DD235648A5 - PROCESS FOR PREPARING THE GLYSIDES OF RESORPINE DERIVATIVES - Google Patents

Abstract

The glycosides of resorufin derivatives have the general formulae Ia and Ib <IMAGE> in which R<1> is hydrogen, R<2>, R<3> and R<5>, which can be identical or different, are hydrogen, halogen or a lower alkyl group, R<4> and R<6>, which can be identical or different, are hydrogen, halogen, a cyano, lower alkyl, lower alkoxy, carboxyl, lower alkoxycarbonyl, carboxy-lower alkyl, lower alkoxycarbonyl-lower alkyl or an optionally mono- or disubstituted carboxamide group or the group -COO-(CH2CH2O)n-R<7> in which R<7> is hydrogen or a lower alkyl group and n is an integer from 1 to 4, where R<6> can additionally be a sulphonyl or nitro group, and Y is a nitrogen or the group NO. These compounds are particularly suitable as chromogenic and fluorogenic substrates for determining the activity of corresponding glycosidases or oligosaccharide-cleaving enzymes. The glycosides are obtained by reacting resorufin derivatives with a mono- or oligosaccharide or a 1-halogeno derivative thereof, whose hydroxyl groups are substituted by customary protective groups. This initially results in per-O-substituted glycosides, which can be converted into the claimed compounds by elimination of the protective groups. The glycosides are used as chromogenic and fluorogenic substrates in diagnostic agents for detecting glycosidases or oligosaccharide-cleaving enzymes. The resorufin derivatives employed as starting materials in the preparation of the compounds are also claimed.

Description

Field of application of the invention

The invention relates to a process for the preparation of the glycosides of resorufin derivatives.

The compounds prepared according to the invention are used in clinical diagnostics, for example for determining the activity of corresponding glycosidases or enzymes which split saccharide chains.

Characteristic of the known technical solutions

Glycosidases fulfill a multiple physiological function in the human and animal organism. Thus, for example, / 3-D-galactosidase plays an important role in the carbohydrate metabolism, since it causes the hydrolysis of lactose. In addition, / 3-D-galactosidase is the key enzyme in the degradation of glycolipids, mucopolysaccharides and glycoproteins. Other physiologically important glycosidases include aD-galactosidase, α-D- and / 3-D-glucosidase and aD mannosidase.

In addition to their physiological significance, the glycosidases have gained in importance in recent years in the diagnostic and biotechnological field. For example, these enzymes are increasingly being used as an indicator enzyme for enzyme immunoassays. Particularly preferred in this context is / 3-D-galactosidase (see, for example, Annals of Clinical Biochemistry 16,221-240 [1979]).

The determination of the activity of glycosidases therefore plays an increasing role in both clinical chemistry and diagnostics. For this purpose, the glycosidase-containing sample is mixed with a suitable substrate in general. The substrate is cleaved by the enzyme. One of the cleavage products is suitably detected. Either the glycine released by the action of the enzyme or the aglycone can be measured. As a rule, the latter is determined. The substrate is often the natural substrates of the enzymes to be detected. However, particularly preferred glycosides are used in which the aglycone is a spectroscopically easily detectable radical. There are a number of glycosidase substrates are known in which after cleavage by the glycosidase, the aglycone can be measured spectroscopically in the visible or UV range and fluorometrically.

So are in Biochem. Z. 333,209 (1960) phenyl-O-D-galactoside, as well as some other aromatic ring-substituted derivatives (e.g., o-nitrophenyl and p-nitrophenyl-β-D-galactoside) as substrates of the 3-D-galactosidase. The phenols liberated by hydrolysis are determined photometrically in the UV range or in the case of the nitrophenols in the short-wave visible wavelength range. Also, an oxidative coupling with aminoantipyrine can be connected as an indicator reaction (Analytical Biochem., 40, 281 [1971]).

For histochemical studies naphthyl-.beta.-D-galactosides are used, such. For example, the 1-naphthyl compound in histochemistry 35, 99 (1973), the 6-bromo-2-naphthyl derivative in J. Biol. Chem. 195,239 (1952) or naphthol-β-D-galactoside (histochemistry 37,89 [1973]). To visualize the resulting naphthols are reacted with various diazonium salts to azo dyes.

The determination of enzyme activities with fluorogenic substrates is widespread, since the sensitivity of fluorometric determinations is often increased by several orders of magnitude compared to the photometric methods. In some cases it is necessary to work with fluorogenic substrates, for example in the investigation of enzymatic activity in cells with automatic cell differentiation devices (cytofluorometry) and in the analysis of immobilized enzymes with flow cytometry. In other cases, for example in the determination of enzymatic labeling of test systems (enzyme immunoassays), the multiplication effect of enzymatic catalysis is considerably enhanced by the use of fluorogenic substrates.

The fluorogenic substrates known to date for β-D-galactosidase and other glycosidases have as fluorophores derivatives of fluorescein, indoxyl or methylumbelliferone. However, these compounds have serious disadvantages for the kinetic analysis of complex systems. The disubstituted derivatives of fluorescein are hydrolyzed in a multi-step reaction sequence. Monosubstituted fluorescein glycosides already fluoresce themselves. Indoxyl derivatives undergo a series of chemical transformations after enzymatic cleavage, which also complicate kinetic analysis. Derivatives of methylumbelliferone must be excited in the UV. This can interfere with the intrinsic fluorescence of biological or synthetic materials. In addition, the UV excitation, especially in laser optics, costly. Most fluorogenic substrates give rise to reaction products that have low solubility so that they are not suitable for the kinetic analysis of enzyme activities, which just require good substrate and product solubility.

Object of the invention

The aim of the invention is the provision of substrates with which various glycosidases can be determined in a simple, rapid and reliable manner and which can possibly be used in both photometric and fluorometric determination methods.

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Explanation of the essence of the invention

The invention has the object zügrunde to find new compounds that are suitable as substrates for the determination of various glycosidases and can meet the need for new substrates with the desired properties.

This problem is solved by the new glycosides of resorufin derivatives, which can be cleaved with the help of glycosidases in the sugar portion and in the resorufin derivatives. The latter are readily water-soluble compounds which have a well-measurable absorption in the visible range and, furthermore, are easily susceptible to fluorescence.

The present invention accordingly glycosides of resorufin derivatives of the general formula I a or I b

Glycoside 0

(I a) (I b)

O-glycoside

in which

R ^{1 is} hydrogen,

R ² , R ³ and R ⁵ , which may be the same or different, are hydrogen, halogen or a lower alkyl group, R ⁴ and R ⁶ , which may be the same or different, represent hydrogen, halogen, a cyano, lower alkyl, lower alkoxy, Carboxy, lower alkoxycarbonyl, carboxy-lower alkyl, lower alkoxycarbonyl-lower alkyl, or an optionally mono- or di-substituted carboxamido group or the group

-COO- (CH ₂ CH ₂ O) _n -R ⁷ ,

in which R ^{7 is} hydrogen or a lower alkyl group and η is an integer from 1 to 4, wherein R ^{6 may} additionally represent a sulfonyl or nitro group, and

Y is a nitrogen or the group N - * O

mean.

The glycosides of resorufin derivatives of the general formula I are novel compounds. They can be prepared by methods known per se from carbohydrate chemistry.

Resorufin derivatives of the general tautomeric formulas Ma and Hb are preferably prepared in a manner known per se

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(II a)

(II b)

in which

R ¹ to R ⁶ and Y have the abovementioned meaning,

with a mono- or oligo-saccharide or an I-halo derivative thereof, wherein in each case all hydroxy groups are substituted by a protective group commonly used in carbohydrate chemistry, converted to per-O-substituted glycosides, from which by deprotection of the known per se Way the glycosides of resorufin derivatives of general formula I are obtained.

