3,5-dihydroxybenzoic acid are dissolved in 200 ml of methanol, 4.8 g of manganese dioxide are added at 0 ° C in portions of 5, 3 ml concentrated sulfuric acid. The reaction mixture is stirred at room temperature for 2 hours, then filtered and ammonia and 200 ml of water are added until the color change to blue 3e. The solution obtained is filtered, 25 ml of concentrated ammonia and 20 g of zinc powder are added to the filtrate under ice-cooling, and the mixture is stirred for a further 15 minutes without further cooling. 200 mg of activated carbon are added, the mixture is filtered, the filtrate is acidified to pH 2 and the precipitated resorufin derivative is centrifuged. Yield: 3.9 gValue = 0.88 (silica gel, solvent system: mixture of n-butanol, glacial acetic acid and water (4: 1: 1). b) N, O, O-triacetyl-8-chlorodihydroresorufin-1-carboxylic acid 3.2 g of the product are obtained from 3.5 g of 8-chloro-resorfin-1-carboxylic acid in an analogous manner to that described in Example 1b). Value = 0.43 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). c) 8-Chloro-resorfin-1-carboxylic acid piperazide trifluoroacetate Starting from 3 g of the triacetyl derivative prepared as in Example 10b), 1.4 g of product are obtained in a manner analogous to that described in Examples 1c) to 1h). CS 273630 Β2 _Rf value = 0.08 (silica, solvent system: mixture of chloroform, methanol, glacial acetic acid (9: 1: 0.1). d) Coupling of 8-chloro-resorfin-1-carboxylic acid piperazide with theophylline-7-propionic acid (2-aminoethyl) amide 190 mg of the product are obtained from 420 mg of N, O, O-triacetyl-8-chlorodihydroresorphin-1-carboxylic acid piperazide and 300 mg of theophylline-7-propionic acid (2-aminoethyl) amide. Example 13 Labeling of immunoglobulin G with N- (4-resorufinylcarbonesDsarcosine) hydrox'-hydroxysuccinimide ester 100 mg of human immunoglobulin G (IgG) are dissolved in 10 ml of 0.1 M potassium phosphate buffer at pH 8.0 and 5 mg of N- (4-resorphinocarbonyl) sarcosine N * -hydroxysuccinimide ester (cf. Example 7e) are added to the obtained solution. The mixture is allowed to stand at room temperature for 12 hours and then chromatographed on an Ultrogel ACA 2002 (LKB) sorbent. The labeled protein elutes before the free low molecular weight resorufin. The degree of labeling is determined by extinction measurements. It is 3, i.e. 3 molecules of the resorufin derivative are bound to the immunoglobulin G molecule. Example 14 Labeling of immunoglobulin G with N- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid N * -Haroxysuccinimide ester a) N- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid 2.0 g of N, O, O-triethyldihydroresorufin-4-carboxylic acid chloride as described in Example 1c) is reacted in an analogous manner to that described in Example 1e) and 0.9 g of piperidine-4-carboxylic acid methyl ester hydrochloride of the acid, analogous to that described in Example 1f) and 1g), the product is deacetylated and oxidized and saponified with sodium hydroxide on N- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid. Yield: 0.9 g. _Rf value = 0.44 (silica gel RP-18, solvent system: mixture of nitromethane and ethanol in a ratio of 4: 1). UV / VIS (0.111 potassium phosphate buffer solution, pH 8.5): = 576.2 nm · b) N-hydroxysuccinimide ester of N- ^(4-i esorufinylkarbonyl) -piperidine-4-carboxylic acid From 200 mg of N- (4-resorphinylcarbonyl) piperidine-4-carboxylic acid, 240 mg of N-hydroxysuccinimide and 468 mg of dicyclohexylcarbodiimide, 190 mg of the product was obtained in an analogous manner to that described in Example 7e). _Rf value = 0.7 (silica gel RP-18, solvent system: 4: 1 nitromethane / ethanol). IR spectrum (KBr technique): 3415 (m), 1814 (w), 1773 (m), 1734 (s), 1626 (m), 1214 (m) cm ¹ c) Labeling of rabbit