DD246120A5 - DIAGNOSTIC IN VITRO AGENT FOR THE DETECTION OF GLYCOSIDAL BINDINGS COLUMN ENZYMES - 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

O-glycoside

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 the

R ^{7 is} hydrogen or a lower alkyl group and

η is an integer from 1 to 4, where

R ^{6 may} additionally represent a sulphonyl or nitro group, and

Y nitrogen or the group N-> 0, are used.

2. In vitro diagnostic agent according to item 1, characterized in that are used for the detection of glycosidases such glycosides of resorufin derivatives according to item 1, wherein the glycoside residue is a ß-D-Galactopyranosid-, aD-Galactopyranosid- / 3 D-glucopyranoside, aD-glucopyranoside or aD-mannopyranoside residue.

3. In vitro diagnostic agent according to item 1, characterized in that for the detection of saccharide-chain-splitting enzymes such glycosides of resorufin derivatives according to item 1 are used, wherein the glycoside group introduces an oligosaccharide having 2 to 10 monosaccharide units.

4. In vitro diagnostic agent according to item 1, characterized in that wetting agents, galenic additives and / or scaffold formers are used as additional aids.

Field of application of the invention

The invention relates to diagnostic in vitro agents for the determination of enzymes which cleave glycosidic bonds. The diagnostic agents are used in clinical diagnostics, for example for determining the activity of glycosidases or of saccharide chain-splitting enzymes.

Characteristic of the known technical solutions

Enzymes that cleave glycosidic bonds 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 the glucosidases 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 in Biochem.Z. 333,209 (1960) phenyl / 3-D-galactoside as well as some other aromatic ring-substituted derivatives (eg o-nitrophenyl- and p-nitrophenyl- / 3-D-galactoside) are described as substrates of β-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. Chem. 195, 239 (1952) or naphthol-.beta.-D-galactoside (histochemistry 37, 89). 1973]). To visualize the resulting naphthols are reacted with various diazonium salts to azo dyes.

For 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 devices for cell differentiation (cytofluorometry) and in the analysis of immobilized enzymes with flow microfluorometry. 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 using fluorogenic substrates.

The previously known fluorogenic substrates for / 3-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 to provide diagnostic in vitro agents with which glycosidic linker-cleaving enzymes, such. B. glycosidases or saccharide chain-cleaving enzymes can be determined in a simple, fast and reliable manner. They should as far as possible be used in both photometric and fluorometric determination methods.

Explanation of the essence of the invention

The invention has for its object to find new in vitro diagnostic tools which are suitable for the determination of glycosidases and saccharide-chain-splitting enzymes.

This object is achieved by the diagnostic agents according to the invention, the glycosides of resorufin derivatives of the general formula Ia) and Ib)

Glycoside 0

(La)

JLbI _

0-glycoside

R ¹ hydrogen ~ '~ ~ "

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

-COO- (CH ₂ CH ₂ O) _n -R ⁷ ,

in the

R ^{7 is} hydrogen or a lower alkyl group and

η is an integer from 1 to 4, where ... ,

R ^{e may} additionally represent a sulphonyl or nitro group and ..

Y is a nitrogen or the group N-> 0 include.

Halogen in the definition of R ¹ to R ⁷ is to be understood as meaning fluorine, chlorine, bromine and iodine, preferably chlorine and bromine. The "lower alkyl group" in the definition R ¹ to R ⁷ 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, Where the methoxy group or the methylene radical are particularly preferred. 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 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. As examples of glycosides according to the invention mentioned:

/ 3-D-Galacto-pyranosides, α-D-galactopyranosides, / 3-D-glucopyranosides, α-D-glucopyranosides and α-D-mannopyranosides. Also suitable as the glycosidic residue are oligosaccharide chains which can be degraded by saccharide chain-splitting enzymes down to the level of a mono- or oligo-saccharide, which in turn can be split off directly from the resorufin skeleton with the corresponding glycosides. As such oligo-saccharide chains are understood in particular chains, which are composed of 2 to 10, preferably from 2 to 7 mono-saccharide units.

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 Enzyn.

It has been found that the substrates used in the diagnostic agent according to the invention 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 1CT ¹² nnol / l. Depending on the requirement of phase separation steps, a distinction is made between homogeneous and heterogeneous test procedure. A further subdivision can be made in competitive and non-competitive test principles.