The reaction of the compounds of the formula II with the per-O-substituted I-halo-saccharides is preferably carried out in the presence of acid scavengers, such as alkali metal hydroxide or carbonate, in aqueous acetone or (under phase transfer conditions) in a water / benzene or water / chloroform Mixture made.

Moreover, this process can be carried out by first converting the resorufin derivatives of the general formula II into the alkali metal salts by means of alkali metal hydroxide or alcoholate or by means of optionally substituted amines and then adding these in dipolar aprotic solvents such as acetone, dimethyl sulfoxide, dichloromethane, Tetrahydrofuran or dimethylformamide with the per-O-substituted I-halo-saccharides.

Furthermore, in the synthesis of the per-O-substituted glycosides from the resorufin derivatives of the general formulas! II and the per-O-substituted I-halo-saccharides additions of individual silver salts or mixtures of silver salts (silver oxide, carbonate, carbonate on Celite, triflate, salicylate) and / or individual mercury salts or mixtures of mercury salts (mercury bromide , cyanide, acetate, oxide), optionally using drying agents such as calcium chloride, molecular sieve or drierite, in solvents such as methylene chloride, chloroform, benzene, toluene or dioxane, proven. For the synthesis of the α-linked glycosides is suitably a compound of general formula II with a saccharide whose hydroxy groups are substituted with a protecting group, particularly preferably with an acetyl group, in the presence of a Lewis acid, such as. As tin tetrachloride, aluminum chloride, preferably zinc chloride, melted (see.

Chem. Ber. 66, 388-383 [1933] and Methods in Carbohydr. Chem. 2,345-347 [1967]). Here is the temperature between 80 and 15O ⁰ C, preferably chosen between 110und 130 ° C.

The resulting per-O-substituted glycosides of denflesorufin derivatives of general formula II are also new compounds.

The cleavage of the protective groups of the per-O-substituted glycosides to the glycosides of the general formula I is by common methods in the carbohydrate chemistry (see, for example, Advances Carbohydrate Chem. 12,157 [1975]), for example, the acyl protecting groups by means of sodium or barium or ammonia in methanol.

By halogen in the definition of R ¹ to R ⁷ is meant fluorine, chlorine, bromine and iodine, preferably chlorine and bromine.

The "lower alkyl group" in the definition of R ¹ to R ⁶ contains 1 to 5, preferably 1 to 3 carbon atoms, with the methyl group being particularly preferred.

The "lower alkoxy group" in the definition of R ⁴ and R ⁶ contains 1 to 5, preferably 1 to 3 carbon atoms, with the methoxy group being particularly preferred.

The "lower alkoxy" or the "lower alkyl" radicals of the lower alkoxycarbonyl, carboxy-lower alkyl and lower alkoxycarbonyl-lower alkyl radicals in the definition of the substituents R ⁴ and R ⁶ likewise have 1 to 5, preferably 1 to 3, carbon atoms wherein the methoxy group or the methylene radical are particularly preferred.

As "protecting group commonly used in carbohydrate chemistry" is particularly suitable acetyl, benzoyl, benzyl or Tri methylsilyl radical.

Suitable substituents of the carboxamido group are alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl radicals in which the alkyl radicals have in each case 1 to 5, preferably 1 to 3, carbon atoms. In a doubly substituted carboxamide function, the two substituents may be closed to a ring which is substituted by heteroatoms, e.g. As oxygen, nitrogen and sulfur, may be interrupted.

As a glycosidic radical which is linked to the resorufin derivatives of the general formula II to give glycosides of the general formula I, all mono- and oligo-saccharides which can be cleaved off again from the resorufin skeleton by the corresponding glycosidases are suitable. Examples of glycosides according to the invention include: 0-D-galactopyranosides α-D-galactopyranosides, / 3-D-glucopyranosides, α-D-glucopyranosides and α-D-mannopyranosides.

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Also suitable as the glycosidic residue are oligo-saccharide chains which can be degraded by saccharide-chain-splitting enzymes down to the level of a monosaccharide or oligo-saccharide, which in turn can be split off directly from the resorufin skeleton with the corresponding glycosides. As such oligosaccharide chains are understood in particular chains, which are composed of 2 to 10, preferably from 2 to 7 mono-saccharide units

 Another object of the invention are the new compounds of the general tautomeric formulas M'a and H'b

(II ¹ a)

(II ¹ b)

in which

R ¹ 'to R ^{6 have the} same meaning as the substituents R ¹ to R ^e , wherein R ¹ to R ^e can not all simultaneously be hydrogen, and

Y has the meaning given above.

These connections are new. They are particularly suitable as an intermediate for the preparation of the glycosides of resorufin derivatives of the general formula I.

The compounds of the general formula ΙΓ can be prepared analogously to processes which are suitable for the preparation of the known resorufin (compound of the general formula II in which R ¹ to R ^{6 in} each case represents a hydrogen atom).

The compounds of the general formula ΙΓ are prepared in an advantageous manner by nitrosoresorcinol derivatives of the general formula III

NO

OH

(III)

with resorcinol derivatives of the general formula IV

(IV)

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in which

R ¹ to R ^{6 have} the abovementioned meaning,

be reacted in the presence of manganese dioxide and sulfuric acid at low temperatures. This results in first compounds of general formula II ', in which Y represents an N-> O group. These substances can easily be converted with zinc powder in the presence of ammonia into compounds of the general formula ΙΓ in which Y is nitrogen.

The reaction of the compounds of general formula III with compounds of general formula IV is usually carried out at a temperature between -10 to -50 ° C, preferably between 0 and 3O ⁰ C.

The reaction proceeds particularly gently when the substances of general formula III and formula IV are mixed at about 0 ^° C. and the reaction mixture can then be warmed to room temperature. The concentration of manganese dioxide should expediently be 0.5 to 5, preferably 1 to 2 mol / l. The sulfuric acid concentration should be 0.5 to 5, preferably 1 to ЗтоІ / І.

The reduction of compounds of the general formula ІГ in which Y represents an N-> O group to compounds of the general formula ІГ in which Y represents a nitrogen, is preferably carried out in ammoniacal solution with zinc dust (see Nietzki et al. , Ber.Dtsch.Chem.Ges. 22, 3020 [1889]). The solvent used is expediently water-alcohol mixtures, preferably a mixture of 1 part of water with 0 to 4 parts of methanol. Per mole of substance to be reduced, 1 to 20, preferably 1 to 5 moles of zinc dust are added in portions. The temperature of the reaction solution is kept at -10 to + 35 ° C, preferably at +5 to + 10 ° C. The exact observance of the temperature range has proven to be necessary for a clear reaction course. Without cooling, the exothermic reaction leads to by-products that are difficult to separate.

Under the chosen mild conditions, the reaction between the substances of the general formula III and the general formula IV proceeds unambiguously and in good yield. The chosen synthetic path is capable of variation. This opens up numerous possibilities of synthesis, in particular with regard to the preparation of unsymmetrically substituted resorufin or also resazurin derivatives.

Resorufin derivatives of the formula ІГ, in which R ⁴ 'or / and R ⁶ ' is a lower alkoxycarbonyl, an optionally mono- or di-substituted carboxamido group or the group -COO- (CH ₂ CH ₂ O) _n -R ⁷ preferably represented by the triacylated dihydroresorufines of the general formula V,

(V)

in the R ¹ , R ² , R ³ and R ^5, the above

Have meaning

R ⁴ * and R ^{6 represent} a carboxy group and R ^{7 represents} hydrogen or a lower alkyl group.

