WO2009080631A2 - Chemo-enzymatic synthesis of a c-terminal aryl amide of an amino acid or peptide - Google Patents

Abstract

The present invention relates to a method for preparing an aryl amide, comprising reacting an aryl amine with a compound selected from N-protected amino acids and optionally N-protected peptides in the presence of a hydrolytic enzyme. The invention further relates to the use of a compound obtained in a method according to the invention in the manufacture of a diagnostic. In addition the invention relates to the use of a compound obtained in a method according to the invention as a substrate for a proteolytic enzyme.

Description

CHEMO-ENZYMATIC SYNTHESIS OF A C-TERMINAL ARYL AMIDE OF AN AMINO ACID OR PEPTIDE

The invention relates to a method for preparing an aryl amide, comprising reacting an aryl amine with a compound selected from N-protected amino acids and optionally N-protected peptides.

C-terminal aryl amides of an amino acid or peptide may for instance be used in enzymology or in clinical diagnostics as substrates of proteolytic enzymes, see Hemker, H. C, ed. (1983) Handbook of Synthetic Substrates for the Coagulation and Fibrinolytic Systems, Martinus Nijhoff Publishers, Boston. For example, such aryl amide may be used as a substrate in a test to determine the activity of a serine protease in blood plasma, e.g. as part of a blood coagulation test.

Methods to prepare such aryl amides via a chemical pathway have been known for a long time but are generally laborious and may suffer from other drawbacks, such as low yield and/or a high degree of racemisation.

For instance, in Analytical Biochemistry 70, 258-262 (1976), Zimmerman et al. describe a method to prepare a fluorogenic aryl amide, as a substrate for chymotrypsin, by reacting N-protected phenyl alanine with dicyclohexylcarbodiimide and 7-amino-4-methylcoumarin in methylene chloride. The yield was only 44 %. In Houben-Weil, Synthesis of Peptides and Peptidomimetics, Part E22a,

4^th Edition, 2004, Thieme, Stuttgart-New York, p 586, the synthesis is described of C- terminal N-protected amino acid aryl amides by a reaction of N-protected amino acids with aryl amines using di-terf-butyldicarbonate and 4-N,N-dimethyl-4-aminopyridine in aprotic solvents or by pre-formation of the mixed anhydride/symmetrical anhydride mixture followed by reaction with an aryl amine. As possible side products urea derivatives are formed. This procedure allows the synthesis of various N-protected amino acid C-terminal aryl amides in yields ranging from 60-90 %. However, the aryl amidation is accompanied by significant racemisation on the α-C atom of the amino acid residue. Such racemisation can only be partly suppressed by the addition of copper(ll)chloride. In other known methods of synthesising C-terminal aryl amides of

N-protected amino acids based on the use of various condensing agents, such as phosphorus oxychloride (e.g. described by Kato et al. Experientia 1978, 34, 319-320), and HMPA/isocyanate (e.g. described by MacKenzie et al. Biochem. J. 1985, 226, 601-606) racemisation is a problem.

Shui-Tein Chen et al. (J. Org. Chem., 1992, 57, 6960-6965) describe a process wherein a C-terminal ester of an amino acid is coupled with an amino compound, e.g. benzyl amine (which is actually not an aryl amine, see also below for a definition of aryl amines), making use of Alcalase as a catalyst in an alcohol comprising up to 5 % water. In one example, an aryl amine (P-NH₂-C₆H₄-NO₂) is coupled to a C-terminal amino acid ester. The yield is low (25 %, after 12 hrs). It is not disclosed to couple an aryl amine to a C-terminal carboxylic acid group of an amino acid or peptide, which C-terminal carboxylic acid group has not been esterified. It would be desirable to provide such a method, for instance because this would allow direct coupling of an aryl amine to a C-terminal carboxylic acid group of an amino acid or peptide, without first having to esterify the C-terminal carboxylic acid group.

An unsuccessful attempt to achieve this has been described in the art, see Y. Kato et al. (Tetrahedron VoI 45, No 18, pp 5743-5754, 1989). An enzymatic aminolysis reaction is described for amines other than aryl amines (3-pentyl amine, neopentyl amine, benzylamine, n-butyl amine), using D-aminopeptidase from a bacterium. In the first paragraph of page 5748 it is stated that aniline (an aryl amine) did not serve as a substrate. The inventors surprisingly found that the amide bond in a reaction between an aryl amine and a compound selected from amino acids and peptides, can be formed by performing the reaction in the presence of a specific enzyme under specific conditions.

Accordingly, in a first aspect the invention is directed to a method for preparing an aryl amide, comprising reacting an aryl amine with a compound selected from N-protected amino acids and optionally N-protected peptides in the presence of a hydrolytic enzyme in a phase comprising less than 2 wt. % water.

The term "substrate" is used herein for a compound with which the aryl amine is reacted, unless specified otherwise. When referred herein to an acid molecule (e.g. a carboxylic acid, a sulphonic acid, an amino acid), this term is meant to include both ionised and non-ionised forms of the molecule, and salts of the acid, unless specified otherwise. Thus, when referred to a carboxylic acid, this is meant to include the protonated carboxylic acid and its conjugated base (the carboxylate), including salts thereof.

In accordance with the invention racemisation on the C-terminal α- carbon of the substrate may be avoided or at least reduced in comparison with the chemical methods mentioned hereinabove.

The invention has the further advantage that the coupling reaction between the aryl amine and the substrate may occur in a stereoselective way. For instance, if the hydrolytic enzyme is a peptidase the reaction usually is L-selective, which means that the aryl amine is more rapidly coupled to the L-enantiomer or the terminal L-diastereomer of the substrate compared to the D-congeners. Hence, a method according to the invention may result in the formation of a stereo-enriched product, compared to the substrate.

In addition, the method of the invention allows forming the amide bond without having to protect reactive side groups of the amino acid or peptide moiety with which the amine is reacted. Such reactive side groups which may remain unprotected are in particular side groups selected from thiol groups, hydroxyl groups, primary amine groups, carboxyl groups, carboxyamide groups, guanidino groups, imidazole groups, indole groups and thioether groups.

