# Patent

## Title
Peptidase substrates

## Abstract

The present invention relates to the use of a compound of the following formula (I) for detecting a peptidase activity and/or a variation in pH:




    
    
        according to which:
        
            Y1 is a peptide, H or an alkyl;
            W1, W2, W3 and W4 are independently H, Br, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl (including the esters or amides thereof) or any combination thereof;
            n=0, 1 or 2;
            X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
            Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
            Z1, Z2, Z3 and Z4 being independently H, Br, Cl, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, including the carboxyl or sulfonyl esters or amides,
        
        
        and salts thereof.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the national stage application under 35 USC 371 of International Application No. PCT/FR2010/051588, filed Jul. 27, 2010, which claims priority to French Patent Application No. 0903759, filed Jul. 30, 2009, the disclosures of which are hereby incorporated by reference.
The present invention relates to novel compounds that can be used as pH indicators and/or as enzyme substrates for the detection of peptidase activity. These substrates can be used in applications comprising an enzymatic hydrolysis step producing a physicochemical signal, in particular in microbiology, biochemistry, immunology, molecular biology, histology, etc. The invention also relates to reaction media containing such substrates, the use of the substrates or of the media for the detection of peptidase activities and/or the differentiation of Gram-positive bacteria with respect to Gram-negative bacteria, and to methods of use.
A very large number of media for the detection of microorganisms currently exists. This detection may be based in particular on the use of particular substrates specific for an enzyme of the microorganism that it is desired to detect. Generally, synthetic substrates of enzymes are formed in such a way that the substrate and the product of its metabolism by the target enzyme have different physicochemical properties, making it possible to distinguish them and to evaluate whether all or part of the substrate has been converted to product by the enzyme. Hydrolase substrates generally consist of a first part specific for the enzymatic activity to be revealed and of a second part which acts as marker, which is generally chromogenic or fluorescent. Thus, in the case of bacteria, through the choice of substrates, depending on whether or not there is a reaction, it is possible to characterize the nature of a microorganism. A peptidase activity can in particular be used to reveal a group, a genus or a species of bacteria. Thus, alanine aminopeptidase activity, for example, makes it possible to differentiate Gram-negative bacteria from Gram-positive bacteria.
Enzymatic chromogenic substrates for the detection of peptidase activity are known from the prior art. Mention may be made in particular of the publication by Manafi (Manafi et al., Microbiol Rev 55(3): 335-348, 1991), which is a review of enzyme substrates used in microbiology. However, the aminopeptidase substrates described release, by hydrolysis, compounds which diffuse in the medium (beta-naphthylamine, 7-amino-4-methylcoumarin). As a result, in a heterogeneous reaction medium (colonies on Petri dishes, histological section, etc.), it is not possible to precisely localize the site of the hydrolysis. Mention may also be made of the substrates described in patent applications WO 98/04735 and WO 99/38995 filed by the applicant. However, although these substrates diffuse little in culture medium, they have certain drawbacks: they are difficult to synthesize, purity is low, yields are low and they are toxic with respect to certain microorganisms.
The present invention therefore proposes the use of novel compounds, either as pH indicators, or as peptidase substrates, which enable the detection of microorganisms. Compared with the existing substrates, these novel compounds are easy to synthesize, and can be used in particular in gelled media for the detection of microorganisms since they produce a coloration which diffuses little or not at all in the reaction medium. In the context of use as an enzyme substrate, this makes it possible to pinpoint a colony or an organelle expressing a peptidase activity among others which do not express it.
Before going any further with the description of the invention, the definitions below are given in order to facilitate the disclosure of the invention.
The term “enzyme substrate” is intended to mean a substrate that can be hydrolyzed by an enzyme into a product that allows the direct or indirect detection of a microorganism, of a cell or of an organelle. This substrate comprises in particular a first part that is specific for the enzymatic activity to be revealed and a second part that acts as a marker.
The compounds according to the invention that are used as substrates are suitable for use in flow cytometry because, since the product of the hydrolysis remains mainly localized in the cell expressing the enzymatic activity, it is possible to specifically count the cells expressing this activity, or even to separate them from the rest of the sample.
The compounds according to the invention that are used as substrates are also very suitable for use in histoenzymology, because, since the product of hydrolysis remains principally localized on the site of the hydrolysis, it is possible specifically to identify the cells or organelles expressing this activity within a tissue.
Owing to their low toxicity, the compounds according to the invention are very suitable, respectively, as pH indicators, or for monitoring peptidase activity in cell culture.
The compounds according to the invention are particularly suitable for use in a detection and/or identification medium since they produce a coloration or a fluorescence which does not diffuse in the reaction medium.
In the present application, the term “coloration” is used to cover a coloration, absorption of light in the visible spectrum, or a fluorescence, and absorption at one wavelength (λex) and emission at a higher wavelength (λem, λem>λex).
The compounds of the invention may be salified, i.e. in the form of a salt, such as chloride, bromide, iodide or trifluoroacetate.
The term “pH indicator” is intended to mean a chemical substance of which the color and/or the fluorescence vary(ies) according to the modifications in pH of the medium, said modifications being optionally linked to the metabolism of the microorganism(s) growing on said medium.