The carboxylic acid function is by literature methods, for. b. with oxalyl chloride / DMF or thionyl chloride / DMF converted into the acid chloride, from which by reaction with any alcohols and amines, the corresponding substituted carboxylic ester or. Carboxamide derivatives are obtained.

By treatment of the resulting acetylated derivatives of general formula III with caustic, preferably 0.1-5 M sodium or potassium hydroxide solution, or with 1-15 M aqueous ammonia and an oxidizing agent, preferably potassium hexacyanoferrate III, with the addition of water-soluble organic solvents, such as 1,4-dioxane or methanol, the corresponding resorufin derivatives of general formula II are obtained.

Suitable alcohol components are, in principle, all possible alcohols. Particular preference is given to diethylene glycol monoethyl ether, triethylene glycol monoethyl ether or simple alcohols, such as methanol or ethanol. The amine component can also be selected from all possible amines. Particularly preferred are amines having a polar group such as morpholine, methoxyethylamine or glycinamide or ammonia, a primary or secondary lower alkylamine. Furthermore, aminocarboxylic acids with protected in the usual way carboxyl function such. As glycine tert-butyl ester, glycine benzyl ester or №-BOC-lysine methyl ester can be used. After cleavage of the protecting groups so obtained Resorufine II or resorufin glycosides I with aliphatic carboxylic acid function.

The acetylated Dihydroresorufine the general formula V is obtained from the corresponding resorufin or resazurin by reaction with a strong reducing agent such. As tin-ll-chloride or chromium-ll-acetate or by electrochemical reduction and subsequent acetylation. For the reduction, the resorufin or resazurin is heated for 10 minutes to 1 hour with 2 to 10 preferably 2 to 6 equivalents of tin (II) chloride in 5 to 35% strength aqueous hydrochloric acid. Upon cooling, the dihydro compound precipitates. The acetylation is carried out in the usual manner with acetic anhydride. Preferably, the compounds of general formula V are prepared in a one-pot process by reductive acetylation. The corresponding resorufin or resazurin is with 2 to 6 equivalents of tin-Il-chloride for 5 min to 5h, preferably 10min to 3h. heated under reflux in acetic anhydride or at RT4 to 16h. touched.

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In the synthesis of the glycosides of resorufin derivatives of general formula II arise non-fluorescent compounds from which by enzymatic cleavage with the help of the corresponding glycosidases again the fluorescent resorufin derivatives are released. The fluorogenic resorufin derivatives according to the invention have very advantageous properties in comparison with the previously known glycosides of other chromogenic or fluorogenic groups. As monosubstituted derivatives, they have kinetics of enzymatic hydrolysis, which can be described very simply by the Michaelis-Menten relationship. For the resorufin β-D-galactopyranoside, for example, a Michaelis constant K _M = 0.38 mmol / l applies. The inhibition of this hydrolysis by the natural substrate lactose is competitive, so that for certain studies, the specific conversion of glycosides of Resorufin derivatives by addition of natural glycosides, such as lactose in the case of / 3-D-galactosidase, can be defined and reversibly changed ,

The glycosides of resorufin derivatives according to the invention are practically unlimited in aqueous solution at + 4 ⁰ C. Even the solubility of the glycosides of the resorufin skeleton is sufficient for most kinetic purposes. By introducing carboxylic acid residues, carboxylic acid derivatives with polar groups or sulfonic acid groups, the solubility of the corresponding glycosides is significantly improved.

Excitation and emission of the products according to the invention are in the visible spectral range with sufficient quantum efficiency. The maximum fluorescence intensity of the resorufin is reached at pH above 7.0, and falls off slowly at lower pHs. The glycosides of resorufin are usually yellow in aqueous solution (A _max at about 470 nm). After the enzymatic reaction, the products usually have a red color (\ _max at about 570 nm), so that the substances are also excellent for photometric determinations and non-instrumental, visual methods are suitable. Another object of the invention is the use of the novel glycosides of resorufin derivatives of the general formula I for determining the activity of corresponding glycosidases, for example α-D and / 3-D-galactosidase, the α-D and / or D-glucosidase and aD-mannosidase.

Resorufin derivatives of the general formula I, in which the glycoside residue represents an oligosaccharide chain, are particularly suitable for the detection of saccharide chain-splitting enzymes, such as α-amylase. In this case, the oligosaccharide chain is cleaved in a characteristic manner by the enzyme to be detected, preferably up to the stage of the monosaccharides, it being possible, if appropriate, to use further auxiliary enzymes. This is then cleaved by the corresponding glycosidase in the manner described above in the resorufin skeleton and the monosaccharide.

Furthermore, diagnostic means for determining the activity of glycosidases are claimed which contain the new glycosides of resorufin derivatives of the general formula I. The use of the glycosides of resorufin derivatives of the general formula I as substrates for the glycosidases leads to significantly more sensitive test systems than hitherto known. The new substrates can be used to determine the activity of glycosidases with advantage in biochemical, biotechnological as well as in the clinical-chemical field. They are more sensitive. This results in several advantages:

a) Lower enzyme activities can be measured.

b) Smaller amounts of sample can be used.

c) The determination of the activity can take place in a much shorter time.

d) The low sample use and the favorable measuring wavelength also reduce the susceptibility of the method by other sample components.

e) The conversion can be measured in carrier matrices with immobilized enzyme.

It has been found that the claimed substrates are suitable for determining the activity of glycosidases of any origin. The diagnostic agents according to the invention with substrates of the general formula I react much more sensitively than the previously known test agents.

The glycosides of resorufin derivatives of the formula I are also suitable for immunological determination methods in which glycosidases are used as indicator enzymes whose activity must be determined after carrying out the immunological reaction. Such immunological detection methods with enzymatic indicator reaction are known in the art as enzyme immunoassays. These methods are used to determine the concentration of proteins, polysaccharides, hormones, drugs and other low-molecular substances in the range of 10 ~ ⁵ to 10 ~ ¹² mmol / l. Depending on the requirement of phase separation steps, a distinction is made between homogeneous and heterogeneous test procedure. Further subdivision can be done in competitive and non-competitive test principles.

However, all test principles work with enzyme antigen or. Enzyme antibody conjugates. The enzymatic indicator reaction is common to all enzyme immunoassays. Indicator enzymes suitable for such purposes are, for example, glycosidases, in particular / 3-D-galactosidase. The determination of the glycosidases in such enzyme immunoassays is usually carried out by adding a suitable substrate, which is enzymatically cleaved and measured in a conventional manner photometrically or fluorometrically.

An improvement of the glycosidase test system thus also leads to considerable advantages in such enzyme immunoassays:

1. The higher sensitivity also allows a further reduction of the detection limits, shorter reaction times and lower sample use and thus also lower interference by other sample components.

2. The more favorable measuring wavelength reduces the susceptibility of the method by insoluble constituents, for example by turbidity in certain reactions.

The diagnostic agent contains in addition to one or more of the substrates of the general formula I, a suitable buffer system and optionally further suitable additives commonly used for such diagnostic agents, such as wetting agents, stabilizers, etc. The diagnostic agent may be in the form of a solution, as a lyophilisate , as a powder mixture, reagent tablet or coated on an absorbent carrier, are present. The diagnostic agent in the form of a solution according to the invention preferably contains all the reagents required for the test. As the solvent, water or mixtures of water with a water-soluble organic solvent such. As methanol, ethanol, acetone or dimethylformamide, in question. For reasons of durability, it may be advantageous to distribute the reagents required for the test to two or more solutions that are mixed only during the actual examination.