It is in particular surprising that the amidation of the substrate with an aryl amine can be accomplished in a high yield and at a relatively high rate, because aryl amines are known to be considerably less reactive in an amidation reaction than alkyl amines. Aryl amines typically are less nucleophilic (the lone pair on the nitrogen being delocalised, as a result of resonance into the aromatic structure). For instance, at least in a number of embodiments, it is possible to obtain the aryl amide in a yield of 90 % or more within 24 hours, or within 16 hours. A method of the invention can advantageously be carried out without removing the aryl amide that has formed from the phase wherein the reaction is carried out before the reaction of amino acid or peptide with aryl amine to aryl amide is completed or stopped. It is surprising that a free carboxylic acid group (or corresponding anion thereof) of a substrate can be amidated to obtain the corresponding amide in a high yield without having to remove formed amide (e.g. by precipitation) during the amidation reaction, despite the fact that the amidation reaction of such a substrate with an aryl amine is thermodynamically highly unfavourable. - A -

Within the context of the present invention the term "organic moiety" is in particular meant to include linear or branched, optionally substituted alkyl, alkenyl or alkynyl groups; optionally substituted cycloalkyl groups which optionally have one or more unsaturated, exocyclic or endocyclic, carbon carbon bonds; optionally substituted aryl groups; and optionally substituted aralkyl groups. Alkyl groups may in particular comprise 1-30 carbons, more in particular 1-12 carbons. Alkenyl or alkynyl groups may in particular comprise 2-30 carbons, more in particular 2-12 carbons. Cycloalkyl groups or aryl groups may in particular comprise 3-50 carbons, more in particular 3-30 carbons, even more in particular 3-18 or 3-12 carbons. Aralkyl groups may in particular comprise 4-51 carbons, more in particular 4-31 carbons, even more in particular 5-19 or 5-13 carbons. An alkyl, alkenyl or alkynyl group optionally comprises one or more heteroatoms in a chain thereof. A cycloalkyl, aryl or aralkyl group optionally comprises one or more heteroatoms in a ring thereof. A heteroatom may in particular be selected from the group of S, N, O, P, Si and halogens, more in particular from S, O and N. If substituted, an organic moiety may in particular be substituted with optionally protected, functional groups comprising one or more heteroatoms, which heteroatoms may in particular be selected from the group of halogens, O, S and N. Examples of substituents include halogens, phosphate groups, sulphates, nitrate groups, hydroxygroups, thiogroups and amine groups. In particular hydroxygroups, thiogroups and amine groups may be unprotected or protected. The term "aryl amine" is used herein to refer to a compound comprising an amine group, which is directly bound to an aromatic ring of an aromatic moiety. The term "aryl amide" is used herein to refer to a compound comprising an amide group, wherein the nitrogen of the amide group is directly bound to an aromatic ring of an aromatic moiety. Thus, an aryl amine may be represented by the formula Ar-NR⁰H, wherin Ar stands for an aromatic ring and R^Q is selected from the group of hydrogen and organic moieties. Accordingly, an aryl amide obtained in accordance with the invention may in general be represented by the formula Ar-NR^Q-(C=O)-R^z, wherein R^z represents the amino acid residue or peptide chain coupled to the aryl amine.

The term "aromatic" as used in this application is meant to refer to a conjugated ring system of unsaturated bonds, lone pairs, or empty orbitals, which exhibit a stabilisation stronger than would be expected by the stabilisation of conjugation (alternate double and single bonds) alone. An aromatic has a delocalised conjugated π-system, a coplanar structure, contributing atoms arranged in one or more rings, and typically 4n+2 delocalised π-electrons (wherein n is a non-negative integer). Examples of aromatics include acridine, anthracene, benzene, benzimidazole, benzofuran, benzopyrazole, benzopyridine, benzothiophene, benzoxazole, carbazole, dibenzofuran, dibenzothiophene, furan, imidazole, isoxadiazole, isoxazole, naphthalene, oxadiazole, oxazole, phenanthrene, phenanthridine, phenanthroline, phenazine, phenothiazine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrole, quinazoline, quinoline, quinoxaline, thiadiazole, thiophene, and xanthen.

The aryl amine can for instance comprise an optionally substituted ring system with at least 2, at least 3, at least 4, at least 5 or at least 6 carbon atoms. One or more heteroatoms, may be present, in particular one or more heteroatoms selected from N, S and O, provided that the aromatic character is maintained. The upper limit for the number of atoms forming the ring system is in principle not critical, as long as the ring system does not hinder the reaction to an unacceptable extent. In particular the number of atoms forming the ring system may be 20 or less, 14 or less, or 12 or less, e.g. 6.

Suitable substituents in particular include substituents selected from the group of F, Cl, Br, I, amino groups, nitro groups, carbonyl groups, imine groups, carboxylic acid groups, sulphonic acid groups and organic moieties. The organic moiety may in particular be selected from the group of optionally substituted Ci_-6 alkyl, optionally substituted C_2-6 alkenyl, optionally substituted C_2-6 alkynyl, optionally substituted C_1-6 alkoxy, optionally substituted C_1-6 alkylthio, optionally substituted C_3-6 cycloalkyl, optionally substituted OC_3-6 cycloalkyl, optionally substituted OCH₂-phenyl, CF₃, OCF₃, SCF₃, OH, NO₂, CN, phenyl, and NR^xR^y . Herein, R^x and R^y are organic moieties which may in particular independently be selected from the group of H, optionally substituted C_1-4 alkyl, optionally substituted cycloalkyl, phenyl, benzyl and phenetyl.

Preferably, the aryl amine is a compound of formula (I) below.

Herein each of R¹, R², R³, R⁴, R⁵ may in particular each independently be chosen from the substitutions mentioned above. It is also possible that one or more of these R-groups, e.g. R₁ and R₂, R₂ and R₃, R₃ and R₄, or R₄ and R₅, respectively, are taken together to form one or more optionally substituted ring structures (including fused rings), optionally comprising one or more heteroatoms. The one or more ring structures can for instance comprise a single 5-, 6-, or 7 membered ring. It is also possible that one or more ring structures comprise one or more fused rings. Said ring structure preferably is an aromatic ring structure, in particular because an aromatic ring structure may contribute to a desirable spectroscopic property, such as increased chromophoric properties or increased fluorescence.

R⁶ preferably is hydrogen, an unsubstituted alkyl group or a substituted alkyl group Said alkyl groups may in particular comprise 1-6 carbon atoms. In a particularly preferred embodiment, R⁶ is selected from hydrogen and methyl. Particularly good results have been achieved with a compound wherein R⁶ represents a hydrogen atom.

The aryl amine can be neutral, cationic, anionic, or zwitterionic. It is preferred that the aryl amine has at least three conjugated π-bonds, such as a π-electron system of a benzene ring. A large conjugated system may be advantageous for providing a moiety that can be detected at a low concentration with a spectroscopic method such as fluorescence spectroscopy or UV-VIS absorption spectroscopy, which may be advantageous for a diagnostic application.

