CS273162B2 - Diagnostic agent for glicosidases activity determination - 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

The invention relates to a diagnostic device for determining the activity of glycosidases.

Glycosidases fulfill multiple physiological functions in human and animal organisms. For example, γ-D-galactosidase plays an important role in the carbohydrate metabolism, since it acts to hydrolyze lactose. In addition, β-D-galactoidase is a key enzyme in the degradation of glycolipids, mucopolysaccharides and glycoproteins. Other physiologically important glycosidases include? -D-galactoidase,? -D- and? -D-glucosidase, as well as? -D-manaosidase,

In addition to being physiologically important, glycosidases have gained importance in the field of diagnostics and biotechnology in recent years. Thus, for example, these enzymes are increasingly used as so-called indicator enzymes in enzymatic immunoassays (enzymimmunoassay). Particularly preferred in this context is β-D-galactosidase (see, for example, Annals of Clinical Biochemistry 16, 221-240 (1979)).

Consequently, the determination of glycoaidase activity plays an increasing role in both clinical chemistry and diagnostics. In general, the glycosidase-containing sample is mixed with a suitable substrate. The substrate is cleaved by the enzyme and one of the cleavage products is suitably determined. It can be determined either by the action of the enzyme liberated glycone or aglycone. As a rule, the aglycone is determined.

Natural substrates of proven enzymes are often suitable as substrates. However, it is particularly preferred to use glycosides in which the aglycone is a spectroscopically readily detectable residue.

A number of substrates for glycosidases are known in which, after cleavage by glycosidase, aglycone can be determined spectroscopically in the visible or ultraviolet region of the spectrum or fluorometrically. ·

Thus, as β-D-galactosidase substrates are described in Biochem. Z. 333. 209 (1960) phenyl-β-D-galactoside and some of its other aromatic-substituted derivatives (e.g. o-nitrophenyl and p-nitrophenyl-β-D-galactoside). Phenols released by hydrolysis and e are determined photometrically in the ultraviolet region. or, in the case of nitrophenols, in the short-wave visible wavelength range of the spectrum. An oxidative coupling with aminoantipyrine (Analytical Biochem. 40. 281 (1971)) may also be added as an indicator reaction.

Ero histooherai assays employ naphthyl -? - D-galactoside, for example, the use of the 1-naphthyl compound is described in Histochemistry 35. 199 (1973), the use of the 6-bromo-2-naphthyl derivative in J. Biol. Chem. 195. 239 (1952) and the use of naphthol -? - D-galactoside in Histochemistry 37. 89 (1973). For visualization ae, the resulting naphthols are reacted to azo dyes with various diazonium salts.

Determination of enzyme activity using fluorogenic substrates is widespread, since the sensitivity of fluorometric assays is several decimal orders higher than photometric methods. In many cases, it is necessary to work with fluorogenic substrates, for example in the determination of enzymatic activity in cells by means of automatic devices for cell differentiation (cytofluorometry) and in the analysis of immobilized enzymes by flow microfluorometry. In other cases, for example in determining enzymatic labeling of systems (enzymatic immunoassay), the multiplier effect of enzymatic catalysis using fluorogenic substrates is greatly enhanced.

The prior art fluorogenic substrates for β-D-galactosidase and other glycosidases contain, as fluorophores, derivatives of fluorescein, indoxyl or methylurabelliferone. However, these compounds have serious disadvantages for the kinetic analysis of complex systems. The monosubstituted fluorescein glycosides themselves already fluoresce themselves. Indoxyl derivatives undergo a number of chemical reactions after their enzymatic cleavage, which also complicate the kinetic analysis. Methylumbelliferone derivatives must be excited by ultraviolet radiation. It can interfere with the intrinsic fluorescence of biological or synthetic substances.

In addition, ultraviolet excitation, especially in the case of laser optics, is expensive. Most fluorogenic substrates provide reaction products with only low solubility, so they are not suitable for kinetic analysis of enzyme activity where good solubility of substrate and product is needed.

Therefore, there remains a need for substrates through which glycosidases can be readily, rapidly and reliably determined, and such substrates should, as far as possible, be applicable to both photometric and fluorometric assay methods. The object of the invention was to satisfy the aforementioned demand.