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For the preparation of the diagnostic agent in the form of a lyophilizate in the total weight of about 5-20mg, preferably about 10mg, a solution is dried, which in addition to all the reagents required for the test conventional scaffold builders such. As polyvinylpyrrolidone, and any other fillers such. As mannitol, sorbitol or xylitol contains. A diagnostic agent in the form of powder mixtures or reagent tablets can be prepared by adding and granulating the ingredients of the test with conventional galenic additives. Additives of this type are z. For example, sugar alcohols such as mannitol, sorbitol or xylitol, or other soluble inert compounds such as Polyettiylenglykole or polyvinylpyrrolidone. The powder mixtures or reagent tablets generally have a final weight of about 30 to 200 mg, preferably 50 to 80 mg.

For the preparation of the diagnostic agent in the form of a test strip is an absorbent carrier, preferably filter paper, cellulose or synthetic fiber fleece with solutions of the necessary, usually used for the preparation of test strips reagents in volatile solvents, such as. As water, methanol, ethanol or acetone impregnated. This can be done in an impregnation step. Often, however, it is useful to carry out the impregnation in several steps, using solutions which each contain a part of the components of the diagnostic agent. For example, in a first step, it may be impregnated with an aqueous solution containing the buffer and other water-soluble additives and then in a second step with a solution containing the glycosidase substrate. The finished test papers can be used as such or adhered to handles in a conventional manner or preferably sealed between plastics and fine mesh networks according to DBP 2118455.

embodiment

The invention is explained in more detail below with reference to some examples.

The following examples illustrate some of the numerous process variants that may be used to synthesize the compounds of this invention, and, by way of example, the use of the novel glycosides of resorufin derivatives to determine the activity of glycosidases. However, they are not intended to limit the subject matter of the invention.

example 1

Resorufin-beta-D-galactopyranoside

a) Presentation of resorufin

Resorufin is made from its oxidation product resazurin, which is commercially available in good purity, based on the description of Nietzki et al. Chem. Ges. 22 (1889) pp. 3020-3038). For this purpose, 10 g (43 mmol) of resazurin (Fluka, Buchs, Switzerland) are heated in a beaker with 50 ml of 25% ammonia solution, 25 ml of 37% sodium hydrogen sulfite solution and 50 ml of water for 30 minutes on the boiling water bath. Another 20 ml of the ammonia solution, 7 ml of the hydrogen sulfite solution and 20 ml of water are added and the mixture is heated for a further 30 minutes. The conversion can be observed by the color change from blue to red or by thin layer chromatography. After the reaction is complete, add 10 mL of 32% brine until pH 5 is reached. The reaction solution is allowed to stand in the refrigerator for one day. Thereafter, the brownish precipitate is filtered off, washed with ice-cold hydrochloric acid solution, pH 4, and dried at 110 ° C. Yield 7.8 g (36.7 mmol, 85% of theory).

b) Glycosylation of resorufin to resorufin β-D-galactopyranoside

The glycosylation at the phenolic hydroxyl group via the tetraacetate intermediate was accomplished by the following procedure.2,13g (10mmol) finely powdered resorufin, 4,12g (10mmol) acetobromo-aD-galactose (Sigma, Munich), 1,16g (5mmol) silver. 1-oxide (Fluka, Buchs, Switzerland), 5 g calcium sulfate (CaSO 4, ₂ H ₂ O, Drierite), a few granules of iodine and 50 μΙ quinoline are stirred in 50 ml of methylene chloride at room temperature for 40 hours. 40% of the resorufin are converted to resorufin-ß-D-galactopyranoside-tetra-acetate. The control of the reaction can in turn be carried out by means of thin-layer chromatography. After separation of the solid components via pleated filters and centrifugation, the methylene chloride is removed in a rotary evaporator. The resorufin β-D-galactopyranoside tetra-acetate remains as a brownish syrup. Yield about 2 g (35% of theory).

2 g of resorufin β-D-galactopyranoside tetra-acetate are taken up in 80 ml of dry methanol without further purification. 0.5 g of sodium are dissolved in small pieces, in an ice bath, in 20 ml of dry methanol. With stirring in an ice bath, 6-8 ml of the methoxide solution are added in portions of 1 ml to the resorufin β-D-galactopyranoside tetra-acetate solution over 30 minutes and the deaeetylation is monitored by thin-layer chromatography. The precipitated, orange-colored resorufin-β-D-galactopyranoside is filtered off with suction and washed with a little ice-cold methanol. Yield about 1 g of crude substance (70% of theory).

Recrystallization from methanol with subsequent drying (not above 6O ⁰ C) provides 0.2 g of pure product. All thin-layer chromatography investigations to control the course of the reaction can be carried out with the system Kieselgel 60 (Merck, Darmstadt) and superplasticizer methylene chloride / methanol (9 / I). The following Rr values apply to this system:

Resorufin 0.4

Resazurin 0.35

Resorufin-jS-D-galactopyranoside tetra-acetate 0.9

Resorufin β-D-galactopyranoside, 0.1.

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Example 2

Resorufin-beta-D-glucoside

2.14 g (10 mmol) of resorufin (prepared according to Example 1a), 4.11 g (10 mmol) of acetobromo-aD-glucose and 3.64 g (10 mmol) of hexadecyltrimethylammonium bromide are stirred for 4 h. heated under reflux in a mixture of 11.5 ml of 1 N sodium hydroxide solution and 50 ml of chloroform. It is then evaporated to dryness and digested with ethyl acetate. The ethyl acetate solution is evaporated again to Tockne. It is taken up with 20 ml of ethanol. The Tetraacetylresorufin-ß-D-glucoside precipitated from this solution is recrystallized from methanol with addition of activated carbon.

For deacetylation is added with 10 ml of methanol. After 2 hours of stirring at room temperature, the precipitated product is filtered off and dried. Yield: 50mg. IH-NMR ([Df-DMSO]: δ = 3.15-3.80 (m, 6H); 4.5 - 6.5 (broad, 4H); 5.12 (d, J = 6.9Hz, 1H); 6.29 (d, J = 2.0Hz, 1H); 6.80 (dd, J = 9.6 and 2.0 Hz, IH); 7.12 (dd, J = 8.5 and 2.0Hz, 1H); 7.16 (d, J = 2.0Hz, 1H); 7.55 (d, J = 9.6Hz, 1H); 7.80 (d, J = 8.5Hz, 1H).

Example 3 Resorufin-1-carboxylic acid methyl ester

50 g of nitrosoresorcinol, 5373 g of 3,5-dihydroxybenzoic acid methyl ester and 28.0 g of manganese dioxide are dissolved or suspended in 500 ml of methanol. With ice cooling, 34.4 ml of concentrated sulfuric acid are added dropwise at 5 to 10 ° C. After removing the ice bath, stirring is continued for a further two hours. Thereafter, 200 ml of aqueous ammonia solution are added with cooling. The precipitate is filtered off via a glass fiber filter. In addition, filtrate is added in portions at 5 to 10 ° C 10g zinc powder. At room temperature, the mixture is stirred until the reduction is complete (TLC check, ethyl acetate / methanol 4: 1 as eluant, reaction time about 1.5 hours). The rotary evaporator is used at 25 ° C bath temperature to Ѵз the volume eigeengt. By acidification with concentrated hydrochloric acid until the color change with cooling, the desired product is precipitated. After washing with dilute hydrochloric acid and drying in vacuo over calcium chloride to obtain resorufin-1-carboxylic acid methyl ester. Yield: 30.9 g (36% of theory).