The aryl amine and/or the aryl amide formed in a method of the invention may be an organic dye molecule, such as a chromophore or a fluorophore. Suitable organic dye molecules include rhodamines, comprising an amino group, and coumarins, comprising an amino group.

The term coumarins refers to compounds of which the aromatic system corresponds to that of coumarin:

wherein at least one of the carbons in the ring, preferably the carbon in the 7-position comprises an amino group substituent and optionally one or more of the other carbons in the ring structure comprise a substituent. Suitable substituents include halogens, in particular F or Cl, and alkyls, which alkyls may be fully or partially halogenated. In particular a substituent may be present selected from methyl, fully or partially halogenated methyl, ethyl and fully or partially halogenated ethyl.

Some suitable examples of aryl amines are given in Table 1 below. The shown counter ion (Q^") may in principle be any ion, for instance Q^" can be selected from Br^", Cl^" and CIO₄ ^". Further suitable aryl amines can for example be found in R. P. Haugland The Handbook: A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen, 10^th edition, 2005.

Table 1

7-amino-4-methylcarbostyryl H₂N -JO

CH₃

7-amino-4-methylcoumarin

7-amino-4-trifluoromethylcoumarin .O

CF₃

7-amino-4-chloromethylcoumaιϊn

acridine-3,6-diamine

o-(6-amino-3-imino-3/-/-xanthen-9-yl)- benzoic acid

(Rhodamine 1 10)

5,9-diaminobenzo[a]phenoxazonium

(Cresyl violet)

Other suitable aryl amines include aniline, p-nitroaniline, 2-naphthylamine, 4-phenylazoaniline, and 6-quinolylamine.

The peptide can be an oligopeptide or a polypeptide. The term "oligopeptide" as used in this application is meant to refer to a peptide based on 2-200 amino acids, in particular based on 2-100, more in particular based on 2-50 amino acids, preferably any linear chain of 2-200 amino acids, more preferably of 2-100, 2-50, 2-30 or 2-10 amino acids. The term polypeptide is in particular used for peptides based on more amino acids than an oligopeptide, as defined herein. N-terminal protection of the amino acid or peptide during the amidation is preferred in order to avoid the undesirable intermolecular or intramolecular side reaction between the unprotected N-terminal NH₂ group of the amino acid or peptide and a C-terminal carboxylic moiety. By protecting the N-terminus of the amino acid or peptide this possible side reaction is avoided and the amino acid or peptide will mainly or only react with the aryl amine.

As used herein, the term amino acid includes proteinogenic and non- proteinogenic amino acids. Likewise, as used herein, the term peptides includes peptides composed of proteinogenic amino acids, non-proteinogenic amino acids and combinations thereof. Proteinogenic amino acids are the amino acids that may be found in proteins and that are coded for by the standard genetic code. The proteinogenic amino acids are glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L-threonine, L-methionine, L-cysteine, L-asparagine, L-glutamine, L-tyrosine, L-tryptophan, L-aspartic acid, L- glutamic acid, L-histidine, L-lysine, L-arginine, L-proline and L-phenylalanine.

Examples of non-proteinogenic amino acids include D-enantiomers of proteinogenic amino acids, D-phenylglycine, L-phenylglycine, D-4-fluoro-phenylalanine and L-4-fluoro-phenylalanine. The substrate with which the aryl amine is reacted may in particular be represented by a compound of formula (II).

Herein P represents H or an N-terminal protecting group. Preferably, P represents an N-terminal protecting group. In particular, in case n is 1 , the N-protective group is generally needed to allow the amidation to proceed well.

In principle, the integer n can have any value of 1 or more. In particular, n can be at least 2, at least 5, at least 10, at least 15, or at least 20. The integer n can be 200 or less, 100 or less, 75 or less, 60 or less, 30 or less, 20 or less, or 10 or less.

Each R^A and each R^B independently represent H, or an amino acid side chain. Thus, it is not required that R^A is the same in all n amino acid units. Similarly, it is not required that R^B is the same in all n amino acid units.

X represents a hydrogen or a cation - preferably a monovalent cation, such as Na⁺ or K⁺.

Suitable N-protecting groups are those N-protecting groups which can be used for the synthesis of (oligo)peptides. Such groups are known to the person skilled in the art. Examples of suitable N-protecting groups include carbonyl type protective groups, for instance Z (benzyloxycarbonyl), Boc (f-butyloxycarbonyl), For (formyl) and PhAc (phenacetyl). The groups For or PhAc may be introduced and cleaved enzymatically using the enzymes Peptide Deformylase and PenG acylase, respectively. Chemical cleavage methods are generally known in the art.

In the context of the invention with 'amino acid side chain' is meant any proteinogenic or non-proteinogenic amino acid side chain. The reactive groups in the amino acid side chains may be protected by amino acid side chain protecting groups or may be unprotected. In a preferred embodiment, the substrate with which the aryl amine is reacted, contains a C-terminal arginine residue of which the side chain is unprotected.

The term "hydrolytic enzyme" as used in this application is meant to refer to enzymes from the classification group E. C. 3. Such enzymes may also popularly be referred to as hydrolases. In principle any hydrolytic enzyme capable of catalysing the amidation reaction can be used.

The hydrolytic enzyme may be used in any form. For example, the hydrolytic enzyme may be used in the form of a dispersion, an emulsion, a solution or in an immobilized form (for instance loaded on a support, e.g. a particulate or monolithic carrier material) - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilised and/or immobilised) cells that naturally or through genetic modification possess hydrolytic activity, or in a lysate of cells with such activity. The hydrolytic enzyme may be obtained or derived from any organism, in particular from an animal, plant, bacterium, a mould, a yeast or fungus.

It will be clear to the person skilled in the art that use can also be made of mutants of naturally occurring (wild-type) enzymes with hydrolytic activity in the process according to the invention. Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild-type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild-type enzyme or so that it encodes an enzyme that is shorter or longer compared to the wild-type and by effecting the expression of the thus modified DNA in a suitable (host) cell. Mutants of the hydrolytic enzyme may have improved properties, for instance with respect to one or more of the following aspects: selectivity towards the substrate, activity, stability, solvent resistance, pH profile, temperature profile, and substrate profile.