This problem is solved by novel glycosides of resorufin derivatives which are cleaved by glycosidases into a sugar residue and a resorufin derivative. The latter are compounds with good water solubility, which exhibit well-measurable absorption in the visible region of the spectrum and can moreover be easily excited for fluorescence.

The present invention provides a diagnostic composition for determining the activity of glycosidases comprising one or more chromogenic and / or fluorogenic substrates, a suitable buffering agent and optionally other commonly used excipients, characterized in that it contains glycosides of resorufin derivatives of the general formula as chromogenic and fluorogenic substrates. la or Ib

wherein the glycoside is a glycoside residue of a monosaccharide,

R ¹ hydrogen, each of R ² , R ⁴ , and R 6, which are the same or different, is hydrogen, halogen or C 1 -C 5 alkyl, each of R 6 and R 6, which are the same or different is hydrogen; halogen; cyano; C 1 -C 5 alkyl; (C 1 -C 5) alkoxy; carboxy; alkoxycarbonyl, carboxyalkyl or alkoxycarbonylalkyl, the alkyl and alkoxy radicals of the following three groups each having from 1 to 5 carbon atoms; or a carboxamido group, optionally mono- or di-substituted by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonylalkyl radicals, in which the alkyl and alkoxy groups each contain from 1 to 5 carbon atoms and, in the presence of two substituents, may be joined together to form an optionally interrupted ring whether with hydrogen, nitrogen or sieves; or a group of the formula -CO- (CH ₂ -CH ₂ O) _n -R ⁷ , wherein T * is hydrogen or an alkyl group and 1 to 5 carbon atoms and n is an integer from 1 to 4.

wherein in addition R 1 is a sulfonyl or nitro group and

Y Ja nitrogen or group ΪΓ—? Q.

The glycosides of the reactorphin derivatives of the formulas Ia and Ib, respectively, are novel compounds. They can be prepared by reacting the reactorphin derivatives of the general tautomeric formulas IIa and IIb.

(Ha))

(lib) *

wherein R ¹ to R ⁴ and Y are as defined above, reacted with the monosaccharide or its 1-halogeno derivative, all hydroxy groups being always substituted by protecting groups customary in carbohydrate chemistry to form per-O-substituted glycosides from which they bake the glycosides of the reactorphin derivatives of formula I in a manner known per se by cleavage of protecting groups.

The reaction of the compounds of formula II with per-O-substituted 1-halosaccharides is preferably carried out in the presence of acid binders such as an alkali metal hydroxide or carbonate in aqueous acetone or (under interfacial transfer conditions) in water / benzene or water / chloroform.

In addition, this process can be carried out by first converting the reactorphinin derivatives of formula (II) to the alkali salts via an alkali metal hydroxide or alkoxide or the optionally substituted amine to the ammonium salts and reacting the salts with per-O-substituted 1-halosaccharides in the dipolar aprotic solvent such as acetone, dimethylsulfoxide, dichloromethane,

CS. 273162 B2 tetrahydrofuran or dimethylformamide.

In addition, the addition of individual silver salts or mixtures of silver salts (silver oxide, silver carbonate, silver carbonate on Celite, silver triflate, salicylate) has proven useful in the synthesis of per-O-subelluted glycosides from resorphin derivatives of formula II and per-O-substituted 1-halogensaccharides. silver) and / or individual mercury salts or mixtures of mercury salts (mercuric bromide, mercuric cyanide, mercuric acetate, mercuric oxide), optionally using a sizing agent such as calcium chloride, molecular sieve or Drierite, in solvents such as methylene chloride, chloroform, benzene, toluene or dioxane. In the synthesis of free-linked glycosides, the compound of formula II is conveniently melted with a saccharide whose hydroxy groups are substituted with protecting groups, preferably acetyl, in the presence of a lewleic acid such as tin tetrachloride, aluminum chloride and preferably zinc chloride (cf. Chem., Ber., 66, 378-383 (1933) and Methods in Carbohydr. Chem., 2, 345-347 (1967). The temperature is selected in the range from 80 to 150 ° C, preferably in the range from 110 to 130 ° C.

The per-O-substituted glycosides of the resorphin derivatives of formula II thus obtained are also novel compounds.