The term “peptidase” is intended to mean an enzyme capable of cleaving, by hydrolysis, the amide group formed between the acyl residue of a peptide and a primary amine. The term “aminopeptidase” is intended to mean an enzyme capable of cleaving, by hydrolysis, the amide group formed between an acyl of an amino acid and a primary amine. In the present application, the term “peptidase” can denote, as appropriate, both a peptidase and an aminopeptidase as defined above.
The term “peptide” is intended to mean a peptide chain comprising from 1 to 10 amino acids, preferentially from 1 to 4 amino acids. Preferentially, the peptide is a di-alanine or tri-alanine.
The term “amino acid” is intended to mean any natural or unnatural amino acid known to those skilled in the art. According to one particular embodiment of the invention, the amino acid is a beta-alanine or L-alanine or D-alanine, or a glycine, pyrroglutamyl, etc.
Said peptide may comprise a blocking agent at its N terminal end. Blocking agents according to the invention comprise any blocking agent known to those skilled in the art which is capable of protecting amines. By way of example, mention may be made of t-butoxycarbonyl (N-tBOC), 9-fluorenyloxycarbonyl, a solubilizing agent such as succinyl, or else a non-metabolizable amino acid, i.e. an unnatural amino acid, such as pipecolic acid or the D form of an amino acid, such as D-phenylalanine. The blocking agents are not systematically present in the compounds of the invention.
The term “alkyl group” is intended to mean a chain of saturated hydrocarbon-based groups, such as, in particular, a C1-C6 alkyl, i.e. a straight or branched alkyl containing from 1 to 6 carbon atoms. By way of example, mention may be made of methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, isopentyl and hexyl.
The term “aryl group” is intended to mean a functional group (or substituent) which derives from an aromatic nucleus such as, in particular, a C6-C10 aromatic nucleus, in particular phenyl, benzyl, 1-naphthyl or 2-naphthyl.
The term “carboxyl group” is intended to mean, in particular, a functional group composed of a carbon atom bonded via a double bond to a first oxygen atom, and via a single bond to a second oxygen atom, which is itself negatively charged or linked to a hydrogen atom. Depending on the pKa of the molecule and on the pH of the medium, the carboxyl group may be in ionized form, i.e. without H bonded to the second oxygen atom, which is then negatively charged.
The term “reaction medium” is intended to mean a medium comprising all the components required for the expression of a metabolism and/or for the growth of microorganisms, of a cell or of an organelle. This reaction medium can be used in flow cytometry, histoenzymology, cell culture, etc., or as a medium for detecting and/or identifying microorganisms.
The reaction medium may comprise one or more components in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, antibiotics, surfactants, buffers, phosphate salts, ammonium salts, sodium salts or metal salts.
The medium may also comprise a dye. By way of indication, mention may be made, as dye, of Evans blue, neutral red, sheep blood, horse blood, an opacifier such as titanium oxide, nitroaniline, malachite green, brilliant green, etc.
The reaction medium may be solid, semisolid or liquid. The term “solid medium” is intended to mean, for example, a gelled medium. Agar is the conventional gelling agent in microbiology for the culturing of microorganisms, but it is possible to use gelatin or agarose. A certain number of preparations are commercially available, for instance Columbia agar, Trypcase-soy agar, MacConkey agar, Sabouraud agar or, more generally, those described in the Handbook of Microbiological Media (CRC Press).
The reaction medium may be a detecting and/or identifying medium, i.e. a revealing medium or a culturing and revealing medium. In the first case the culturing of the microorganisms is carried out before inoculation and, in the second case, the detecting and/or identifying medium also constitutes the culture medium.
The term “biological sample” is intended to mean a clinical sample, derived from a specimen of biological fluid, or a food sample, derived from any type of food, or a cosmetic or pharmaceutical sample derived from any cosmetic or pharmaceutical preparation. This sample may thus be liquid or solid and mention may be made, in a nonlimiting manner, of a clinical sample of blood, plasma, urine, or feces, of nose, throat, skin, wound or cerebrospinal fluid specimens, a food sample from water, from drinks such as milk or a fruit juice; from yogurt, from meat, from eggs, from vegetables, from mayonnaise, from cheese; from fish, etc., a food sample derived from an animal feed, such as, in particular, a sample derived from animal meal. The sample may also be derived from a clinical environment specimen, a livestock specimen or a food, cosmetic or pharmaceutical production specimen. The term “environment specimen” is intended to mean in particular a surface, liquid, raw-material or product specimen. The term “sample” is therefore intended to mean both the specimen in itself (swab, stools, foods, etc.) and colonies of microorganisms resulting from said specimen (for example after isolation on a gelled culture medium) or a medium containing microorganisms resulting from said specimen (for example an enrichment broth inoculated with said specimen).
For the purpose of the present invention, the term “microorganism” covers bacteria, yeasts, molds, and more generally, organisms which in general are single-cell organisms, and invisible to the naked eye, and which can be multiplied or manipulated in the laboratory.
By way of Gram-negative bacteria, mention may be made of the bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia, Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella. 
By way of Gram-positive bacteria, mention may be made of the bacteria of the following genera: Aerococcus, Enterococcus, Streptococcus, Staphylococcus, Bacillus, Lactobacillus, Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Falkamia, Gemella, Pediococcus, Mycobacterium and Corynebacterium. 
By way of yeasts, mention may be made of the yeasts of the following genera: Candida, Cryptococcus, Saccharomyces and Trichosporon. 
Preferentially, the microorganism is Escherichia coli, Serratia marcescens, Enterobacter cloacae or Enterococcus faecalis. 
In this respect, the invention relates to the use of a compound of the following formula (I) for detecting a peptidase activity and/or a variation in pH:




according to which:

    
    
        Y1 is a peptide, H or an alkyl;
        W1, W2, W3 and W4 are independently H, Br, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl (including the esters or amides thereof) or any combination thereof;
        n=0, 1 or 2;
        X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
        Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
        Z1, Z2, Z3 and Z4 being independently H, Br, Cl, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, including the carboxyl or sulfonyl esters or amides,

and salts thereof.

    
    


When said compound of formula (I) is used for detecting only a variation of pH, Y1 is H or an alkyl.
When said compound of formula (I) is used as an enzyme substrate for the detection of a peptidase activity, Y1 is a peptide.
When said compound of formula (I) is used as an enzyme substrate for the detection of a peptidase activity and of a variation in pH, Y1 is a peptide.
According to one preferred embodiment of the invention, Y1 is a peptide, preferentially chosen from glycine, alanine and pyroglutamyl, preferentially glycine.
According to one preferred embodiment of the invention, n=1.
According to one preferred embodiment of the invention, W1, W2, W3 and W4 are independently H.
According to one preferred embodiment of the invention, X is S or CZ5Z6, preferentially CCH3CH3.
According to one preferred embodiment of the invention, Y is N, N+C3H7 or N+CH3.
According to one preferred embodiment of the invention, Z1, Z2, Z3 and Z4 are H.
According to one preferred embodiment of the invention, said compound is chosen from L-alanyl-2-(4′-amidostyryl)benzothiazole TFA, β-alanyl-2-(4′-amidostyryl)benzothiazole TFA, L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride, glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide, pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide and L-alanyl-2-(4′-amidostyryl)-1,3,3-trimethylindolinium dibromide.
According to one preferred embodiment of the invention, said compound is chosen from:

    
    
        β-alanyl-2-(4′-amidostyryl)benzothiazole TFA salt
        L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride
        glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide
        pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide
        L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide.
    