When referred to an enzyme from a particular source, recombinant enzymes originating from a first organism, but actually produced in a (genetically modified) second organism, are specifically meant to be included as enzymes from that first organism. Examples of organisms from which the enzyme may be derived include Trichoderma sp., such as from Trichoderma reeseϊ, Rhizopus sp., such as from Rhizopus oryzae; Bacillus sp., such as from Baccillus licheniformis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus alkalophilus, or Bacillus halodurans; Aspergillus sp., such as from Aspergillus oryzae or Aspergillus niger, Streptomyces sp., such as from Streptomyces caespitosus or Streptomyces griseus; Candida sp.; fungi; Humicola sp.; Rhizoctonia sp.; Cytophagia; Mucor sp.; Carica, in particular Carica papaya; and animal tissue, in particular from pancreas, such as from porcine pancreas, bovine pancreas or sheep pancreas. Preferably, one or more hydrolytic enzymes are used selected from the group of carboxylic ester hydrolases (E. C. 3.1.1 ), thiolester hydrolases (E. C. 3.1.2) and peptidases (E. C. 3.4).

In particular a peptidase (E. C. 3.4) may be used. Preferred peptidases are peptidases selected from the group of serine-type carboxypeptidases (E. C. 3.4.16), metallocarboxypeptidases (E. C. 3.4.17), cysteine-type carboxypeptidases (E. C. 3.4.18), serine endopeptidases (E. C. 3.4.21 ), cysteine endopeptidases (E. C. 3.4.22), aspartic endopeptidases (E. C. 3.4.23) and metalloendopeptidases (E. C. 3.4.24).

Particularly preferred is a hydrolytic enzyme selected from the group of serine endopeptidases (E. C. 3.4.21 ) and cysteine endopeptidases (E. C. 3.4.22). Of the cysteine endopeptidases papain is particularly preferred

(E.C. 3.4.22.2).

Of the carboxylic ester hydrolases, carboxyl esterases (E.C. 3.1.1.1 ) and lipases (E.C. 3.1.1.3) are preferred. The lipase may in particular be from Candida sp., more in particular from Candida Antarctica. In particular, good results have been achieved with Candida Antarctica lipase B, e.g. obtainable under the trademark Novozym 435 (from Novozymes).

In particular, good results have been achieved with a subtilisine (E.C. 3.4.21.62). Various subtilisins are known in the art, see e.g. US-A-5 316 935 and the references cited therein. For instance, the subtilisin may be selected from among the group consisting of mature, Class I subtilisins. In particular, the modification may at least comprise an amino acid substitution at amino acid sequence position 181 of said Class I subtilisin enzyme which corresponds substantially to position 181 of the mature subtilisin BPN', said substitution consisting of N181S, as described in US-A-5 316 935.

Particularly preferred is subtilisine Carlsberg.

It has surprisingly been found that the reaction can efficiently be carried out by using Alcalase^®, available from Novozymes (Bagsvaerd, Denmark). Alcalase^® is a cheap and industrially available proteolytic enzyme mixture produced by Bacillus licheniformis (containing subtilisine Carlsberg as a major enzyme component).

Commercially available enzyme, such as Alcalase^®, may be provided by the supplier as a liquid, in particular an aqueous liquid. Other suitable hydrolases may be selected from the group of the following commercially available products, and functional analogues of such enzymes.

Novozymes (Bagsvaerd, Denmark) offers ovozyme, liquanase, Alcalase^® , Alcalase-ultra^® (in particular effective at alkaline pH), duramyl, esperase, kannase, savinase, savinase ultra, termamyl, termamyl ultra, novobate, polarzyme, neutrase, novoline, pyrase, novocor (bacterial alkaline proteases).

Proteinase-K is available from New England Biolabs (Ipswich (MA), USA).

Novo Nordisk Biochem North America lnc (Franklinton NC, USA) offers Protease Bacillus species (Esperase 6.0 T; Savinase 6.0 T), Protease Bacillus subtilis (Neutrase 1.5 MG), Protease Bacillus licheniformis (Alcalase 3.0 T).

Amano International Enzyme Co (Troy, Va, USA) offers Protease Bacillus subtilis (Proleather; Protease N) and Protease Aspergillus oryzae (Prozyme 6).

In case the enzyme is provided as a liquid, the enzyme is preferably first isolated from undesired liquid. For instance a high concentration of water may be undesired,. This may suitably be accomplished by precipitating and/or drying. Precipitation may be accomplished using an alcohol, such as terf-butanol or another organic liquid, e.g. an ether which may be used as reaction medium wherein the amidation can be carried out. The precipitate may subsequently by isolated from the liquid, e.g., by filtration or decantation. The enzyme may thereafter by resuspended in a liquid, for instance a tertiary alcohol, such as terf-butanol or another tertiary alcohol or an ether, for instance methyl tert-butylether (MTBE). The amount of enzyme used may be chosen within wide limits, depending upon its catalytic activity under reaction conditions, the desired conversion and the desired reaction time. Usually, the amount is at least 0.001 wt. %, at least 0.01 wt. %, at least 0.1 wt. %, at least 1 wt. %, at least 10 wt. % or at least 20 wt. %, based on the weight of the substrate. For practical reasons the amount is usually 1000 wt. % or less, in particular 750 wt. % or less, 500 wt. % or less, 200 wt. % or less, 100 wt. % or less, 50 wt. % or less, or 25 wt. % or less, based on the weight of the substrate.

The method of the invention is typically carried out under substantially non-aqueous conditions. As the skilled person will understand, a small amount of water may be desired, depending upon the enzyme, to enable the enzyme to properly perform its catalytic activity.

With substantially non-aqueous is meant that the reaction medium is free of water or contains a minor amount of water, namely an amount of less than 2 wt.% water, based on the weight of liquids in the reaction medium. The reaction medium may be dispersed in a second liquid phase or another liquid phase may be dispersed in the reaction medium. In case of a dual or multiphase system, the specified water content is based on the weight of liquids in the phase wherein the amidation reaction (at least predominantly) takes place.

In particular - at least at the beginning of the amidation reaction - the water concentration may be less than 1.5 wt. %. Advantageously, a method may be carried out in a phase containing - at least at the beginning of the amidation reaction - less than 1.0 wt. % water, in particular 0.5 wt. % or less water, more in particular 0.2 wt. % or less water, for instance about 0.09 wt. % or less water, whilst still retaining substantial desired enzyme activity. For a good catalytic activity the presence of a trace of water, e.g. of at least 0.01 wt. %, based on the liquid phase, may be desired. In particular, the water concentration may be at least 0.02 wt. % or at least 0.05 wt. %.

