# 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;
        U and V are N, N+R or CZ4, R being H, 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/FR2010/051599, filed Jul. 28, 2010, which claims priority to French Patent Application No. 0903755, 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 “petide” 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 chosen from Escherichia coli, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus faecalis. 
In this respect, the invention relates to the use of a compound of the following formula (I), as an enzyme substrate for the detection of 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;
        U and V are N, N+R or CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl. Preferentially, if U is CZ4, V is N or N+R; if V is CZ4, U is N or N+R;
        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 for the detection of a peptidase activity, Y1 is a peptide.
When said compound of formula (I) is used 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 and alanine, preferentially alanine.
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, U is CZ4, preferentially CH.
According to one preferred embodiment of the invention, V is N+R, preferentially 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: glycyl-4-(4′-amidostyryl)-N-methylpyridinium chloride, L-alanyl-4-(4′-amidostyryl)-N-methylpyridinium TFA, β-alanyl-4-(4′-amidostyryl)-N-methylpyridinium chloride and L-alanyl-4-(4′-amidostyryl)pyridine perchlorate (unquaternized).
According to one preferred embodiment of the invention,

    
    
        said peptine is alanine;
        n=1;
        W1, W2, W3 and W4 are independently H;
        U is CZ4, preferentially CH;
        V is N+R, preferentially N+CH3;
        Z1, Z2, Z3 and Z4 are H.
    
    


The invention also relates to a method for the detection in microorganisms of a peptidase activity and/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;
        U and V are N, N+R or CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl. Preferentially, if U is CZ4, V is N or N+R; if V is CZ4, U is N or N+R;
        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 and alanine, preferentially alanine.
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, U is CZ4, preferentially CH.
According to one preferred embodiment of the invention, V is N+R, preferentially 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: glycyl-4-(4′-amidostyryl)-N-methylpyridinium chloride, L-alanyl-4-(4′-amidostyryl)-N-methylpyridinium TFA, β-alanyl-4-(4′-amidostyryl)-N-methylpyridinium chloride and L-alanyl-4-(4′-amidostyryl)pyridine perchlorate (unquaternized).
According to one preferred embodiment of the invention,

    
    
        said peptine is alanine;
        n=1;
        W1, W2, W3 and W4 are independently H;
        U is CZ4, preferentially CH;
        V is N+R, preferentially N+CH3;
        Z1, Z2, Z3 and Z4 are H.
    
    


Preferentially, the microorganism is chosen from Escherichia coli, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa 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), as an enzyme substrate for the detection of a peptidase activity and/or of 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;
        U and V are N, N+R or CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl. Preferentially, if U is CZ4, V is N or N+R; if V is CZ4, U is N or N+R;
        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 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 and alanine, preferentially alanine.
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, U is CZ4, preferentially CH.
According to one preferred embodiment of the invention, V is N+R, preferentially 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: glycyl-4-(4′-amidostyryl)-N-methylpyridinium chloride, L-alanyl-4-(4′-amidostyryl)-N-methylpyridinium TFA, β-alanyl-4-(4′-amidostyryl)-N-methylpyridinium chloride and L-alanyl-4-(4′-amidostyryl)pyridine perchlorate (unquaternized).
According to one preferred embodiment of the invention,

    
    
        said peptide is alanine;
        n=1;
        W1, W2, W3 and W4 are independently H;
        U is CZ4, preferentially CH;
        V is N+R, preferentially N+CH3;
        Z1, Z2, Z3 and Z4 are H.
    
    


Preferentially, the microorganism is chosen from Escherichia coli, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus faecalis. 
The invention also 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;
        U and V are N, N+R or CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl. Preferentially, if U is CZ4, V is N or N+R; if V is CZ4, U is N or N+R;
        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 sole 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 and alanine, preferentially alanine.
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, U is CZ4, preferentially CH.
According to one preferred embodiment of the invention, V is N+R, preferentially 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: glycyl-4-(4′-amidostyryl)-N-methylpyridinium chloride, L-alanyl-4-(4′-amidostyryl)-N-methylpyridinium TFA, β-alanyl-4-(4′-amidostyryl)-N-methylpyridinium chloride and L-alanyl-4-(4′-amidostyryl)pyridine perchlorate (unquaternized).
According to one preferred embodiment of the invention,

    
    
        said peptine is alanine;
        n=1;
        W1, W2, W3 and W4 are independently H;
        U is CZ4, preferentially CH;
        V is N+R, preferentially N+CH3;
        Z1, Z2, Z3 and Z4 are H.
    
    


Preferentially, the microorganism is chosen from Escherichia coli, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa and 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.
The examples hereinafter are given by way of illustration.


EXAMPLE 1
Substrate Synthesis
4-(4′-Aminostyryl)pyridine was prepared according to the method of Royer (R. Royer, Journal of the Chemical Society, 1947, 560-561).
The aminoacylation of these compounds 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 similar manner to that mentioned above:
dichlorinated glycine-N-methyl-4-(4′-aminostyryl)pyridinium.
1H-NMR: (DMSO) δ 3.83 (2H, s, >CH2), 4.25 (3H, s, NCH), 7.43 (1H, d, J=16 Hz, ═CH—), 7.78 (4H, s, Ar—H), 7.99 (1H, d, J=16 Hz, ═CH—), 8.18 (2H, d, J=5 Hz, Py—H), 8.28 (1H, broad s, >NH), 8.87 (2H, d, J=5 Hz, Py-H).
EXAMPLE 2
Use of Substrates of Formula I According to the Invention for Detecting a Variation in pH
a) pH Indicator
4-4′-Aminostyrylpyridine was synthesized as described in example 1.
b) Preparation of the Medium