1 H-NMR: ([Df-DMSO]: δ = 3.98 (s, 3H); 6.47 (d, J = 2.2Hz, 1H); 6.85-6.95 (m, 2H); 7.09 (d, J = 2.2Hz, 1H); 7.60 (d, J = 9.6Hz, 1H)

Fluorescence: Absorption: \ _max = 570 nm

Emission: A _max = 588nm

 In an analogous manner you get out

a) 3,5-Dihydroxybenzoic acid and nitrosoresorcinol via resazurin-1-carboxylic acid resorufin-1-carboxylic acid UV / VIS (0.1 M potassium phosphate buffer, pH 7.5): λ _max = 569 nm

b) 4-0-Methyl-gallic acid methyl ester and nitrosoresorcinol over 4-methoxy-resazurin-1-carboxylic acid methyl ester 4-methoxy-resorufin-1-carboxylic acid methyl ester

UV / VIS (0.1M potassium phosphate buffer, pH 7.5): k _max = 592nm

c) 3,5-dihydroxybenzoic acid methyl ester and 4-chloro-6-nitrosoresorcinol over 8-chloro-resazurin-1-carboxylic acid methyl ester 8-chloro-resorufin-1-carboxylic acid methyl ester

d) 4-O-Methylgallussäuremethylester and 4-bromo-6-nitrosoresorcin over

S-Brpm ^ -methoxy-resazurin-1-carboxylic acid methyl ester S-bromo-methoxy-resorufin-1-carboxylic acid methyl ester

e) 5-nitroresorcinol and nitrosoresorcinol via 1-nitro-resazurin 1-nitro-resorufin

f) resorcinol-5-sulfonic acid and nitrosoresorcin via Resazurin-1-sulfonic acid resorufin-1-sulfonic acid

Example 4 Resazurin-4-carboxylic acid

1.60 mg (10.5 mmol) of nitrosoresorcinol, 1.55 g (10.0 mmol) of 2,6-dihydroxybenzoic acid and 0.86 g (10 mmol) of manganese dioxide are taken up in 20 ml of methanol and cooled to 0 ° C. These are 1.06ml conc. Sulfuric acid added dropwise. It is continued for 2 hours without cooling. The precipitated red product is filtered off, washed with methanol and dried. Yield:

2.3 g (85% of theory).

UV / VIS ( _0.1M potassium phosphate _buffer pH 7.5): Л _тах = 614nm (ε = 48cm ² тоГ ¹ ) After acidification: X _max = 522nm (ε = 32cm ² тоГ ¹ )

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Example 5 Resorufin-4-carboxylic acid

2.3 g of resazurin-4-carboxylic acid (prepared according to Example 4) are dissolved in 20 ml of water and 5 ml of 25% ammonia. 5g of zinc dust are added to the blue solution with ice cooling. Thereafter, the ice-cooling is removed, so that the solution gradually warms up to room temperature. The reduction can be easily recognized by the color change from blue to dark violet or by means of thin-layer chromatography (eluent: methanol / ethyl acetate 1: 1). The excess zinc powder is filtered off. The reaction solution is washed with 5 ml of glacial acetic acid and conc. Hydrochloric acid acidified. The precipitated product is filtered off, washed with dilute hydrochloric acid and dried in vacuo over calcium chloride. Yield: 1.8 g (82% of theory)

 UV / VIS:

(0,1 M potassium phosphate _buffer , pH 7,5): Х _тах = 579,4пт (е = 48,6ст ² тоГ ¹ ); After acidification: X _max = 485.9 nm (ε = 34.7 cm ² тоГ ¹ ).

Fluorescence: Absorption: Л _тах = 579 nm

Emission: A _max = 593 nm.

Example 6 i-IVethylresorufin-4-carboxylic acid

840 mg of 2,6-dihydroxy-4-methylbenzoic acid, 760 mg of nitrosoresorcinol, 430 mg of manganese dioxide and 0.53 ml of sulfuric acid are reacted analogously to Example 4 to give i-methylresazurin-4-carboxylic acid. Yield: 0.8g.

The resulting i-methylresazurin-4-carboxylic acid is reduced analogously to Example 5 to i-methylresorufin-4-carboxylic acid.

Yield: 0.4g.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5): A _max = 571 nm.

Example 7 Resorufin-4-carboxylic acid morpholide

1) N.CO-triacetyldihydroresorufin ^ -carboxylic acid

Option A)

5 g (19.4 mmol) of resorufin-4-carboxylic acid or 5.3 g (19.4 mmol) of resazurin-4-carboxylic acid are dissolved in 100 ml of 10% hydrochloric acid for 0.5 h. heated under reflux with 7 g (38 mmol) of tin (II) chloride. The solution turns green. The mixture is allowed to cool, filtered from the precipitated Eihydroresorufin-4-carboxylic acid under nitrogen and dried in vacuo over phosphorus pentoxide. The crude product thus obtained is refluxed for 30 minutes with 30 ml of acetic anhydride and 20 mg of sodium acetate. The reaction mixture is added to 200 ml of ice-water and stirred for 14 h. The precipitate is recrystallized from ethanol / water. This gives 4.8 g (65%) of the desired compound.

Melting point: 197-199 ° C

TLC (silica gel, eluant chloroform / methanol / glacial acetic acid 9: 1: 0.1),

Variant b)

5.1 g (20 mmol) resorufin-4-carboxylic acid are stirred in 20 ml of acetic anhydride with 11 g (60 mmol) of tin-II-chlorid1 hrs. At 80 ⁰ C.

It is poured into 230 ml of ice-water, stirred for 1 hour, filtered and worked up analogously to variant a).

Yield: 5.4g (71%)

2) N.O.O-triacetyldihydroresorufin ^ -carboxylic acid chloride 3.85g (ЮттоІ)

NOO-Triacetyldihydroresorufin ^ -carboxylic acid are mixed with 5.4 ml (60 mmol) of oxalyl chloride and cooled to -10 ⁰ C. To this is added a drop of dimethylformamide and the reaction mixture is allowed to warm to room temperature with stirring. The starting material dissolves under gas evolution. After completion of the evolution of gas, the mixture is stirred for 0.5 h., Evaporates, takes each 3 times with 20 ml of dry methylene chloride and evaporated to dryness. This gives 4 g of crude product, which is further processed without further purification.

TLC (silica gel, eluant chloroform / methanol / glacial acetic acid 9: 1 : 0, V)

colorless stain, which turns red after a few hours.

3) N.Cso-triacetyldihydroresorufin ^ -carboxylic acid morpholide

11.3 g (31.4 mmol) of acid chloride tube are dissolved in 150 ml of dry methylene chloride. Drip 8.7 ml (63 mmol) of triethylamine and then 3.3 ml (37.7 mmol) of morpholine. The mixture is stirred for 2 h., The solution was washed with 1% citric acid, sodium bicarbonate solution and water, the organic phase is dried over magnesium sulfate and evaporated. The residue is crystallized from ethanol

 Yield: 8.1 g (63%), m.p .: 133-135 ° C (decomposition)

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4) Resorufin ^ -carboxylic acid morpholide

3.7 g of OmmoDTriacetyldihydroresorufin ^ -carboxylic acid morpholide are taken up with 250 ml of methanol and 250 ml of water. To this are added 36 ml of 1 N sodium hydroxide solution and 6.0 g (18 mmol) of potassium hexacyanoferrate III and stirred for 14 h. at room temperature. After acidification with hydrochloric acid to pH 3 is evaporated to dryness and digested with acetone. The dye solution is filtered through 500 ml of silica gel with acetone as eluant. Evaporation of the dye-containing eluate gives 2.3 g (80%) of product.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5): K _mm = 575 nm, ε = 55000 cm ² mol " ¹ DC (flow agent, see under 2) R f = 0.52.