    


According to one preferred embodiment of the invention, said peptide is glycine, n=1; W1, W2, W3 and W4 are independently H; X is NH; Y is N; Z1, Z2, Z3 and Z4 are H.
The invention also relates to a method for the detection in microorganisms of a peptidase activity or of a variation in pH, characterized in that it comprises, or consists of, the following steps:

    
    
        a) providing a detecting and/or identifying medium comprising a compound of the following formula (I):
    
    






according to which:

    
    
        Y1 is a peptide, H or an alkyl;
        W1, W2, W3 and W4 are independently H, Br, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl (including the esters or amides thereof) or any combination thereof;
        n=0, 1 or 2;
        X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
        Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
        Z1, Z2, Z3 and Z4 being independently H, Br, Cl, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, including the carboxyl or sulfonyl esters or amides,

and salts thereof,

        b) inoculating the medium with a biological sample to be tested,
        c) leaving to incubate, and
        d) revealing the presence of at least one peptidase activity or a variation in pH.
    
    


The inoculation of the microorganisms can be carried out by any of the inoculation techniques known to those skilled in the art. An incubation step can be carried out at a temperature for which the enzymatic activity that it is desired to detect is optimal, which those skilled in the art can readily choose according to the enzymatic activity to be detected. Step d) can be carried out by visual examination or by colorimetry or fluorimetry. During step d), it is possible to reveal the presence of the peptidase activity or the variation in pH, alone or in combination with at least one other enzymatic activity.
When said method is a method for detecting only a variation in pH, Y1 is H or an alkyl.
When said method is a method for the detection in microorganisms of a peptidase activity, Y1 is a peptide.
When said method is a method for the detection in microorganisms of a peptidase activity and of a variation in pH, Y1 is a peptide.
According to one preferred embodiment of the invention, Y1 is a peptide, preferentially chosen from glycine, alanine, and pyroglutamyl, preferentially glycine.
According to one preferred embodiment of the invention, n=1.
According to one preferred embodiment of the invention, W1, W2, W3 and W4 are independently H.
According to one preferred embodiment of the invention, X is S or CZ5Z6, preferentially CCH3CH3.
According to one preferred embodiment of the invention, Y is N, N+C3H7 or N+CH3.
According to one preferred embodiment of the invention, Z1, Z2, Z3 and Z4 are H.
According to one preferred embodiment of the invention, said compound is chosen from: L-alanyl-2-(4′-amidostyryl)benzothiazole TFA, β-alanyl-2-(4′-amidostyryl)benzothiazole TFA, L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride, glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide, pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide and L-alanyl-2-(4′-amidostyryl)-1,3,3-trimethylindolinium dibromide.
According to one preferred embodiment of the invention, said compound is chosen from:

    
    
        β-alanyl-2-(4′-amidostyryl)benzothiazole TFA salt
        L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride
        glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide
        pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide
        L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide.
    
    


According to one preferred embodiment of the invention, said peptide is glycine, n=1; W1, W2, W3 and W4 are independently H; X is NH; Y is N; Z1, Z2, Z3 and Z4 are H.
Preferentially, the microorganism is chosen from Escherichia coli, Serratia marcescens, Enterobacter cloacae and Enterococcus faecalis. 
The invention also relates to a method for differentiating bacteria in terms of whether they belong to Gram-positive bacteria or to Gram-negative bacteria, characterized in that it comprises or consists of the following steps:

    
    
        a) providing a detecting and/or identifying medium comprising a compound of the following formula (I):
    
    






according to which:

    
    
        Y1 is a peptide, H or an alkyl;
        W1, W2, W3 and W4 are independently H, Br, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl (including the esters or amides thereof) or any combination thereof;
        n=0, 1 or 2;
        X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
        Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
        Z1, Z2, Z3 and Z4, being independently H, Br, Cl, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, including the carboxyl or sulfonyl esters or amides,

and salts thereof,

        b) inoculating the medium with a biological sample to be tested,
        c) leaving to incubate, and
        d) revealing the presence of at least one peptidase activity.
    
    