All test principles, however, 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 usually photometrically or else

measured by fluorometry.

An improvement of the glycosidase test system, therefore, also leads to significant 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.

For the preparation of the diagnostic agent in the form of a lyophilizate in the total weight of about 5-20 mg, preferably about 10 mg, a solution is dried, which in addition to all the reagents required for the test usual scaffolding, such as. 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. As sugar alcohols, such as mannitol, sorbitol or xylitol, or other soluble inert compounds, such as polyethylene glycols 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, an absorbent carrier, preferably filter paper, cellulose or synthetic fiber fleece, is provided with solutions of the requisite reagents commonly used to make test strips in volatile solvents, e.g. 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 manner known per se or preferably sealed between plastics and fine-mesh networks in accordance with DBP 2118455.

embodiment

The invention is explained in more detail below with reference to some examples. However, they are not intended to limit the inventive concept.

example 1

Determination of the activity of / 3-D-galactosidase

a) Preparation of the solutions used:

Buffer solution: HEPES 100mmol / l

Sodium chloride 154mmol / l

Magnesium L-aspartate 2mmol / l

BSA 10 g / l

Tween-20, 0.5 g / l

pH value (with sodium hydroxide solution 7.3

set) (37 ⁰ C)

Reagent solution 1:

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

Reagent solution 2:

In the buffer solution described above, 3.5 mmol / l of resorufin-9-carboxylic acid-Jβ-D-galactopyranoside are dissolved

Reagent solution 3:

In the buffer solution described above, 1.0 mmol / l of resorufin-iJ-carboxylic acid methyl ester-β-D-galactopyranoside is dissolved. «K ·

enzyme solution

Commercially available / 3-D-galactosidase from Escherichia coli is dissolved in the buffer solution mentioned above. 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.

950μ, Ι Reagents are mixed in a 1 cm cuvette at 37 ⁰ 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 2

Detection of 0-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 polyvinyl lie to silica gel. ·

12 ml of water 0.4 g Triton X-100

50 mg of 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 6 mm. These are adhered by adhesive tape to a 400 / xm strong 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. For this purpose, too, a calibration curve is usually created on the basis of samples with known / 3-D-galactosidase activity, with the aid of which an unknown / 3-D-galactosidase activity of a sample can be determined.

Example 3

  Determination of the activity of free and conjugated ßO-D-galactosidase

a) Preparation of the solutions used Buffer solution: HEPES lOOmmol / l

Sodium chloride 154mmol / l

Magnesium L-aspartate 2mmol / l

BSA 10 g / l

Tween-20 0.5 g / l

pH value (with sodium hydroxide solution 7.3

set). '(37 ⁰ C)

reagent:

In the buffer solution described above, 0.8 mmol / L of resorufin / 3-D-galactopyranoside is dissolved.

Enzyme solution: Commercially available / 3-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 information)

Enzyme conjugate solution:

A / 3-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 to give an approximately comparable activity to the enzyme solution described above.

b) Performance of the measurement:

The measurement is photometric at 578nm. 950μΙ reagent solution are each mixed in a 1 cm cuvette at 37 ° C mit 50 ^ l enzyme solution or with 50 / xl enzyme-conjugate solution. As a measure of the reaction, the extingtion increase 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 / 3-D-galactosidase antibody conjugate 92 mExt / min. gl ·

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

It follows that the described compounds are equally suitable as substrates for both, free glycosidase and glycosidase conjugates. Thus, the novel substrates can not 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. , ".

Example 4

Determination of α-amylase activity

Filter paper is impregnated with resorufin maltoheptaoside (prepared according to Example 17) dissolved in citrate buffer, pH 6. That so

Reagent paper obtained is added sequentially with the a-amylase-containing sample and with a- and ß-glucosidase.

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

With the aid of samples having a 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 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid.

BSA bovine serum albumin, (bovineserum albumin)

Tween20 polyoxyethylene (20) sorbitan monolaurate

Claims (1)

Invention claim: Diagnostic in vitro agent for the detection of glycosidic bond-cleaving enzymes containing one or more chromogens and / or fluoragenic substrates, a suitable buffer substance, optionally other commonly used excipients and possibly auxiliary enzymes, characterized in that as chromogenic and fluorogenic substrates glycosides of resorufin Derivatives of the general formula Ia) or Ib) Glycoside-O (loaf)