immunoglobulin G with N- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid N'-hydroxysuccinimide ester max CS 273630 32 To 10 mg of rabbit immunoglobulin G dissolved in 1 ml of a 0.1 M potassium phosphate buffer solution at pH 8.5 is added 100 µl of a solution of 1.9 mg of N- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid N-hydroxy-succinimide ester in 1 ml 1,4-dioxane and the mixture was allowed to stand at room temperature for 2 hours. This corresponds to a ratio of 6.4 moles of resorufin derivative to 1 mol of rabbit immunoglobulin G. Chromatography on AGA 202 and using a 0.1 M potassium phosphate buffer solution at pH 8.5 as the eluent yielded a protein fraction having an absorption rate of ^A 578 ^{/ A} 280 ° ^C corresponding to a loading rate of 3.4 mol resorufin per l mole of immunoglobulin G. If, in an analogous experiment, 20 µl of the activated resorufin solution is added to 10 mg of rabbit immunoglobulin G, then a device grade of 0.8 is obtained at 1.05 mol of dye offered per 1 mol of immunoglobulin G. The absorption maximum of resorufin-labeled immunoglobulin G is 578 nm. The solution fluoresces intensely light red. When exposed to daylight resorufin-labeled immunoglobulin G for one month, the fluorescence value drops to 59% of its original value, while for analogously produced fluorescein-isothiocyanate-labeled immunoglobulin G to 12% and Texas red-labeled immunoglobulin G to 12%. Example 15 Determination of diphenylhydantoin in human serum using ΡΙΡΙΑ To 1950 (µL of 0.1 M sodium phosphate buffer solution (pH 7.8) was added 5 µL of sample) 5 µl of antibody solution (2) and 25 µl of diphenylhydantoin-resorufin solution from Ui <3) (1) After incubation for 5 minutes at 37 ° C, fluorescence polarization is measured. (excitation wavelength: 575 nm, emission wavelength 594 nm, measuring instrument: fluorescence spectrometer 650-108, Hitachi). 1 g of the triacetyl derivative described in Example 8d) is reacted in an analogous manner to that described in Examples 1g) to 1h). Yield: 0.43 g. f) Coupling of 2- (1-diphenylhydantoinyl) acetic acid H-hydroxysuccinimide ester with 6-methylresorfin-4-carboxylic acid piperazide 222 mg of the compound obtained in Example 8e) was treated with 200 mg of 2- (1-diphenylhydantoinyl) acetic acid N-hydroxysuccinimide ester. Yield: 250 mg. UV / VIS (0.1 molar potassium phosphate buffer, pH 8.0): = 584 nm. max Fluorescence emission: η = 600 nm. yv max Example 9 Coupling of 9-hydroxy-5-benzo [alpha] 7-phenoxazone-8-carboxylic acid piperazide with N-hydroxysuccinimide ester / 2- (1-diphenylhydantoinyl) acetic acid a) 12-Hydroxy-5-benzoic acid / phenoxazone-8-carboxylic acid 2.84 g of 1,3-dihydroxy-4-nitrosonaphthalene, 2.31 g of 2,6-dihydroxybenzoic acid, 1.29 g of burel and 1.6 ml of concentrated sulfuric acid are reacted in a manner analogous to that described in Example 1a. ). Yield: 2.8 g. The value of E _f = 0.63 (silica gel, solvent system: mixture of n-butanol, glacial acetic acid and water (4: 1: 1). b) 12-acetyl-5,9-dialethylbenzol / phenoxazone-8-carboxylic acid In an analogous manner to that described in Example 8c), 1.8 g of the triacetylated dihydro compound is obtained from 2.4 g of 9-hydroxy-5-benzo [a] phenoxazone-8-carboxylic acid 12-oxide. E-value = 0.31 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). c) N'-butoxycarbonylpiperazide 12-acetyl-5,9-diacetoxybenzoZa / phenoxazine-8-carboxylic acid Δ = 1.36 (s, 9H); 2.81 (a, 4H); 2.9-3.2 (a, 2H); 4.5-4.9 (m, 1H); 7.03 (s, 2 H); 7.63 (d, J = 9Hz, 1 H); 7.90 (s, 2 H); 9.2 (s, 1 H). Example 4 Coupling of resorufin-4-carboxylic acid piperazide with 3-O- [3- (N-succinimidooxycarbonyl) propyl] -stradiol In analogy to Example 1i), 295 mg of the product was obtained from 212. mg of resorufin-4-carboxylic acid trifluoroacetate and 220 mg of 3-O- [3- (N-succinimidyloxycarbonyl) propyl] ^-3 stradiol. E value «0.58 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). 