In principle the pH used may be chosen within wide limits. It may in particular be chosen to be about neutral. If desired, alkaline or acidic conditions may be used, depending on the enzyme. A method according to the invention can be carried out without adding further pH modulating additives. If desired, the pH may be adjusted using an acidic compound and/or an alkaline compound or buffered with a suitable combination of an acid/base pair. Suitable acids and bases are in particular those soluble in the reaction medium, e.g. from the group of ammonia and alcohol-soluble acids, e.g. acetic acid and formic acid. The pH of the reaction may be controlled by using an automated pH-stat system. Optimal pH conditions can easily be identified by a person skilled in the art through routine experimentation.

In principle the temperature used is not critical, as long as the enzyme shows substantial activity. Generally, the temperature may be at least 0 ⁰C, in particular at least 15 ⁰C. A desired maximum temperature depends upon the enzyme. In general such maximum temperature is known in the art, e.g. indicated in a product data sheet in case of a commercially available enzyme, or can be determined routinely based on common general knowledge and the information disclosed herein. The temperature is usually 70⁰C or less, in particular 60⁰C or less or 50 °C or less. In particular if a thermophilic hydrolytic enzyme is used, the temperature may be chosen relatively high, for instance in the range of 40 to 90⁰C. Optimal temperature conditions can be identified for a specific enzyme by a person skilled in the art through routine experimentation, based on common general knowledge and the information disclosed herein. For instance, for subtilisin, in particular subtilisin Carlsberg (e.g. in Alcalase) the temperature may advantageously be in the range of 25-60°C. In an advantageous method, water that is formed, in particular during amidation, may be removed continuously or intermittently. In principle removal may be accomplished in a manner known in the art.

Good results have in particular been achieved using molecular sieves. Also very suitable for the removal is evaporation, such as azeotropic removal using vacuum or distillation.

The products obtained in accordance with the invention may for instance be used in enzymology or in clinical diagnostics as substrates of proteolytic enzymes, see, e.g., Hemker, H. C, ed. (1983) Handbook of Synthetic Substrates for the Coagulation and Fibrinolytic Systems, Martinus Nijhoff Publishers, Boston. Accordingly, the invention further relates to the use of a compound obtained in a method according to the invention as a diagnostic and/or as a substrate for a proteolytic enzyme. Examples of proteolytic enzymes for which a compound obtained in accordance with the invention can be used as a substrate for said proteolytic enzyme include trypsin, urokinase, thrombin, activated protein C, plasmin. Suitable amino acids or peptides upon which the amide is based can be determined, depending upon the specific enzyme for which the aryl amide is a substrate, based on common general knowledge, references cited herein, the information described in the present description and/or claims, and/or on information provided in diagnostic catalogues, e.g. catalogues from Molecular Probes (e.g. http:/probes/invitrogen. com/handbook) or Pentapharm (Asch, Switzerland), e.g. the 2005 version of the Catalogue "Haemostatis kits, Substrates, Inhibitors, Snake Venom proteins, Biochemicals", available via the Internet http:/www.pentapharm.com/graphics/pentapharm/download/diagnostics/Katalog2005.pdf.

For use of a compound obtained in accordance with the invention in a method involving a spectroscopic detection, the amine and amide preferably have different spectroscopic properties, such as different absorption and/or fluorescence properties. In an advantageous embodiment, the amine has an absorption maximum at a different wavelength than the amide, for instance the amine may be a chromophore, i.e. a compound having an (strong) absorption maximum in the visible range of the light spectrum, whereas the amide is essentially colourless, or vice versa. A compound that is colourless, but after reaction generates a coloured compound is called chromogenic, e.g. an aryl amide that upon cleavage releases an coloured aryl amine is called chromogenic. In an advantageous embodiment, the amine is fluorescent whereas the amide is essentially non-fluorescent, or if the amide is fluorescent it has an excitation and/or emission wavelength at a different wavelength, that allows selective detection, or vice versa. In an embodiment, the aryl amide is fluorogenic, i.e. the aryl amide is essentially non-fluorescent but upon cleavage of the amide bond provides a fluorescent aryl amine.

In an embodiment, the aryl amide is amperogenic, which means that upon hydrolysis of the amide bond an aryl amine is formed that can be reduced or oxidised and that thereby can be detected amperometrically. Suitable aryl amines for such a method include hydroxy- aryl amines, e.g. 3-chloro-4-hydroxy aniline. The invention will now be illustrated by the following examples. EXAMPLES

Materials and Methods

Unless stated otherwise, chemicals were obtained from commercial sources and used without further purification. 3A molsieves (8 to 12 mesh, Acros) were activated under reduced pressure at 200⁰C. f-BuOH was stored on these activated molsieves and pre-heated to liquid (45°C) before use. Before use, 3 g Alcalase-CLEA (Codexis, 650 AGEU/g, containing 3.5 wt% water), was suspended in 100 ml. f-BuOH and crushed with a spatula. After filtration, the Alcalase-CLEA was re-suspended in 50 mL MTBE followed by filtration. CaI-B was purchased from Novozymes (immobilized

Novozym-435^®, batch LC 200204). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer and chemical shifts are given in ppm (δ) relative to TMS (0.00 ppm) or DMSO-c/₆ (2.50 ppm). Thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F ₂5₄ plates (Merck); spots were visualized using UV light, ninhydrin or permanganate solution. Column chromatography was carried out using silica gel, Merck grade 9385 60 A. Analytical HPLC diagrams were recorded on an HP1090 Liquid Chromatograph, using a reversed-phase column (Inertsil ODS-3, C18, 5 μm particle size, 150 x 4.6 mm) at 40⁰C. UV detection was performed at 220 nm using a UV-VIS 204 Linear spectrometer (Varian). The gradient program was: 0-25 min linear gradient ramp from 5% to 98% eluent B and from 25.1-30 min 5% eluent B (eluent A: 0.5 mL/L methane sulfonic acid (MSA) in H₂O; eluent B: 0.5 mL/L MSA in acetonitrile). The flow was 1 mL/min from 0-25.1 min and 2 mL/min from 25.2-29.8 min, then back to 1 mL/min until stop at 30 min. Injection volumes were 20 μL. Melting points were determined on an automated Mettler FP81 HT MBC cell coupled to a Mettler FP80HT central processor; samples were measured in triplo using a temperature gradient of 1 °C/min from 150⁰C to 250⁰C. IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer. Preparative HPLC was performed on a Varian PrepStar system using a Pursuit XRs column (C18, 10 μm particle size, 500 x 41.4 mm) at ambient temperature. UV detection was performed at 220 nm and 254 nm using a UV-VIS Varian ProStar spectrometer. The elution was performed with 85% eluent B and 15% eluent A (eluent A: 0.1 mL/L formic acid in H₂O; eluent B: 0.1 mL/L formic acid in acetonitrile) with a flow rate of 80 mL/min, injection volumes of 10 mL and total run times of 30 min. Pure fractions were pooled and concentrated in vacuo and co-evaporated with toluene (25 ml_, 2x) and CHCI₃ (25 mL, 2x).

