WO2003062267A2

WO2003062267A2 - Enzyme substrates for the determination of activity of staphylococcus sp. extracellular protease

Info

Publication number: WO2003062267A2
Application number: PCT/GB2003/000120
Authority: WO
Inventors: Manoj Kumar Ramjee
Original assignee: Amura Therapeutics Limited
Priority date: 2002-01-17
Filing date: 2003-01-16
Publication date: 2003-07-31
Also published as: AU2003201663A1; WO2003062267A3

Abstract

Compounds of general formula (I): A) A compound of general formula (I) Y-AA1-AA2-AA3-AA4-R-Glu-NH2, Y-A1-A2-A3-A4-R-Glu-NH2 wherein Y and R are reporter groups; A1 is either the D-isomer or the L-isomer form of isoleucine; each of A2 and A3 is independently Ala or 2-aminobutyric acid (Abu); A4 is Pro, L-3-hydroxyproline (Pro(3-OH)), L-4-hydroxyproline (Pro (4-OH)), L-tetrahydroisoquinoline-1-carboxylic acid (THIQ), L-tetrahydroisoquinoline-3-carboxylic acid (Tic), L-pipecolic acid (Pip), 1-amino-1-cyclopentane carboxylic acid (1-ACP) or D-Pro; and any one of A1, A2, A3 and A4 can be replaced by any other amino acid group (Xaa); and the other protein based substrates are cleavable by Staphyococcus sp. extracellular cysteine protease and are therefore likely to be of use in monitoring enzyme activity for purposes such as investigating candidate enzyme modulators and screening for therapeutic agents.

Description

ENZYME SUBSTRATES

Staphylococcus sp.

Staphylococcus aureus, a gram-positive bacterium (Kuroda, M., et al., (2001), Lancet, 357, 1218-1219), produces a wide range of extracellular products, examples of which include nucleases, hyaluronidases, staphylokinase (Arvidson, S., et al., (1973), Contrib Microbiol Immunol, 1, 406-412), lipase, phosphatase (Arvidson, S., et al., (1971), Acta Pathol Microbiol Scand [BJ Microbiol Immunol, 79, (3), 399- 405), peptidases (i.e. proteases) (Arvidson, S., (1973), Acta Pathol Microbiol Scand [BJ Microbiol Immunol, 81 (5), 538-544; Tirunarayanan, M. O., (1966), Acta Pathol Microbiol Scand, 68 (2), 273-280) together with a host of other toxins (Vesterberg, O., et al., (1967), Biochim Biophys Acta, 133 (3), 435-445; Wadstrom, T. and Vesterberg, O., (1971), Acta Pathol Microbiol Scand [BJ Microbiol Immunol, 19 (2), 248-264). It is generally accepted that one or more these products aid in the infection process by various mechanisms including the acquisition of nutrients, binding to host cellular receptors, neutralising host defences, etc. As such many of these extracellular products have been categorised as potential virulence factors. Of the many factors present, peptidases have been implicated as one of the major virulence factors and as such this has been an intensive area of research.