As indicated above, the inoculation of the microorganisms can be carried out by any of the inoculation techniques known to those skilled in the art. An incubation step can be carried out at a temperature for which the enzymatic activity that it is desired to detect is optimal, which those skilled in the art can readily choose according to the enzymatic activity to be detected. Step d) can be carried out by visual examination or by colorimetry or fluorimetry. During step d), it is possible to reveal the presence of the peptidase activity, alone or in combination with other enzymatic activities. In certain cases, it may be advantageous to perform step d) in the presence of an acid, such as acetic acid.
When said method is a method for detecting only a sole variation in pH, Y1 is H or an alkyl.
When said method is a method for the detection in microorganisms of a peptidase activity, Y1 is a peptide.
When said method is a method for the detection in microorganisms of a peptidase activity and a variation in pH, Y1 is a peptide.
According to one preferred embodiment of the invention, Y1 is a peptide, preferentially chosen from glycine, alanine and pyroglutamyl, preferentially glycine.
According to one preferred embodiment of the invention, n=1.
According to one preferred embodiment of the invention, W1, W2, W3 and W4 are independently H.
According to one preferred embodiment of the invention, X is S or CZ5Z6, preferentially CCH3CH3.
According to one preferred embodiment of the invention, Y is N, N+C3H7 or N+CH3.
According to one preferred embodiment of the invention, Z1, Z2, Z3 and Z4 are H.
According to one preferred embodiment of the invention, said compound is chosen from L-alanyl-2-(4′-amidostyryl)benzothiazole TFA, β-alanyl-2-(4′-amidostyryl)benzothiazole TFA, L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride, glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide, pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide and L-alanyl-2-(4′-amidostyryl)-1,3,3-trimethylindolinium dibromide.
According to one preferred embodiment of the invention, said compound is chosen from:

    
    
        β-alanyl-2-(4′-amidostyryl)benzothiazole TFA salt
        L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride
        glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide
        pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide
        L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide.
    
    


According to one preferred embodiment of the invention, said peptide is glycine, n=1; W1, W2, W3 and W4 are independently H; X is NH; Y is N; Z1, Z2, Z3 and Z4 are H.
Preferentially, the microorganism is Escherichia coli, Serratia marcescens, Enterobacter cloacae or Enterococcus faecalis. 
The invention finally relates to a medium for detecting and/or identifying microorganisms, comprising a compound of the following formula (I) for detecting a peptidase activity and/or a variation in pH:




according to which:

    
    
        Y1 is a peptide, H or an alkyl;
        W1, W2, W3 and W4 are independently H, Br, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl (including the esters or amides thereof) or any combination thereof;
        n=0, 1 or 2;
        X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
        Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
        Z1, Z2, Z3 and Z4 being independently H, Br, Cl, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, including the carboxyl or sulfonyl esters or amides,

and salts thereof.

    
    


When said medium is a medium for detecting only a variation in pH, Y1 is H or an alkyl.
When said medium is a medium for detecting a peptidase activity, Y1 is a peptide.
When said medium is a medium for detecting a peptidase activity and a variation in pH, Y1 is a peptide.
According to one preferred embodiment of the invention, Y1 is a peptide, preferentially chosen from glycine, alanine and pyroglutamyl, preferentially glycine.
According to one preferred embodiment of the invention, n=1.
According to one preferred embodiment of the invention, W1, W2, W3 and W4 are independently H.
According to one preferred embodiment of the invention, X is S or CZ5Z6, preferentially CCH3CH3.
According to one preferred embodiment of the invention, Y is N, N+C3H7 or N+CH3.
According to one preferred embodiment of the invention, Z1, Z2, Z3 and Z4 are H.
According to one preferred embodiment of the invention, said compound is chosen from: L-alanyl-2-(4′-amidostyryl)benzothiazole TFA, β-alanyl-2-(4′-amidostyryl)benzothiazole TFA, L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride, glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide, pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide and L-alanyl-2-(4′-amidostyryl)-1,3,3-trimethylindolinium dibromide.
According to one preferred embodiment of the invention, said compound is chosen from:

    
    
        β-alanyl-2-(4′-amidostyryl)benzothiazole TFA salt
        L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride
        glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide
        pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide
        L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide.
    
    