Composition
in g/l



















Peptones
6.25



NaCl
5



Lactose
10



Sucrose
10



4,4′-Aminostyrylpyridine
0.05



5-Bromo-4-chloro-3-indoxyl-β-D-glucopyranoside
0.05



Agar
13










The medium hereinafter is autoclaved then distributed into Petri dishes 90 mm in diameter, in a proportion of 20 ml per dish.
c) Inoculation and Incubation
Seven strains of microorganisms originating from the NCTC collection and belonging to various species of bacteria are inoculated onto these media by quadrant streaking of 10 μl of a suspension at 0.5 McFarland diluted to a 1/20. The media are incubated for 24 hours at 37° C., then the colonies formed are examined visually.
d) Reading of the Results
The results obtained are given in table 1.







TABLE 1







Color of the colonies in the presence of the


pH indicator according to the invention











Species
NCTC number
Colony color







Citrobacter freundii
  9750
Yellow



Klebsiella pneumoniae
10 896
Green



Enterobacter cloacae
11 936
Green



Enterobacter aerogenes
10 102
Green



Escherichia coli
12 076
Yellow



Morganella morganii
  235
Colorless



Shigella boydii
  9327
Colorless










The medium according to the invention makes it possible to potentially distinguish 4 groups (3 among the strains tested in this experiment) of microorganisms depending on whether or not they acidify the medium via lactose and/or sucrose metabolism: pH indicator turns yellow, and whether or not they hydrolyze 5-bromo-4-chloro-3-indoxyl-β-D-glucopyranoside: blue coloration of the colonies. The colonies belonging to the species Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter aerogenes which express the 2 activities appear green (mixture of yellow and blue), those of Escherichia coli and Citrobacter freundii which acidify the medium via lactose and/or sucrose metabolism appear yellow and those of Morganella morganii and Shigella boydii which express neither of the two activities appear colorless.
EXAMPLE 3
Use of Substrates of Formula I According to the Invention for Detecting a Peptidase Activity
a) Peptidase Substrate
β-Alanyl-4-(4′-amidostyryl)-N-methyl-pyridinium chloride (β-Ala-ASPM+) was synthesized as described in example 1.
b) Preparation of the Medium
40 mg of each of the substrates were dissolved in 4 ml of dimethyl sulfoxide (80 mg for Asn-ASQM+) and added to 400 ml of previously autoclaved Columbia medium. The 6 media were 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, with or without the addition of acid and under a UV lamp at 365 nm.
d) Reading of the Results
The results obtained are given in table 2.







TABLE 2







Color of the colonies in the presence of peptidase


substrates according to the invention








Species
β-Ala-ASPM+










Collection number
Grow.
Col.
Fluo.





Escherichia coli NCTC 10 418
2
Green
Pale





yellow


Serratia marcescens NCTC 10 211
2
Orange
Pale





yellow


Pseudomonas aeruginosa NCTC 10 662
2
Orange
Pale





yellow


Yersinia enterocolitica NCTC 11 176
2
Pale green


Salmonella typhimurium NCTC 74
1
Pale green


Enterobacter cloacae NCTC 11 936
2
Green


Providencia rettgeri NCTC 7 475
2
Green


Bacillus subtilis NCTC 9 372
2
Pale green


Enterococcus faecalis NCTC 775
2
Pale green


Enterococcus faecium NCTC 7 171
2
Pale green


Staphylococcus epidermidis NCTC 11 047
2
Green


Staphylococcus aureus NCTC 6 571
2
Green


Staphylococcus aureus NCTC 11 939
2
Green


Streptococcus pyogenes NCTC 8 306
1
Pale green


Listeria monocytogenes NCTC 11 994
2
Pale green


Candida albicans ATCC 90 028
2
Pale green


Candida glabrata NCPF 3 943
1
Pale green





Grow.: growth, Fluo.: fluorescence of the colonies, Col.: colour of the colonies


NG: No growth, 0.5: weak growth, 1: moderate growth, 2: good growth, —: no color/fluorescence






e) Conclusion
On the media according to the invention, 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.
By virtue of the various structural variations in the peptidase substrates according to the invention, it is possible to distinguish various groups of microorganisms. Furthermore, 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, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl, carboxyl esters or carboxyl amides or any combination thereof,
n=0, 1 or 2;
U and V are N, N+R, CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, wherein if U is CZ4, then V is N or N+R; or if V is CZ4, then U is N or N+R;
Z1, Z2, Z3 and Z4 being independently H, Br, Cl, 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 U is CZ4.

6. The method of claim 1, wherein V is N+R.

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 glycyl-4-(4′-amidostyryl)-N-methylpyridinium chloride, L-alanyl-4-(4′-amidostyryl)-N-methylpyridinium TFA, beta-alanyl-4-(4′-amidostyryl)-N-methylpyridinium chloride and L-alanyl-4-(4′-amidostyryl)pyridine perchlorate (unquaternized).

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, Cl, F, I, alkyl, alkoxy, thiomethyl, perfluoroalkyl, nitro, cyano, carboxyl, carboxyl esters or carboxyl amides or any combination thereof,
n=0, 1 or 2;
U and V are N, N+R, CZ4, R being H, alkyl, aralkyl, aryl, alkanoyl or alkylsulfonyl, wherein if U is CZ4, then V is N or N+R; or if V is CZ4, then U is N or N+R;
Z1, Z2, Z3 and Z4 being independently H, Br, Cl, 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 and glycine.