The cleavage of protecting groups from per-O-substituted glycosides to form glycosides of formula I is carried out by methods common in carbohydrate chemistry (see, for example, Advanoes Carbohydrate Chem. 12, 157 (1975)), for acyl groups, for example, sodium methoxide or barium methoxide or ammonia in methanol,

7

The term halogen in the definition of R to R * means fluorine, chlorine, bromine and iodine, preferably chlorine and bromine.

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

The alkoxy group in the definition of R 1 and R 1 preferably contains 1 to 3 carbon atoms, with methoxy being most preferred,

The alkoxy or alkyl radicals of the alkoxycarbonyl, carboxyalkyl and alkoxycarbonylalkyl radicals in the definition of the substituents R @ 1 to R @ 3 also preferably contain from 1 to 3 carbon atoms, with methoxy or methylene being particularly preferred.

Acetyl, benzoyl, benzyl or trimethylsilyl are particularly suitable as the protective group customary in ohemil carbohydrates.

Alkyl, alkoxyalkyl, carboxyalkyl and alkoxycarbonylalkyl groups which are suitable as carboxamido substituents preferably contain from 1 to 3 carbon atoms.

Any monosaccharide which can be cleaved from the resorphin backbone by the corresponding glycosidase is suitable as the glycosidic residue which is attached to the resorufin derivatives of the formula II to form the glycosides of the formula I. Examples of glycosides which can be used according to the invention are? -D-galactopyranoside,? -D-galactopyranoside,? -D-glucopyranoside,? -D-glucopyranoside and -D-mannopyranoside. The use of glycosides of resorufin derivatives of the formula I as substrates for glycosidases leads to significantly more sensitive test systems as compared to previously known systems. The novel substrates may be used to determine the activity of glycosidases, preferably in both the biochemical and biotechnological as well as in the clinioco-hemi-hi region. They are more sensitive and therefore offer numerous advantages:

(a) Lower enzyme activities may be measured.

(b) Smaller quantities of substances may be used as samples.

c) The activity will be determined in a considerably shorter time.

(d) The use of a small sample and the favorable wavelength of the measurements also reduce the susceptibility of other sample components to interfering with the method.

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

The substrates of the invention have been shown to be suitable for determining glycosidase activity of any origin. The diagnostic compositions of the invention containing substrates of formula (X) react much more sensitively than the prior art test compositions.

The glycoaids of the resorufin derivatives of formula (I) are also suitable for immunoassays in which glycosidases are used as indicator enzymes whose activity after immunological reaction has to be determined. These immunoassay methods using enzymatic indicator reactions are referred to as enzymiwmunoassay (hereinafter enzymatic immunoassays). These methods are used for determining the concentration of proteins, polysaccharides, hormones, drugs e other low molecular weight substances present in konoentraci ranging from IO ^"5 to ^{about 10} moles / 1st Depending on the phase separation requirements, a distinction is made between homogeneous and heterogeneous testing. The further distribution of the tests is based on whether they are conducted on a competitive or non-competitive basis.

However, all test principles work with enzyme-antigen or enzyme-antibody conjugates. The enzymatic indicator reaction is common to all enzymatic immunoassays. Suitable indicator enzymes for this purpose are, for example, glycosidases, in particular β-D-galactosidase. The determination of glycosidases in these enzymatic immunoassays is carried out in a conventional manner by adding a suitable substrate which is enzymatically cleaved and then photometrically or fluorometrically measured in a conventional manner.

Consequently, the improvement of glycosidase assay systems also results in substantial advantages in the following enzymatic immunoassays:

1. Higher sensitivity also makes it possible to achieve a further reduction of the detection limit, furthermore to reduce the reaction time and reduce the amount of the sample, thereby also reducing the interference of the method by other sample components.

2. More favorable wavelengths reduce the susceptibility of the method to insoluble components, such as haze, in certain embodiments of the reaction.

The diagnostic composition comprises, in addition to one or more substrates of Formula I, a suitable buffer system and, optionally, other additives commonly used in diagnostic agents such as wetting agents, stabilizers, etc. The diagnostic composition may be in the form of a solution, lyophilisate, powder mixture, reagent tablet or test strip. in which the substances are applied in an absorbent carrier.