¹ H-NMR ([Df-DMSO]: δ = 3.3-3.8 (m, 8H); 6.50 (d, J = 2Hz, 1H); 6.64 (d, J = 10Hz, 1H); 6.76 (dd, J = 10 and 2Hz, 1H), 7.44 and 7.51 (each d, J = 10Hz, 2H7).

Example 8

Methyl tetraacetylresorufin-i-carboxylate beta-D-galactopyranoside and tetraacetylresorufin-S-carboxylic acid methyl ester beta-D-galactopyranoside

6.8 g of resorufin-1-carboxylic acid methyl ester (prepared according to Example 3), 5.75 g of silver l-oxide, 6.75 g of silver carbonate, 15 g of molecular sieve 4A and 10 g ± bromotetraacetylgalactose are treated with 250 ml of absolute chloroform for 4 h. stirred at room temperature. After addition of a further 5 g of α-bromo-tetraacetyl-galactose, stirring is continued overnight. The reaction mixture is filtered through a glass fiber filter and evaporated. The oily crude product is chromatographed on 21 silica gel with chloroform / ethyl acetate 2: 1 as eluant. 2.5 g of a yellow fraction are eluted with Rf = 0.28 (HPTLC silica gel, same solvent).

By stirring with methanol, tetraacetylresorufin-S-carboxylic acid methyl ester-β-D-galactopyranoside is obtained in the form of orange crystals.

Yield 1.5g.

¹ H-NMR ([D] _6- DMSO): δ = 1.95,2.03,2.04 and 2.14 (each s, 12H); 3.88 (s, 3H); 4.11 (d, J = 7Hz, 2H); 4.51 (t, J = 7Hz, 2H); 5.24 (m, 2H); 5.36 (m, 1H); 5.69 (m, 1H); 6.31 (d, J = 2Hz, 1H); 6.97 (d, J = 2Hz, 1H); 7.04 (dd, J = 8.8 and 2.4Hz, 1H); 7.14 (d, J = 2.4Hz, 1H); 7.78 (dd, J = 8.8Hz, 1H).

Thereafter, 1.6 g of a likewise yellow fraction are eluted with Rf = 0.24. Tetraacetylresorufin-1-carboxylic acid methyl ester-.beta.-D-galactopyranoside crystallizes from methanol in the form of yellow-orange crystals.

Yield: 1.2g.

¹ H NMR ([D] ₆ -DMSO): δ = 1.95.2.02.2.04 and 2.14 (e, 12H); 3.90 (s, 3H); 4.09 (m, 2H); 4.51 (m, 1H) 5.24 (d, J = 7Hz, 1H), 5.25 (m, 1H), 5.36 (m, 1H), 5.71 (m, 1H), 6.50 (d, J = 2Hz, 1H), 6.83 (dd, J = 10 and 2 Hz, 1 H); 7.20 and 7.24 (each d, J = 2 Hz, 2 H); 7.47 (d, J = IOHz, 1H).

Between the pure products, a mixed fraction can be eluted from which can be obtained by recrystallization from methanol 2.5g crystals of a mixture of the two isomeric compounds.

In an analogous manner, obtained from a-Bromtetraacetylgalactose and

a) 4-Methoxy-resorufin-i-carboxylic acid methyl ester

Methyl tetraacetyl-methoxy-resorufin-1-carboxylate-β-D-galactopyranoside and tetraacetyl-e-methoxy-resorufin-S-carboxylic acid methyl ester-zS-D-galactopyranoside.

b) 8-chloro-resorufin-1-carboxylic acid methyl ester

Tetraacetyl-8-chloro-resoruf-1-carboxylate-β-D-galactopyranoside and tetraacetyl-1'-chloro-resorufin-S-carboxylic acid methyl ester-jS-D-galactopyranoside.

Example 9

Tetraacetylresorufin-4-carboxylic acid morpholid- / 3-D-galactopyranosidundTetraacetγlresorufin-6-carboxylic acid morpholide-ß-D-galactopyranoside

3.26 g (ЮттоІ) of resorufin-4-carboxylic acid morpholide are galactosidated analogously to Example 8. The crude product is chromatographed on 11 silica gel with ethyl acetate / acetone 3j 1 as eluant. This gives 0.9 g of tetraacetylresorufin-6-carboxylic acid morpholide-β-D-galactopyranoside,

TLC (silica gel, flow agent see Example 7)

and 0.4 g of tetraacetylresorufin -carboxylic acid morpholide-β-D-galactopyranoside, TLC (silica gel, same eluant)

and 1.2 g of a mixed fraction of both isomers.

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Example 10 Resorufin-i-carboxylic acid methyl ester β-D-galactopyranoside

1.2 g of tetraacetylresorufin-S-carboxylic acid methyl ester-jS-D-galactopyranoside analogously to Example 2 with sodium methylate / methanol deacetylated. Yield: 0.8g.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

λ, ™ = 464nm (ε = 21,8cm ² , гпоГ ¹ )

 After cleavage with / 3-galactosidase, the anion of resorufin-1-carboxylic acid methyl ester is obtained:

Amax = 572 nm {ε = 65.4 cm ² mol " ¹ ).

¹ H NMR ([D] ₆ -DMSO): δ = 3.20 = 3.80 (m, 6H); 3.91 (s, 3H); 5.09 (d, J = 7.5Hz, 1H); 6.30 (d, J = 2.1 Hz, 1 H); 6.81 (dd, J = 9.8 and 2.1 Hz, 1 H); 7.30 (m, 2H); 7.51 (d, J = 9.8Hz, 1H); OH protons very broad by 5. In an analogous manner obtained by deacetylation of the corresponding tetraacetates

a) Resorufin-i-carboxylic acid methyl ester β-D-galactopyranoside ¹ H NMR ([D] ₆ -DMSO): = 3.4-3.7 (m, 6H); 5.09 (d, J = 7.5Hz, 1H); 6.34 (d, J = 2Hz, 1H); 6.97 (d, J = 2Hz, 1H); 7.08-7.17 (m, 2H); 7.75 (d, J = 10Hz, 1H); OH: very wide, around 5ppm.

b) 4-Methoxy-resorufin-1-carboxylic acid methyl ester / 3-D-galactopyranoside

c) Methyl e-methoxy-resorufin-g-carboxylate-β-D-galactopyranoside

d) S-chloro-resorufin-i-carboxylic acid methyl ester β-D-galactopyranoside

e) 2-Chloro-resorufin-9-carboxylic acid methyl ester - ^ - D-galactopyranoside

f) resorufin-e-carboxylic acid morpholide-jS-D-galactopyranoside Rf (ethyl acetate / isopropanol / water 9: 4: 2): 0.3 After cleavage with / 3-galactosidase:

UV / VIS (0.1M potassium phosphate buffer, pH 7.5): _max = 574.6nm fluorescence emission: _max = 593nm

g) resorufin-4-carboxylic acid morpholide-β-galactopyranoside Rf (ethyl acetate / isopropanol / water 9: 4: 2): 0.3 After cleavage with / 3-galactosidase:

UV / VIS (0.1M potassium phosphate buffer, pH 7.5): _max = 574.6nm fluorescence emission: _max = 593nm

Example 11 Resorufin S-carboxylic acid jS-D-galactopyranoside triethylammonium salt

266 mg of resorufin-g-carboxylic acid methyl ester β-D-galactopyranoside are taken up in 50 ml of water and 20 ml of 1,4-dioxane.