3-0- / 3- (N-sukcinimidoxykarbonyl) propyl / - ^{ES tr} 8diol is usually obtained from 3-0-karboxypropylestradiolu (oestradiol and bromobutyrate analogously to the procedure described in Lübke et al Immunologische Teste fur niedermolekulare Wirkstoffe , G. Thieme Verlag, Stuttgart, p. 94) and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. Example 5 Coupling of resorufin-4-carboxylic acid piperazide with N- / 3- (N-succinimidoxycarbonyl) propyl / phenobarbital In analogy to Example 1i), 220 mg of the product is obtained from 212 mg of resorufin-4-carboxylic acid piperazide trifluoroacetate and 205 mg of N- [3- (N-succinimidoxycarbonyl) propyl] phenobarbital. H 2. = 0.45 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). N- [3- (N-succinimidoxycarbonyl) propyl] -phenobarbital is obtained in a conventional manner from phenobarbital-1-butyric acid (T. Nistikawa et al., Clin. Chim. Acta 91 (1979), p. 59) and N- hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. Example 6 Coupling of resorufin-4-carboxylic acid piperazide with theophylline-7-propionic acid N-hydroxysuccinimide ester From 212 mg of piperazide trifluoroacetate reeorufin-4-carboxylic acid and 175 mg Theophylline-7-propionic acid N-hydroxysuccinimide ester was obtained in an analogous manner to that described in Example II), 200 mg of the product. Rf value = 0.44 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). Theophylline-7-propionic acid N-hydroxysuccinimide ester is obtained in conventional manner from theophylline-7-propionic acid (T. Nistikawa et al., Chem. Pharm. Bull. 27 (1979) p. 893) and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. Example 7 Coupling of N * -hydroxysuccinimide ester of N- (4-resorufinylcarbonyl) sarcosine with 1- (2-aminoethyl) diphenylhydantoin a) N, O, O-Triacetyldihydroresorufin-4-carboxylic acid tert-butoxycarbonylmethyl-methylamide 10 g of the acid chloride described in Example 1c) are reacted in a manner analogous to Example 1e) with a target. sarcosine butyl ester. Yield: 7.5 g. Rf value = 0.77 (silica gel, solvent system: 9: 1: 0.1 chloroform / methanol / glacial acetic acid), b) N-Acetyldihydroresorufin-4-carboxylic acid (tert-butoxycarbonylmethyl) methylamide 7.5 g of the product described in Example 7a) is deacetylated in an analogous manner to that described in Example 1 f). Yield: 5.2 g. E-value = 0.56 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). c) Resorufin-4-carboxylic acid tert-butoxycarbonylmethyl) amide 4.5 g of the product described in Example 7b) were reacted in a manner analogous to that described in Example 1g). ' Yield: 2.6 g. Rf value = 0.64 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). d) Resorufin-4-carboxylic acid (carboxymethyl) methylamide 0.55 g of the product described in Example 7 o) was allowed to stand for 1 hour at room temperature in 6 ml of trifluoroacetic acid. Then the reaction mixture is evaporated to dryness and the residue is triturated with 3 ether and the product is filtered off. Yield: 0.45 g. Rf value = 0.11 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). e) N- (4-resorufinylksrbonyl) sarcosine N-hydroxy succinimide ester 200 mg of the product of Example 7d) was stirred with 72 mg of N-hydroxysuccinimide and 138 mg of dicyclohexylcarbodiimide in 40 ml of tetrahydrofuran for 14 hours. The precipitated urea was filtered off, the filtrate was evaporated and the residue was chromatographed on silica gel (RP IS) eluting with 4: 1 nitromethane / ethanol. Yield: 150 mg _Rf value = 0.79 (silica gel, SP-18, solvent system: 4: 1 nitromethane / ethanol). f) Coupling of N- (4-resorphinylcarbonyl) sarcosine N * -hydroxysuccinimide