The solution according to the invention preferably contains all the reagents required for the test. Suitable solvents are water or mixtures of water with water-soluble organic solvents, such as methanol, ethanol, acetone or dimethylformamide. For reasons of shelf life, it may be advantageous to separate the reagents necessary for carrying out the test into two or more solutions, which are only mixed together during the test.

For the preparation of a diagnostic composition in the form of a lyophilisate with a total weight of 5 to 20 mg, preferably about 10 mg, a solution is dried which, in addition to all the reagents required for carrying out the test, also contains conventional carriers such as polyvinylpyrrolidone and optionally other carriers such as mannitol. , sorbitol or xylite.

The diagnostic composition in the form of a powder mixture or reagent tablet can be prepared by mixing the components required to carry out the test with conventional galenic ingredients and granulating the mixture. Additives of this type are, for example, sugar alcohols such as mannitol, sorbitol or xylite or other soluble inert compounds such as polyethylene glycols or polyvinylpyrrolidone. Powder mixtures or reagent tablets usually have a final weight of about 30 to about 200 mg, preferably 50 to 80 mg.

In the manufacture of a test strip diagnostic device, the absorbent carrier, preferably filter paper, cellulose or synthetic fiber web, is impregnated with the solution required for the test strip conventionally used reagents in a readily volatile solvent such as water, methanol, ethanol or acetone. The impregnation can be carried out in a single stage, but it is often expedient to perform the impregnation in several stages, using solutions which always contain part of the components of the diagnostic agent. Thus, for example, the impregnation can be carried out in the first step with an aqueous solution containing a buffer and other water-soluble additives and then in the second step impregnated with a solution containing a glycosidase substrate. The finished test papers may be used as such or may be glued in a known manner to the handles or may advantageously be glued between the plastic and the fine mesh as described in DE 2 118 455.

The following examples further illustrate compositions of the invention. The examples do not limit the invention in any way.

Example 1

Determination of β-D-galactosidase activity a) Preparation of used solutions

Buffer solution: HEPES 100 mmol / l sodium chloride 154 mmol / l

Magnesium I-aspartate 2 mmol / L

BSA 10g / 1

Tneen 20 0.5 g / l pH (adjusted with sodium hydroxide 7.3 (37 ° C)

Reagent solution 1: 1

0.8 mmol / l of [beta] -D-galactopyranoside resorufin is dissolved in the buffer described above.

Reagent solution 2:

3.5 mmol / l D-galactopyranoside of resorufin-9-carboxylic acid are dissolved in the buffer described above.

Reagent solution 3:

1.0 mmol / 1-D-galactopyranoside of resorufin-9-carboxylic acid methyl ester is dissolved in the buffer described above.

Enzyme solution:

The commercially available β-D-galactosidase produced using Esoheriohia coli is dissolved in the buffer solution described above. The activity of this solution is about 0.08 U / ml (based on the manufacturer's data).

(b) Performing measurements

The measurement is performed photometrically at 579 nm.

950 µl of reagent solution was mixed with 50 µL of enzyme solution in a 1 cm cuvette at 37 ° C.

The measure of the reaction is the increase in extinction relative to the Sas' unit (mEXT / min). This value is calculated from the measured extension by dividing the reaction time.

The data obtained are shown in the following table:

Reagent solution no

Reaction (mExt / rain)

Example 2

Detection of β-galactosidase by indicator film

Mixture of an aqueous solution containing 0,5 M potassium phosphates and 0,05 M magnesium chloride, pH 7,3,

0.13 g sodium alginate, g 50% polyvinyl propionate dispersion 10 g silica gel, ml water,

0.4 g of Triton X-10 and mg of resorufin- [beta] -D-galactopyranoside dissolved in 5 ml of methanol are mixed together to form a homogeneous mass. This mass is applied to a 0.1 mm thick polycarbonate film (Pokalon, Fa. Lonza) with a slot width of 0.2 mm. The coating is dried at 50 [deg.] C. and the film is cut into 6x6 mm pieces which are glued onto a 400 l polystyrene film using adhesive tape.

The test strips thus obtained are briefly immersed in a test solution containing β-D-galactosidase. After a waiting time of two minutes at room temperature, a red reaction color develops on the strip, the intensity of which is dependent on the concentration of [beta] -D-galactosidase in the test sample. Using test solutions with a predetermined known concentration of β-D-galactosidase, a color scale can be obtained to determine the unknown content of β-D-galactosidase in the test samples.