For this purpose, 5 ml of 0.1 N sodium hydroxide solution are added in portions of 0.5 ml, wherein the pH of the solution must not rise above 12.5. The reaction mixture is added to 6 ml of DEAE Sephadex, carbonate form. It is washed with 180ml of water.

Thereafter, the product is eluted with 0.1 M triethylammonium carbonate buffer, pH 7.5. The eluate is evaporated in vacuo.

Thereafter, it is evaporated several times with ethanol. Yield: 110 mg of resorufin-g-carboxylic acid β-D-galactopyranoside triethylammonium salt.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

λ, ™ = 465 η m After cleavage with galactosidase:

Example 12 Resazurin β-D-galactopyranoside

12.6 g of resazurin sodium salt are dissolved in 65 ml of 1 N sodium hydroxide solution and 80 ml of water. To this are added 20.6 g of acetobrom-a-D-galactose and 18.2 g of hexadecyltrimethylammonium bromide in 150 ml of chloroform. The mixture is 3h. heated to reflux. It is then evaporated to dryness, and digested with ethyl acetate. The ethyl acetate solution is evaporated down again and chromatographed on 500 g of silica gel (eluent: methylene chloride / ethyl acetate 3: 1). 2.13 g of tetraacetylresazurin-β-D-galactopyranoside with Rf = 0.5 (HPTLC silica gel, the same flow agent) are obtained.

For deacetylation, 2.13 g of tetraacetyl derivative are dried with 0.1 g of sodium methylate in 100 liters of absolute methanol for 1 hour at room temperature and over calcium chloride in vacuo. Yield: 1.0g.

Rf = 0.62 (HPTLC silica gel, ethyl acetate / isopropanol / water 9: 4: 2).

¹ H NMR ([D] ₆ -DMSO): δ = 3.30-3.90 (m, 6H); 4.54 (br d, J = 4.4Hz, 1H); 4.67 (br, t, J = 4.4Hz, 1H); 5.07 (d, J = 7Hz, 1H); 5.27 (br d, J = 4.4Hz, 1H); 6.15 (d, J = 2Hz, 1H); 6.63 (dd, J = 9.6 and 2Hz, 1H); 7.10 (dd, J = 9 and 2Hz, 1H); 7.2 (m, 2H); 7.96 (d, J = 9.6Hz, 1H); 8.07 (d, J = 9Hz, 1H).

Example 13 e-chloro-resazurin-carboxylic acid

4,3g (ЗОттоІ) 4-Chloro-resorcinol are dissolved in 20ml ethanol. After addition of 2.4 g of potassium hydroxide, it is cooled to 5 ° C. While cooling, 2.8 ml of isopentyl nitrite are added dropwise. Thereafter, stirring is continued overnight at room temperature. The

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Precipitate is filtered off, dissolved in water and acidified. The yellow precipitate is filtered again and dried at 4O ⁰ C in vacuo. Yield: 2.5 g (48%) of 4-chloro-e-nitrosoresorcinol.

R _f : 0.28 (HPTLC, silica gel, eluent: methanol / ethyl acetate 1: 3).

1,83g ^ e-Chloro-nitrosoresorcin, 1.55 g 2,6-dihydroxybenzoic acid, 0.86 g of manganese dioxide and 1.06 ml of sulfuric acid are added to 20 ml of methanol and stirred for 2 hours at 0 ⁰ C and 14 hours at room temperature. A red precipitate is obtained, which is filtered off and dried. Yield: 2.45 g (80%) of e-chloro-resazurin-carboxylic acid.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

In an analogous manner you get out

a) 2-methyl-resorcinol over the 2-methyl-6-nitrosoresorcinol e-methyl-resazurin ^ -carboxylic acid

b) 4-methyl-resorcinol via the 4-methyl-6-nitrosoresorcinol e-methyl-resazurin ^ -carboxylic acid

c) 4-bromo-resorcinol via the 4-bromo-6-nitrosoresorcinol e-bromo-resazurin ^ -carboxylic acid.

Example 14 S-chloro-resorufin-t-carboxylic acid

2 g of e-chloro-resazurin-carboxylic acid (prepared according to Example 10) are dissolved in 20 ml of water and 5 ml of 25% ammonia.

With ice-cooling, zinc powder is added until the color changes to red-violet. The excess zinc is filtered off. After acidification, the precipitated product is filtered off and dried in vacuo at 40 ⁰ C over calcium chloride. Yield: 1.5 g (79%) of e-chloro-resorufin ^ -carboxylic acid.

UV / VIS (0.1 M potassium phosphate buffer, pH 7.5):

λη3χ = 585.5 nm.

In an analogous manner you get out

a) o-methyl-resazurin-carboxylic acid e-methyl-resorufin-carboxylic acid,

b) e-methyl-resazurin ^ -carboxylic acid e-methyl-resorufin ^ -carboxylic acid,

c) e-bromo-resazurin ^ -carboxylic acid e-bromo-resorufin ^ -carboxylic acid.

Example 15 Resorufin-S-carboxylic acid ethyl ester a-D-galactopyranoside

3.9 g of pentaacetylgalactose are mixed with 0.1 g of anhydrous zinc chloride and heated to 125.degree. It is stirred for 20 minutes at 10 Torr. To the melt is added 1 g of resorufin-1-carboxylic acid methyl ester. The mixture is stirred for 1 hour at 41 ⁰ C and then chromatographed in the manner described in Example 7 on silica gel with chloroform / ethyl acetate 2: 1.

Yield: гбтдТеігаасеіуІ-гезогиЯп-Э-сагЬопзаиге-теіЬуІезіег-а-О-даІасЮругапозігі (yellow oil) Rf = 0.22 (HPTLC, silica gel, same solvent).

The deacetylation to resorufin-g-carboxylic acid methyl ester a-D-gaiactopyranoside is carried out analogously to Example 10.

Example 16

Resorufin-S-carboxylic acid O.e-dioxaoctyD-ester-jS-D-galactopyranoside

0.6 g of tetraacetylresorufin-g-carboxylic acid methyl ester β-D-galactopyranoside are mixed with 50 ml of diethylene glycol monoethyl ether and a spatula tip of sodium hydride and stirred for 15 minutes at room temperature. Thereafter, 100 ml of acetone are added. The precipitate is filtered off and dried. Yield: 56 mg Resorufin ^ -carboxylic acid O-dioxaocty O-ester-β-D-galactopyranoside Rf: 0.54 (silica gel HPTLC, eluent: ethyl acetate / isopropanol 9: 4)

Example 17 Resorufin maltoheptaoside

Amylase (E.C. 2.4.1.19), for example amylase from Bacillus macerans, in addition to hydrolytic and cyclizing action, also has glycosyl-transferring properties which can be exploited for the synthesis of oligosaccharides and derivatives (Methods in Carbohydrate Chemistry II, 347).

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680mg Amy cheese from Bacillus macerans DSM

(Lyophilisate, 0.46U / mg sample, 28.5% protein content), 500 mg resorcinol glucoside, 3.5 g cyclodextrin and 70 ml Soerensen phosphate buffer (pSH6.2, 0.01 M) are mixed. The mixture is incubated for 24 hours at 37 ° C. For purification, α- and resulting β- cyclodextrin are then first separated off via the tetrachlorethylene inclusion compound. Chromatography on crosslinked dextran (Sephadex LH 20) gives 50 mg of lyophilizate of highly active resorufinyl maltoheptaoside in the amylase assay.