ester with 1- (2-aminoethyl) diphenylhydantoin 125 mg of the N-hydroxysuccinimide ester of Example 7e) is stirred for 1 hour with 90 mg of 1- (2-aminoethyl) diphenylhydantoin in 40 ml of a mixture of dioxane and potassium phosphate buffer at pH 8.5 in 1: 1 ratio. The dioxane is completely evaporated, ammonia is added to the residue until complete color change, the mixture is filtered and the product precipitates from the filtrate by addition of hydrochloric acid. Yield: 110 mg. B-value = 0.78 (silica gel, solvent system: mixture of n-butanol, glacial acetic acid and water (4: 1: 1). UV / VIS (0.1 molar solution of potassium phosphate buffer, pH 8.0): = 575 nm. max Fluorescence emission: = 592 nm. J / ^maX Example 8 Coupling of 6-methylresorufin-4-carboxylic acid piperazide with N-hydroxysuccinimide ester / 2- (1-diphenylhydantoinyl) / acetic acid (a) 2-methyl-4-nitrosoresorcin 19.8 g of 2-methylresorcin and 13.4 g of potassium hydroxide are dissolved in 120 ml of ethanol and the solution is cooled to 5 ° C. 24 ml of isopentyl nitrite are added dropwise to the cooled solution, the mixture is stirred for 3 hours and the precipitate is filtered off. Stir the yellow solid into 200 mL of 5N sulfuric acid. A pale yellow product precipitated. Yield: 22 g. The value of E _f = 0.53 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). b) 6-methylresazurine-4-carboxylic acid 15.3 g of 2-methyl-4-nitrosoresorcin, 15.4 g of 2,6-dihydroxybenzoic acid, 8.8 g of burel and 11 ml of concentrated sulfuric acid are reacted in an analogous manner as described in Example 1a). Yield: 28.7 g. Value = 0.15 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). e) H, O, O-triacetyl-6-methyldihydroresorufin-4-carboxylic acid From 10 g of 6-methylresazurine-4-carboxylic acid, 19.8 g of tin (II) chloride, 20 ml of acetic anhydride and 150 ml of glacial acetic acid, a directly triacetylated leuco compound is obtained in an analogous manner to that described in Example 1b). The crude product is purified by boiling in acetone. Yield: 7.3 g. Value = 0.51 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). III spectrum (perdeuterated dimethylsulfoxide; = 2.10, 2.25, 2.29, 2.33 (each s, 12H), 7.00, 7.09, 7.50 and 7.74 ppm (each d, J = 8.8 Hz, 4H), d) N, O-Triacetyl-6-methyldihydroresorufin-4-carboxylic acid N'-butoxycarbonylpiperazide In an analogous manner to that described in Examples 1d) to 1e), 3 g of the product are obtained from 5 g of H, O, O-triacetyl-6-methyldihydroresorufin-4-carboxylic acid, 10.7 ml of oxalyl chloride and 2 g of H-butoxycarbonylpiperazine. Value E ^. = 0.57 (silica gel, solvent system: ethyl acetate). e) 6-Methylresorufin-4-carboxylic acid piperazide trifluoroacetate 1) 2 g (9.5 mmol) of N-hydroxysuccinimide. The solution was cooled to 10 ° C and then a solution of 2.3 g (9.9 mmol) of dicyclohexylcarbodiimide in 40 ml of ethylene glycol dimethyl ether was added dropwise. After stirring at room temperature for 2 hours, the precipitated dicyclohexylurea is filtered off and the filtrate is evaporated under reduced pressure at 40 ° C. The residue is triturated with isopropyl alcohol and the suspension is filtered. The product is dried in a desiccator at room temperature. Yield: 9.19 g = 94% of theory (total yield based on thyroxine = 81%). EI value = 0.6 (high performance liquid chromatography HPTLC-EP 18; solvent system: 8: 2 acetonitrile / water). @ 1 H NMR Spectrum: (DMSO): 1 g of the tert-butoxycarbonyl derivative described in Example 1g) is left to stand for 15 minutes in 20 ml of trifluoroacetic acid. The solution was evaporated, the residue was digested with ether and the product was filtered off. Yield: 0.96 g. Rf value. = 0.92 (silica gel, solvent system; mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). i) Coupling of resorufin-4-carboxylic