Example 1 : Synthesis of Cbz-Phe-anilide from Cbz-Phe-OH

Alcalase-CLEA

500 mg Alcalase-CLEA was added to a mixture containing 4.5 mL MTBE, 500 μL aniline (4.48 mmol), 134 mg Cbz-Phe-OH (0.45 mmol) and 200 mg 3 A molsieves. This mixture was shaken at 50⁰C with 150 rpm for 16h. The reaction mixture was filtrated over a P4 glass filter and the solids were washed with ethyl acetate (50 mL, 3x). Distilled water (150 mL) was added to the combined filtrate and under vigorous stirring the pH was adjusted to 3.0 with 1 N aqueous HCI solution. After an additional 10 min stirring the two layers were separated. This procedure of washing the organic layer with an aqueous phase of pH 3 was repeated twice. Subsequently, the organic phase was washed with saturated aqueous NaHCO₃ (100 mL) and brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. The residue was diluted with n-heptane (2 mL) and the resulting white crystals were isolated by filtration and washed with n-heptane (2 x 5 mL) giving Cbz-Phe-anilide as a white solid in 93% yield (156 mg). R_f(anal. HPLC) 19.65 min, purity > 99%; R^EtOAc/n-hexane, 1/1 , v/v) 0.55; Mp: 169°C; ¹H NMR (300 MHz, CDCI₃) δ = 3.05-3.10 (2xdd, 2 H, CpH₂), 4.44-4.51 (m, 1 H, C₀H), 5.02 (s, 2 H, CH₂OCO), 5.43 (d, 1 H, NH), 7.01-7.25 (m, 15 H, C_ArH), 7.59 (s, 1 H, NH); ¹³C NMR (75 MHz, CDCI₃) δ = 38.6, 57.2, 67.3, 120.1 , 124.6, 127.2, 128.0, 128.3, 128.6, 128.9, 129.3, 136.0, 136.3, 137.1 , 169.1 , 182.0; IR v^/cm^"1 = 3285, 1686, 1654, 1600, 1531 , 1494, 1445, 1284, 1260, 1243. Example 2: Synthesis of Cbz-Phe-p-nitro-anilide from Cbz-Phe-OH

500 mg Alcalase-CLEA was added to a mixture containing 5 mL acetone, 100 mg p-nitro-aniline (3.6 mmol), 100 mg Cbz-Phe-OH (0.33 mmol) and 200 mg 3 A molsieves. This mixture was shaken at 50⁰C with 150 rpm for 16h. The reaction mixture was filtrated over a P4 glass filter and the solids were washed with ethyl acetate (50 ml_, 3x). The combined filtrates were concentrated in vacuo. Cbz-Phe-p-nitro-anilide was obtained after preparative HPLC purification in 65% yield as a white solid (90 mg). R_f(anal. HPLC) 20.29 min, purity 99%; R^(EtO Ac/n-hexane, 1/1 , v/v) 0.58; Mp: 159°C; ¹H NMR (300 MHz, CDCI₃) δ = 3.03 (d, J = 6.9 Hz, 2 H, CpH₂), 4.57 (m, 1 H, C₀H), 4.98 (s, 2 H, CH₂OCO), 5.37 (d, J = 7.8 Hz, 1 H, NH), 7.07-7.22 (m, 10 H, C_ArH), 7.33 (d, J = 9.3 Hz, 2 H, C_ArH), 7.96 (d, J = 9.0 Hz, 2 H, C_ArH), 8.37 (s, 1 H, NH); ¹³C NMR (75 MHz, CDCI₃) δ = 38.0, 57.3, 67.6, 119.2, 124.9, 127.4, 127.9, 128.5, 128.6, 129.0, 129.2, 135.8, 143.0, 143.7, 156.6, 169.8; IR v^/cm^"1 = 3305, 3270, 3033, 1696, 1681 , 1533, 1509, 1495, 1347, 1249, 1214.

Example 3: Synthesis of Cbz-Ala-anilide from Cbz-Ala using Candida antarctica lipase-B

100 mg CaI-B was added to a mixture containing 4.5 mL MTBE, 500 μL aniline, 100 mg Cbz-Ala-OH (0.45 mmol) and 200 mg molsieves. This mixture was shaken at 50⁰C with 150 rpm for 16h. The reaction mixture was filtrated over a P4 glass filter and the solids were washed with ethyl acetate (50 mL, 3x). Distilled water (150 mL) was added to the combined filtrates and under vigorous stirring the pH was adjusted to 3.0 with 1 N aqueous HCI solution. After an additional 10 min stirring the two layers were separated. This procedure of washing the organic layer with an aqueous phase of pH 3 was repeated twice. Subsequently, the organic phase was washed with saturated aqueous NaHCO₃ (100 ml.) and brine (100 ml_), dried (Na₂SO₄) and concentrated in vacuo. The residue was diluted with n-heptane (2 ml.) and the resulting yellow crystals were isolated by filtration and washed with n-heptane (2 x 5 ml.) giving Cbz-Ala-anilide as a yellow solid in 92% yield (116 mg). R_f(anal. HPLC) 15.43 min, purity 98%; R^EtOAc/n-hexane, 1/1 , v/v) 0.43; Mp: 152 ⁰C; ¹H NMR (300 MHz, DMSO-d₆) δ = 1.29 (d, J = 7.2 Hz, 3 H, CH₃), 4.20 (m, 1 H, C₀H), 5.03 (s, 2 H, CH₂OCO), 7.04 (t, J = 7.2 Hz, 1 H, C_ArH), 7.10-7.65 (m, 10 H, C_ArH and NH), 9.96 (s, 1 H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ = 17.9, 50.7, 65.3, 1 19.1 , 123.1 , 127.6, 127.7, 128.2, 128.6, 136.9, 138.9, 155.7, 171.4; IR v_max/cm^"1 = 3296, 1686, 1665, 1533, 1490, 1455, 1285, 1257, 1248, 1213, 1058.