According to one preferred embodiment of the invention, said peptide is glycine, n=1; W1, W2, W3 and W4 are independently H; X is NH; Y is N; Z1, Z2, Z3 and Z4 are H.
Preferentially, the microorganism is Escherichia coli, Serratia marcescens, Enterobacter cloacae or Enterococcus faecalis. 
Preferentially, said reaction medium is a medium for detecting and/or identifying microorganisms, said medium comprising at least one molecule used as an enzyme substrate or a pH indicator as defined above.
Preferentially, said compound, enzyme substrate or pH indicator, is at a concentration of between 1 and 1000 mg/l, preferably between 10 and 500 mg/l.
According to one particular embodiment of the invention, said detecting and/or identifying medium according to the invention also comprises at least one other enzyme substrate, specific for an enzymatic activity other than the peptidase activity detected by the molecule according to the invention.
According to another particular embodiment of the invention, said detecting and/or identifying medium according to the invention also comprises at least one substrate specific for an enzymatic activity other than that demonstrated by the variation in pH.
The enzymatic metabolism of the other substrate(s) generates a detectable signal, which is different from the signal generated by the compound according to the invention used as an enzyme substrate or as a pH indicator, for instance different colored or fluorescent products, for enabling the demonstration, such as the detection and/or the identification and/or the quantification, or one or more microorganisms. As other specific substrate, any other substrate conventionally used in the detection of microorganisms may be used. The concentration of the other specific enzyme substrate is generally between 0.01 and 1 g/l. Those skilled in the art will be able readily to determine such a concentration according to the substrate used. By way of indication, it is possible to combine the compounds according to the invention with peptidase, osidase, esterase or reductase enzyme substrates. In particular, it is possible to combine a substrate according to the invention for which the peptide is a β-alanine with an osidase substrate, such as 5-bromo-4-chloro-3-indolyl-β-glucoside, or alizarin-β-galactoside. It is also possible to combine a substrate according to the invention for which the peptide is L-alanine with an esterase substrate, such as 5-bromo-6-chloro-3-indoxyl octanoate or 5-bromo-3-indoxyl phosphate.
According to one particular embodiment of the invention, said detecting and/or identifying medium according to the invention also comprises at least one other enzyme substrate specific for the peptidase activity or specific for the enzymatic activity detected by a compound according to the invention used as a pH indicator. Through the particular choice of substrates, it is therefore possible to identify groups of microorganisms expressing the same enzymatic activity. The concentration of other specific enzyme substrate is generally between 0.01 and 1 g/l. Those skilled in the art will be able to readily determine such a concentration according to the substrate used. In particular, it is possible to combine a substrate according to the invention for which the peptide is an L-alanine with an L-alanine aminopeptidase substrate described in application WO 2006030119, such as L-alanine-pentyl-resorufamine.
According to one particular embodiment of the invention, said detecting and/or identifying medium according to the invention also comprises at least one metabolic indicator, specific for a metabolic activity other than that detected by the compound according to the invention used as a substrate or as a pH indicator.
This metabolic indicator can in particular be a carbon or nitrogen source optionally combined with a reagent which reveals its metabolism. According to one particular embodiment, the carbon or nitrogen source is combined with a pH indicator other than the compound according to the invention used in this respect. According to another particular embodiment, the carbon or nitrogen source is combined with a cation. According to another particular embodiment, the metabolic indicator makes it possible to detect a tryptophanase activity and combines tryptophan and a reagent which makes it possible to detect the production of indole.


EXAMPLE 1
Substrate Synthesis
2-(4′Aminostyryl)benzothiazole was prepared according to the method of Grozinger et al. (K. G. Grozinger, R. J. Sorcek and J. T. Olivier, European Journal of Medicinal Chemistry—Chim. Ther., 1985, 20, 487-491).
The aminoacylation of this compound in order to obtain 4-(4′-aminostyryl)aryl with a protected amino acid was carried out according to the mixed anhydride method. Another example of a typical substrate obtained in a manner similar to that mentioned above:
L-alanine-N-methyl-2-(4′aminostyryl)benzothiazolium dichloride. 1H-NMR: (DMSO) δ 1.48 (3H, d, J=7 Hz, —CH3), 4.15 (1H, m, >CH3—), 4.33 (3H, s, N—CH3), 7.75-8.45 (11H, Ar—H, —CH═, >NH).
EXAMPLE 2
Use of Substrates of Formula I According to the Invention for Detecting a Peptidase Activity
a) Peptidase Substrates
Glycyl-2-(4′-aminostyryl)-N-methyl-benzothiazolinium iodide (Gly-ASBM+) was synthesized as described in example 1.
b) Preparation of the Medium
A weight of 40 mg of substrate was dissolved in 4 ml of dimethyl sulfoxide and added to 400 ml of previously autoclaved Columbia medium. The medium was distributed into Petri dishes 90 mm in diameter, in a proportion of 20 ml per dish.
c) Inoculation and Incubation
Seventeen strains of microorganisms stemming from collections and belonging to various species of bacteria and yeasts are inoculated in spots of approximately 10 000 colony-forming units.
The media are incubated for 24 hours at 37° C., and then the colonies formed are examined visually.
d) Reading of the Results
The results obtained are given in the following table.