Example 18 Determination of the activity of / 3-D-galactosidase

a) Preparation of the solutions used:

Buffer solution: HEPES lOOmmol / l sodium chloride 154mmol / l Magnesium L-aspartate 2 mmol / l BSA 10 g / l Tween-20 0.5 g / l pH value (adjusted with sodium hydroxide solution) 7.3 (37 ° C)

Reagent solution 1:

In the buffer solution described above, 0.8 mmol / l of resorufin β-galactopyranoside is dissolved.

Reagent solution 2:

In the buffer solution described above, 3.5 mmol / l of resorufin-S-carboxylic acid β-D-galactopyranoside are dissolved.

Reagent solution 3:

In the buffer solution described above, 1.0 mmol / L of resorufin-S-carboxylic acid methyl ester β-D-galactopyranoside is dissolved.

enzyme solution

Commercially available β-D-galactosidase from Escherichia coli is dissolved in the above-mentioned buffer solution. The activity of this solution is about 0.08 U / ml (based on the manufacturer's instructions).

b) carrying out the measurements

The measurement is photometric at 579nm.

950μΙ reagent are mixed in a 1 cm cuvette at 37 ° C with 50μΙ enzyme solution. As a measure of the reaction, the increase in extinction per unit time in (mExt / min) is determined. It is calculated from the measured absorbance by division with the reaction time.

The following table lists the measured values found:

Reagent No. Reaction

[MExt / min] -

1 85 2 46 3 76

Example 19 Detection of / 3-D-galactosidase using an indicator film

To a homogeneous mass are processed:

5 ml of an aqueous solution with 0.5 M potassium phosphate and 0.05 M magnesium chloride, pH 7.3.

0.13g sodium alginate 8g of a 50% dispersion of polyvinylpropionate

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10g silica gel

12 ml of water

0.4g Triton X-100

50 mg resorufin-β-D-galactopyranoside, dissolved in 5 ml of methanol.

This mass is applied to a 0.1 mm thick polycarbonate film (Pokalon, Lonza) with a gap width of 0.2 mm.

The coating is dried at 50 ° C and then cut into pieces from 6 to 6mm. These are adhered by adhesive tape to a 400 ^ m thick polystyrene film.

The test strips thus obtained are immersed in the test solution containing / 3-D-galactosidase for a short time. After a waiting time of 2 minutes at room temperature, a red reaction color has developed, the intensity of which depends on the concentration of / 3-D-galactosidase in the test solution. With the help of test solutions with a specific, known / 3-D-galactosidase content, a color scale can be obtained by means of which the unknown content of / 3-D-galactosidase in a sample can be determined.

The test strips described above can also be used to kinetically determine the / 3-D-galactosidase activity present in a sample by means of reflection photometry. Also for this purpose, a calibration curve is prepared in the usual way on the basis of samples with known / 3-D-galactosidase activity, with the help of an unknown / 3-D-galactosidase activity of a sample can be determined.

Example 20 Determination of the activity of free and conjugated / 3-D-galactosidase

a) Preparation of the solutions used

Buffer solution: HEPES 100 mmol / l sodium chloride 154mmol / l Magnesium L-aspartate 2 mmol / l BSA 10g / l Tween-20 0.5 g / l pH value (adjusted with sodium hydroxide solution) 7.3 (37 ⁰ C)

reagent:

In the above-described buffer solution, 0.8 mmol / L of resorufin-β-D-galactopyranoside is dissolved.

Enzyme solution:

Commercially available β-D-galactosidase from Escherichia coli is dissolved in buffer. The activity of this solution is about 0.08 U / ml (based on the manufacturer's instructions).

Enzyme conjugate solution:

A beta-D-galactosidase antibody preparation is used. The preparation of such enzyme-antibody conjugates is known. She is in Biochem, for example. Biophys. Acta 612, 40-49 (1980). The preparation is diluted in buffer so as to give an approximately comparable activity to the enzyme solution described above.

b) Performance of the measurement:

The measurement is photometric at 578nm. 950 μΙ reagent solution are each mixed in a 1 cm cuvette at 37 ⁰ C with 50μΙ enzyme solution or with 50μΙ enzyme-conjugate solution. As a measure of the reaction, the increase in extinction per unit time in [mExt / min] is determined.

For the reaction with the free / 3-D-galactosidase 85 mExt / min; measured for the reaction with β-D-galactosidase antibody conjugate 92mExt / min.

Both measurements show that a very well-measurable extinction difference is found with both free and conjugated / 3-D-galactosidase.

It follows that the compounds described are equally suitable as substrates for both free glycosidase and glycosidase conjugates. The new substrates can thus not only be used as diagnostic agents for the determination of free glycosidases. They are also advantageously used in immunological methods of determination in which a glycosidase is used as an indicator enzyme.

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Example 21 Determination of α-amylase activity

Filter paper is impregnated with resorufin maltoheptaoside (prepared according to Example 17) dissolved in citrate buffer, pH 6. The reagent paper thus obtained is sequentially mixed with the a-amylase-containing sample and with a- and / or 3-glucosidase.

After a few minutes, a clearly visible red color is to be detected, whose intensity or amylase concentration in the sample is proportional.

By means of samples with known a-amylase concentration, a calibration curve can be determined, by means of which the unknown a-amylase content of a sample can be determined.

In the above examples, the following abbreviations were used:

HEPES 21- [4- (2-hydroxyethyl) -1-piperazinyl] -ethanesulfonic acid

BSA bovine serum albumin, (bovineserum albumin)

Tween 20 polyoxyethylene (20) sorbitan monolaurate

Claims (3)

1-744 Invention claim: 1. A process for preparing the glycosides of resorufin derivatives of the general formulas Ia and Ib Glycoaid-0 (La) (Ib) O-glycoside in which R ^{1 is} hydrogen, R ² , R ³ and R ⁵ , which may be the same or different, are hydrogen, halogen or a lower alkyl group, R ⁴ and R ⁶ , which may be the same or different, represent hydrogen, halogen, a cyano, lower alkyl, lower alkoxy, carboxy, lower alkoxycarbonyl, carboxy-lower alkyl, lower alkoxycarbonyl-lower alkyl or optionally mono- or di-substituted carboxamido or the group COO- (CH ₂ CH ₂ O) _n -R ⁷ in which R ^{7 is} hydrogen or a lower alkyl group and η is an integer from 1 to 4, wherein R ^{6 may} additionally represent a sulfonyl or nitro group, and Y is nitrogen or the group N -> 0, characterized in that in a conventional manner compounds of the general tautomeric formula Il a and Il b (II a) R " (II b) in which R ¹ to R ⁶ and Y have the abovementioned meaning, with a mono- or oligosaccharide or a 1-halo-derivative thereof, wherein in each case all hydroxy groups are substituted by a protective group customary in the carbohydrate chemistry, converted to per-O-substituted glycosides, from which by deprotection of the protective groups in a conventional manner the glycosides of resorufin derivatives of the general formulas Ia and Ib, respectively. -2- 744 94 2. Method according to item 1, characterized in that glycosides are prepared according to item 1, wherein the glycoside residue is a / 3-D-galactopyranoside, aD-galactopyranoside - / 3-D-glucopyranoside,
aD-glucopyranoside- introduces the aD-mannopyranoside residue. 3. The method according to item 1, characterized in that the glycoside residue presents an oligosaccharide having 2 to 10 mono-saccharide units.