Example 4: Synthesis of Cbz-Asp-α-anilide from Cbz-Asp-OH

Chemical synthesis of Cbz-Asp-α-anilide as reference compound: 182 μl_ aniline (2 mmol, 2 equiv.) was added to a mixture of 4 ml. pyridine and 86 μl_ PCI₃ (1 mmol, 1 equiv.) and stirred for 1 h at ambient temperature. Subsequently, a solution of 323 mg Z-Asp(O^fBu)-OH (1 mmol, 1 equiv.) in 2.5 ml. pyridine was added and the mixture stirred at 40 ⁰C for 4 h. The reaction mixture was concentrated in vacuo and addition of water (50 ml.) to the residue resulted in presipitation. Precipitates were filtered off and washed with 20 ml. water (2x). The solid was resuspended in 1O mL TFA to which a drop of water had been added and the mixture was stirred for 3 h at ambient temperature, concentrated in vacuo and the residue redissolved in 50 mL DCM and 50 mL water. The water layer was adjusted to pH = 8 with 1 N aqueous NaOH and the mixture was stirred vigorously for 10 min followed by separation of the two layers.. The water layer was adjusted to pH = 2 using 1 N aqueaous HCI, crystals were filtered off and washed with 20 mL water (2x). After co-evaporation with toluene (20 mL, 2x) and CHCI₃ (20 mL, 2x), Cbz- Asp-α-anilide was obtained as a white solid in 81 % yield (276 mg). R_f(anal. HPLC), 14.37 min, purity; 98% R^MeOH/DCM/AcOH, 15/84/1 , v/v) 0.16; Mp: 179 ⁰C; ¹H NMR (300 MHz, DMSO-de) δ = 2.55-2.83 (m, 2 H, CpH₂), 4.59 (m, 1 H, C₀H), 5.10 (s, 2 H, CH₂OCO), 7.11 (t, J = 7.2 Hz, 1 H, C_ArH), 7.25-7.45 (m, 7 H, C_ArH), 7.66 (d, J = 7.8 Hz, 2 H, C_ArH), 7.75 (d, J = 7.8 Hz, 1 H, NH), 10.11 (s, 1 H, NH), 12.41 (s, 1 H, COOH); ¹³C NMR (75 MHz, DMSO-d₆) δ = 36.2, 52.0, 65.4, 1 19.3, 123.2, 127.6, 127.7, 128.2, 128.5, 136.8, 138.8, 155.7, 169.5, 171.4; IR v_max /crτr¹ = 3292, 3064, 1693, 1678, 1531 , 1444, 1276, 1243.

Enzymatic synthesis of Cbz-Asp-α-anilide:

Alcalase-CLEA

500 mg Alcalase-CLEA was added to a mixture containing 4.5 mL MTBE, 500 μL aniline (4.5 mmol, 10 equiv.), 120 mg Cbz-Asp-OH (0.45 mmol, 1 equiv.) and 200 mg 3 A molsieves. The reaction mixture was shaken at 50⁰C with 150 rpm for 16 h and filtrated over a P4 glass filter and the solids were washed with ethyl acetate (50 ml_, 3x) and aqueous HCI (50 ml_, pH = 1 , 3x). The layers of the combined filtrates were separated and the organic layer concentrated in vacuo. After preparative HPLC purification, Cbz-Asp-α-anilide was obtained in 91% yield as a white solid (140 mg). The spectroscopic and physical data were as given above.

Example 5: Synthesis of Cbz-Ser-anilide from Cbz-Ser-OH

Alcalase-CLEA

500 mg Alcalase-CLEA was added to a mixture containing 4.5 mL MTBE, 500 μL aniline (4.5 mmol, 10 equiv.), 108 mg Cbz-Ser-OH (0.45 mmol, 1 equiv.) and 200 mg 3 A molsieves. The reaction mixture was shaken at 50⁰C with 150 rpm for 16 h and filtrated over a P4 glass filter and the solids were washed with ethyl acetate (50 mL, 3x) and aqueous HCI (50 mL, pH = 1 , 3x). The layers of the combined filtrates were separated and the organic layer concentrated in vacuo. After preparative HPLC purification, Cbz-Ser-anilide was obtained in 93% yield as a white solid (131 mg). R_f(anal. HPLC) 20.36 min, purity 98%; R^MeOH/DCM, 1/20, v/v) 0.19; Mp: 160 °C; ¹H NMR (300 MHz, DMSO-de) δ = 4.28 (m, 2 H, CpH₂), 4.57 (m, 1 H, C₀H), 5.11 (s, 2 H, CH₂OCO), 7.12 (t, J = 6.9 Hz, 1 H, C_ArH), 7.20-7.50 (m, 7 H, C_ArH), 7.64 (d, J = 7.5 Hz, 2 H, C_ArH), 7.86 (d, J = 7.8 Hz, 1 H, NH), 10.24 (s, 1 H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ = 55.2, 55.3, 64.3, 65.7, 119.5, 123.6, 127.6, 127.7, 128.2, 128.6, 136.6, 138.4, 155.8, 167.1 IR v_ma_x/cm^"1 = 3282, 3061 , 1694, 1677, 1668, 1601 , 1537, 1498, 1445, 1298, 1250.