Species
Gly-ASBM+












Collection number
Growth
Color














Escherichia coli
2
Pale



NCTC 10 418

orange



Serratia marcescens
2
Orange



NCTC 10 211





Pseudomonas aeruginosa
2
—



NCTC 10 662





Yersinia enterocolitica
2
—



NCTC 11 176





Salmonella typhimurium
2
—



NCTC 74





Enterobacter cloacae
2
Pale



NCTC 11 936

orange



Providencia rettgeri
2
—



NCTC 7 475





Bacillus subtilis
2
—



NCTC 9 372





Enterococcus faecalis
1
Pale



NCTC 775

orange



Enterococcus faecium
1
—



NCTC 7 171





Staphylococcus
1
—



epidermidis





NCTC 11 047





Staphylococcus aureus
2
—



NCTC 6 571





Staphylococcus aureus
1
—



NCTC 11 939





Streptococcus pyogenes
1
—



NCTC 8 306





Listeria monocytogenes
1
—



NCTC 11 994





Candida albicans
1
—



ATCC 90 028





Candida glabrata
0.5
—



NCPF 3 943









e) Conclusion
It is possible to detect peptidase activities of microorganisms by virtue of the fluorescence or of the coloration of the colonies. The substrate according to the invention allows the growth of microorganisms of any type: Gram-negative bacteria, Gram-positive bacteria, yeasts, etc. Since the color does not diffuse in the reaction medium, it is possible to distinguish the cells or colonies expressing the peptidase activity from those not expressing it, and to count said cells or colonies.

## Claims

1. A method of detecting in microorganisms of a peptidase activity, comprising the following steps:
a) having available a detection and/or identification medium comprising a compound of formula (I)




wherein:
Y1 is a peptide, H or an alkyl;
W1, W2, W3 and W4 are independently H, Br, CI, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl, carboxyl esters or carboxyl amides or any combination thereof,
n=0, 1 or 2;
X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
Z1, Z2, Z3 and Z4 being independently H, Br, CI, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, sulfonyl esters or sulfonyl amides or salts thereof;
b) inoculating the medium with a biological sample to be tested,
c) allowing to incubate, and
d) detecting the presence of at least one peptidase activity;
wherein the compound is also an indicator of pH variation.

2. The method of claim 1, wherein Y1 is a peptide.

3. The method of claim 1, wherein n=1.

4. The method of claim 1, wherein W1, W2, W3 and W4 are independently H.

5. The method of claim 1, wherein X is S or CZ5Z6.

6. The method of claim 1, wherein Y is N, N+C3H7 or N+CH N+CH3.

7. The method of claim 1, wherein Z1, Z2, Z3 and Z4 are independently H.

8. The method of claim 1, wherein the said compound of formula (I) is selected from the group consisting of: L-alanyl-2-(4′-amidostyryl)benzothiazole TFA, beta-alanyl-2-(4′-amidostyryl)benzothiazole TFA, L-alanyl-2-(4′-amidostyryl)-N-methylbenzothiazolium chloride, glycyl-2-(4′-aminostyryl)-N-methylbenzothiazolium iodide, pyroglutamyl-2-(4′-amidostyryl)-N-methylbenzothiazolium iodide, L-alanyl-2-(4′-amidostyryl)-N-propylbenzothiazolium iodide and L-alanyl-2-(4′-amidostyryl)-1,3,3-trimethylindolinium dibromide.

9. A method for differentiating bacteria in terms of whether they belong to Gram-positive bacteria or to Gram-negative bacteria, comprising the following steps:
a) having available a detection and/or identification medium comprising a compound of formula (I)




wherein:
Y1 is a peptide, H or an alkyl;
W1, W2, W3 and W4 are independently H, Br, CI, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl, carboxyl esters or carboxyl amides or any combination thereof,
n=0, 1 or 2;
X is NR, CZ5Z6, S or O, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, Z5 and Z6 being an alkyl;
Y is N or N+R, R being alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl;
Z1, Z2, Z3 and Z4 being independently H, Br, CI, F, I, alkyl, aryl, alkoxy, perfluoroalkyl, nitro, cyano, carboxyl, sulfonyl, sulfonyl esters or sulfonyl amides or salts thereof;
b) inoculating the medium with a biological sample to be tested,
c) allowing to incubate, and
d) detecting the presence of at least one peptidase activity.

10. The method of claim 1, wherein Y1 is selected from the group consisting of alanine, glycine and pyroglutamyl.