acid piperazide with 3- (1-diphenylhydantoinyl) -propionic acid N-hydroxysuccinimide ester 191 mg of piperazide trifluoroacetate described in Example 1h) and 210 mg of 3- (1-diphenylhydantoinyl) propionic acid N-hydroxysuccinimide ester (prepared from diphenylhydantoin sodium salt and 3-bromopropionic acid ethyl ester analogously to the procedure described by Cooc et al., Res. Communications in Chemical Pathology and Pharmacology J5 (1973), page 7.67) was stirred for 15 hours in 20 ml dioxane and 20 ml 0.1 M potassium phosphate buffer pH 8.5. The precipitated product was filtered off, the filtrate was evaporated and the residue was chromatographed on silica gel. (RP18) using isopropyl alcohol as the eluent to give a further crop of product, and the product was recrystallized from ethyl acetate-methanol to give a total of 250 mg of the coupling product. Rf value = 0.61 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0.1). ¹ H-NMR spectrum (perdeuterated dimethylsulfoxide): (7- 2.6-2.8 (m, 2H); 3.0-3.8 (m, IOH); 6.74 (d, J = 2.2Hz, 1H); 6.82 (d, J = 9.5Hz, 1H); 6.91 (double d, J = 9.5 and 2.2 Hz); 7.25-7.33 (m, 10H); 7.55 and 7.66 ppm (each d, J = 9.5 Hz, 2H). UV / VIS (0.1 M potassium phosphate buffer pH 7.5): X max = ^{576 '8} Fluorescence emission: = 592 nm. X max Example 2 Coupling of resorufin-4-carboxylic acid piperazide with N-hydroxysuccinimidoester 3-fl-Diphenylhydantoinylacetic acid In analogy to Example 1 a) 210 mg of the desired product are obtained from 365 mg of resorufin-4-carboxylic acid trifluoroacetate trifluoroacetate and 339 mg of 3- (1-diphenylhydantoin-acetic acid N-hydroxy-succinamide). Rj value = 0.82 (silica gel, solvent system: mixture of n-butanol, glacial acetic acid and water (4: 1: 1). @ 1 H NMR Spectrum: (DMSO): (T = 3.2-4.5 (m, 1H); 6.60 (d, J = 2.4 Hz, 1H); 6.71 (d, J = 9.5 Hz, 1H); 6.80 (double d, J = 9.05 and 2.4 Hz, 1H); 7.39 (s, 10H); 7.52 (d, J = 9.5Hz, 1H); 7.61 (d, J = 9.0 Hz, 1H); 9.65 ppm (s, 1 H). UV / VIS (0, 1 potassium phosphate buffer solution, pH 8.0): = 575.4 nm Λ ^max Fluorescence emission: = 592 nm. Example 3 Coupling of resorufin-4-carboxylic acid piperazide with N-hydroxy-succinimide ester of N-tert-butoxycarbonyl-L-thyroxine In analogy to the procedure described in Example 1i), resorufin-4-carboxylic acid piperazide trifluoroacetate trifluoroacetate and 419 mg K-tert-N-hydroxysuccinimide ester are obtained. butoxycarbonyl-L-thyrosine yields 320 mg of product. Rf value = 0.58 (silica gel, solvent system: mixture of chloroform, methanol and glacial acetic acid (9: 1: 0,1) The N-tert-butoxycarbonyl-thyroxine H-hydroxy succinimide ester is obtained as follows: a) N-tert-butoxycarbonylthyroxine A solution of 10 g (12.5 mmol) of L-thyroxine sodium monohydrate in 300 ml of dioxane / water (2: 1) and 15 ml of 1N sodium hydroxide solution was added with the addition of 3 g (13.75 mmol) of di-tert. butyldicarbonate (BOOgO) and the mixture was stirred at room temperature to avoid light. The pH is adjusted to 2 by addition of 2M potassium bisulfate solution, extraction is performed with ethyl acetate, the ethyl acetate extract is washed with water, dried over sodium sulfate and evaporated. The solid residue is triturated with petroleum ether, the suspension is filtered and the product is dried in an excolator. Yield: 9.45 g = 6% of theory. R, -. = 0.6 (silica gel, solvent 3 system): mixture of chloroform, ligroin and acetic acid in a ratio of 6: 3: 1). b) N-BOC-thyroxine N-hydroxysuccinimideater To a solution of 8.8 g of L-BOC-thyroxine in 200 ml of ethylene glycol dimethyl ether is added 2. A process according to claim 1 wherein the bifunctional compound having from 1 to 5 carbon atoms is piperazine, 1,2-ethylenediamine, glycine, sarcosine, [beta] -slanine or piperidine-4-carboxylic acid.