Example 6: Synthesis of Cbz-Phe-Leu-anilide from Cbz-Phe-Leu-OH

500 mg Alcalase-CLEA was added to a mixture of 100 mg Cbz-Phe-Leu-

OH (0.243 mmol), 4.8 ml. of MTBE, 200 μl_ aniline (2.2 mmol, 204 mg, 9 equiv.) and 100 mg 3 A molsieves. The mixture was shaken at 50 ⁰C with 150 rpm for 16 h and filtrated over a P4 glass filter and the solids were washed with ethyl acetate (20 ml_, 2x). Water (20 ml.) was added to the combined filtrates and under vigorous stirring the pH was adjusted to 3.0 with 1 N aqueous HCI solution. After an additional 10 min stirring the two layers were separated. This procedure of washing the organic layer with an aqueous phase of pH 3 was repeated once. The organic phase was then washed with saturated aqueous NaHCO₃ (20 ml_, 2x) and brine (20 ml_), dried (Na₂SO₄) and concentrated in vacuo. The residue was diluted with n-heptane (5 ml.) and the resulting white crystals were isolated by filtration and washed with n-heptane (2 x 5 ml.) giving Z-Phe-Leu-anilide (108 mg, 0.308 mmol) in 91 % yield based on Cbz-Phe-Leu-OH. R_f(anal. HPLC) 21.08 min, purity 99%; R^EtOAc/n-hexane, 1/1 , v/v) 0.52; Mp: 225 ⁰C; ¹H NMR (300 MHz, CDCI₃) δ = 0.81 (d, J = 6.0 Hz, 6 H, C_δLΘUH₃), 1.35-1.55 (m, 2 H, C_γLΘUH and C_pLeuH), 1.65- 1.75 (m, 1 H, C_βLΘUH), 3.00 (m, 2 H, C_pPheH₂), 4.34-4.50 (m, 2 H, C_αPhΘH and C_αLΘUH), 5.00 (s, 2 H, CH₂OCO), 5.21 (d, J = 6.6 Hz, 1 H, NH), 6.25 (d, J = 7.2 Hz, 1 H, NH), 7.00-7.30 (m, 15 H, C_ArH), 7.47 (d, J = 7.5 Hz, 2 H, C_ArH), 8.25 (s, 1 H, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ = 21.7, 22.4, 24.5, 38.4, 41.0, 50.6, 51.9, 55.7, 66.6, 126.5, 127.6, 127.8, 128.2, 129.2, 136.1 , 136.3, 155.9, 170.9, 172.7; IR v_max/crτr¹ = 3281 , 1692, 1641 , 1531 , 1495, 1447, 1285, 1258, 1234.

Example 7: Synthesis of Cbz-protected phenylalanine arylamides starting from Cbz- protected phenylalanine

General procedure:

500 mg Alcalase-CLEA was added to a mixture containing 4.5 ml_ MTBE, 500 mg arylamine, 100 mg Cbz-Phe-OH (0.33 mmol) and 200 mg 3 A molsieves. Mixtures were shaken at 50⁰C with 150 rpm for 16h. The reaction mixture was filtered over a P4 glass filter and the solids were washed with ethyl acetate (50 ml_, 3x). The combined filtrate was concentrated in vacuo followed by preparative HPLC purification.

Table 1 : Cbz-protected phenylalanine arylamides; yields.

Product

P rod u ct

Cbz-Phe-p-methoxyanilide was obtained in 88% yield as an off-white solid (1 17 mg).

Cbz-Phe-o-fluoro-anilide was obtained in 85% yield as a white solid (110 mg).

Cbz-Phe-m-fluoro-anilide was obtained in 79% yield as a white solid (102 mg).

Cbz-Phe-p-fluoro-anilide was obtained in 81 % yield as a white solid (105 mg).

Cbz-Phe-o-cyano-anilide was obtained in 70% yield as an off-white solid (92 mg).

Cbz-Phe-m-cyano-anilide was obtained in 84% yield as an off-white solid (1 10 mg).

P rod u ct

Cbz-Phe-p-cyano-anilide was obtained in 65% yield as an off-white solid (85 mg).

Using MeOH/DCM, 1/20, v/v. 2Using EtOAc/n-hexane, 1/1 , v/v.

Table 3: Η NMR (300 MHz, DMSO-C/R) data of Cbz-protected phenylalanine arylamides.

Product

Cbz-Phe-4-methylcoumarin- 2.43 (s, 3 H, CH ..93 (dd, J = 13.5 and 4 5 Hz, 1 H,

7-amide CpH₂), 3 .09 (dd, J = 13.5 and 10.5 Hz, 1 H, CpH₂) , 4.48

(m, 1 H, C₀H), 5 .01 (s, 2 H, CH₂OCO), 6.30 (s, 1 H,

C_ArH), 7 21-7.38 (m , 10 H, C_ArH), 7.50 (d, J = 8.4 Hz, 1

H, C_ArH) , 7.74-7 .85 (m, 3 H, C_ArH and NH), 10.58 (s, 1

H, NH)

Table 4: ¹X NMR (75 MHz, DMSO-C/R) data of Cbz-protected phenylalanine arylamides.

Table 5: IR v_maχ/cm^"1 data of Cbz-protected phenylalanine arylamides.

Claims

1. Method for preparing an aryl amide, comprising reacting an aryl amine with a compound selected from N-protected amino acids and optionally N-protected peptides, in the presence of a hydrolytic enzyme in a phase comprising less than

2 wt. % water.

2. Method according to claim 1 , wherein the aryl amine comprises an optionally substituted ring system with 2-50 carbon atoms, in particular 3-30 carbon atoms, more in particular 3-20 carbon atoms. 3. Method according to claim 1 or 2, wherein the aryl amine is represented by a compound of formula (I)

wherein R¹, R², R³, R⁴, and R⁵ are each independently H, F, Cl, Br, I, an amino group, a carbonyl group, imine group, a sulphonic acid group, a nitro group or an organic moiety, wherein optionally one or more pairs of said R-groups together form an optionally substituted ring structure, optionally comprising one or more heteroatoms, and wherein R⁶ represents hydrogen, an unsubstituted alkyl or a substituted alkyl.

Method according to any one of the preceding claims, wherein the aryl amide or the aryl amine is fluorogenic, chromogenic or amperogenic. Method according to any one of the preceding claims, wherein the compound with which the aryl amine is reacted, contains a C-terminal arginine residue. Method according to any one of the preceding claims, wherein the aryl amine is selected from the group of aniline; p-nitroaniline; 7-amino-coumarins, in particular 7-amino-4-methylcoumarin; 2-naphthylamine; 4-phenylazoaniline; rhodamines containing an amino group; cresyl violet; acιϊdine-3,6-diamine; 6-quinolylamine, and hydroxyanilines.

7. Method according to any one of the preceding claims, wherein the hydrolytic enzyme is chosen from the group of carboxylic ester hydrolases, thioester hydrolases and peptidases.

8. Method according to claim 7, wherein the enzyme is a peptidase selected from the group of serine-type carboxypeptidases, metallocarboxypeptidases, cysteine-type carboxypeptidases, serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases and metalloendopeptidases, in particular from serine endopeptidases.

9. Method according to claim 8, wherein the enzyme is a serine endopeptidase, preferably a subtilisin, more preferably subtilisin Carlsberg.

10. Method according to claim 8, wherein the enzyme is a cysteine endopeptidase, preferably papain. 11. Method according to claim 7, wherein the enzyme is a lipase, preferably a Candida Antarctica lipase, preferably CAL-B.

12. Use of a compound obtained in a method according to any one of the preceding claims in the manufacture of a diagnostic.

13. Use of a compound obtained in a method according to any one of the claims 1 -1 1 as a substrate for a proteolytic enzyme.