The above test strips can also be used for kinetic determination of β-D-galactosidase activity in a sample by reflection photometry. In this case too, a calibration curve can be constructed using samples of known β-D-galactosidase activity, by means of which unknown values of β-D-galactosidase activity in the test samples can be determined.

Example 3

Determination of free and conjugated β-D-galactosidase activity a) Preparation of used solutions

Buffer solution: HEPES 100 mmol / l sodium chloride L-aspartic magnesium BSA

Tween-20

154 mmol / l 2 mmol / l g / l

0.5 g / l

PH value (adjusted with sodium hydroxide) 7.3 (37 ° C)

Reagent solution:

0.8 mol / L of resorufin-4-D-galactopyranoside is dissolved in the buffer described above.

Enzyme solution:

Commercially available β-D-galactosidase produced using Esoheriohia ooli is dissolved in the buffer solution described above. The activity of the resulting solution is about 0.08 U / ml (based on the manufacturer's data).

Enzyme Conjugate Solution:

A preparation containing the β-D-galactosidase conjugate and the antibody is used. A method for making such a conjugate is known and is described, for example, in Biochem. Biophys. Acta 612, 40-49 (1980). The preparation is diluted with a buffer solution to a concentration such that it has approximately comparable activity to the enzyme solution described above.

(b) Conducting measurements:

The measurement is performed photometrically at 578 nm. 950 μΙ of reagent solution is mixed with 50 μl of enzyme solution or 50 μιΐ of enzyme conjugate solution at 37 ° C in a 1-cuvette. The measure of reaction is the increase in extinction per unit of time and is expressed in mExt / min.

Reaction with free? -D-galactosidase gives a value of 85 mExt / min and reaction with? -D-galtosidase-antibody conjugate gives a value of 92 mExt / min.

Both measured values show that both free and conjugated A-D-galactosidase produce a very measurable difference in extinction,

Accordingly, the disclosed compounds are suitable as substrates equally well for both free glycosidases and glycosidase conjugates. The novel substrates can therefore be used not only as diagnostic agents for the determination of free glycosidases, but also, very advantageously, for immunological assays in which glycosidases are used as indicator enzymes.

Claims (3)

SUBJECT OF THE INVENTION A diagnostic composition for determining the activity of glycosidases comprising one or more of the halogenated and / or fluorogenic substrates, a suitable buffering agent and optionally other customary excipients, characterized in that the chromogenic and fluorogenic substrates comprise glycosides of resorphin derivatives of the formula Ta or Ib (Ia) ) / A ° S 273162 B2 (Xb) wherein the glycoside is a glycoside residue of a monosaobaride R 6 is hydrogen, each of R 6, R 6 and R 6 which are the same or different, hydrogen, halogen or C 1 -C 5 alkyl, each of R 6 and R 6 which are the same or different; various are hydrogen, halogen, cyano, (C 1 -C 5) alkyl, (C 1 -C 5) alkoxy; carboxy, alkoxycarbonyl, carboxyalkyl or alkoxycarbonylalkyl, in the latter three groups alkyl and alkoxy radicals having from 1 to 5 carbon atoms, or carboxamido, optionally mono- or di-substituted by alkyl, alkoxyalkyl, carboxyalkyl or alkoxycarbonylalkyl, the groups each contain from 1 to 5 carbon atoms, and in the case of the presence of two substituents, they may be joined together to form a ring, which may also be interrupted by oxygen, nitrogen or sulfur, or a group of the formula - COO - (CH ₂ -CH ₂ O) _n - R ⁷ wherein R ⁷ is hydrogen or alkyl of 1 to 5 carbon atoms and n is an integer of 1 to 4, wherein in addition R 6 is sulfonyl or nitro and Y is nitrogen or group Ν '-> 0. 2. The diagnostic composition according to claim 1, wherein the resorufin derivative comprises as a glycoside residue a &lt; RTI ID = 0.0 &gt; -D-galactopyranoside, -D-galactopyranoside, &lt; / RTI &gt; -D-glucopyranoside, 06-D-glucopyranoside . Diagnostic device according to claim 1 or 2, characterized in that it contains wetting agents, galenic additives and / or carcass carriers as additional auxiliaries.