Extracellular proteolytic activity in S. aureus culture supernatant was reported as early as the 1940s (Abe, T., (1947), Tohoku JExp Med, 49, 27-32; Abe, T., (1947), Tohoku J Exp Med, 49, 33-38). Subsequent studies have established that the peptidase activity was due to a number of peptidases, with representatives from the major mechanistic classes (Dubin, G., (2002), Biol. Chem., 383, 1075-1086). Examples of S. aureus extracellular peptidases include serine peptidases (Arvidson, S., et al., (1973), Biochim Biophys Acta, 302 (1), 135-148; Drapeau, G. R., et al, (1972), JBiol Chem, 247 (20), 6720-6726; Nath, G. M., et al, (1999), Biochemistry, 38 (32), 10239-10246), meiallopeptidases (Arvidson, S., (1973), Biochim Biophys Acta, 302 (1), 149-157; Drapeau, G. R., (1978), J Bacteriol, 136 (2), 607-613) and cysteine peptidases (Arvidson, S., et al, (1973), Biochim Biophys Acta, 302 (1), 135- 148; Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667; Takeuchi, S., et al, (1999), Vet Microbiol, 67 (3), 195-202). To date, at least one example of each has been reported to have been purified and characterised, plus the three-dimensional x-ray crystallographic structure of an example of each class has also been solved (Gehrig, L. M., et al, (1985), J Mol Biol, 185 (3), 651; Banbula, A., et al, (1998), Structure, 6 (9), 1185-1193; Hofmann, B., et al, (1993), Acta Crystallographa, section A (supplement), 102). Of the three main examples, the serine peptidases have been the most extensively studied of the peptidases and although slightly different characteristics have been described for various preparations, a unifying feature of this enzyme class has been the over-riding preference for glutamate as the PI ¹ residue in the substrate (Houmard, J., (1976), Eur J Biochem, 68 (2), 621-627; Houmard, J. and Drapeau, G. R., (1972), Proc Natl Acad Sci U S A, 69 (12), 3506- 3509). The N8 protease has been classified as glutamyl endopeptidase I (Stennicke, H. R. and Breddam, K., (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 243-246), but it has also been referred to as V8 protease, Ssp A (ref), exfoliative toxins (due to their involvement in staphylococcal scalded-skin syndrome (SSSS) (Nath, G. M., et al, (1999), Biochemistry, 38 (32), 10239-10246)) and/or staphylococcal superantigens, because of the ability of the peptidase to stimulate T-cell responses (Monday, S. R., et al, (1999), J Immunol, 162 (8), 4550- 4559).

Metallopeptidases, generally referred to as aureolysin(s) (Potempa, J., et al, (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 1540-1542), of at least two different types, have also been described (Arvidson, S., (1973), Biochim Biophys Acta, 302 (1), 149-157; Drapeau, G. R., (1978), J Bacteriol, 136 (2), 607-613). The role of these peptidases has been implicated in the activation of the staphylococcal protease (Drapeau, G. R., (1978), J Bacteriol, 136 (2), 607-613), cell matrix-and tissue-degradation (Banbula, A., et al, (1998), Structure, 6 (9), 1185-1193) as well as housekeeping functions (Sabat, A., et al. , (2000), Infect Immun, 68 (2), 973-976).

^a Nomenclature is based on amino acid preferences within residue-binding sub-sites spanning the peptidase active-site, with cleavage occurring between PI and PI ' residues (Schechter, I. and Berger, Cysteine peptidases, classified as staphylopain (Potempa, J., et al, (1998), Handbook of Proteolytic Enzymes, London, Academic Press, 669-671), have also been reported (Arvidson, S., et al, (1973), Biochim Biophys Acta, 302 (1), 135-148; Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667; Takeuchi, S., et al, (1999), Vet Microbiol, 67 (3), 195-202; Takeuchi, S. and Suto, T., (1974), Natl lnstAnim Health Q (Tokyo), 14 (3), 121-128; Bjorklind, A. and Jornvall, H., (1974), Biochim Biophys Acta, 370 (2), 524-529). However, in many cases the use of slightly different organisms, varying culture conditions and differences in biochemical techniques has lead to different descriptions of biochemical characteristics of this class of extracellular S. aureus peptidases. Since in most cases the N-terminal amino acid sequence of the purified protein was not determined, an absolute identification of the peptidase was not reported. This in turn has translated into differences in nomenclature (e.g. staphopain {Scp} (Hofmann, B., et al, (1993), Acta Crystallographa, section A ((supplement)), 102), cysteine protease {SspB} (Karlsson, A., et al, (2001), Infect Immun, 69 (8), 4742-4748; Kuroda, M., et al, (2001), Lancet, 357 (9264), 1225-1240)). Although it was assumed that a single cysteine peptidase activity was present in the extracellular fraction, it has recently been reported that more than one cysteine peptidase was present (Golonka, E., et al, (2001), 2nd General Meeting of the International Proteolysis Society, Freising, Germany, International Proteolysis Society; Dubin, G., (2002), Biol. Chem., 383, 1075-1086). Similarly, there have also been reports of more than one 'staphopain' present in the extracellular fraction of the closely related organism Staphylococcus epidermis (Oleksy, A., et al, (2001), 2nd General Meeting of the International Proteolysis Society, Freising, Germany, International Proteolysis Society; Dubin, G., et al, (2001), 2nd General Meeting of the International Proteolysis Society, Freising, Germany, International Proteolysis Society). The presence of more than one cysteine peptidase activity may help to explain some of the confusion surrounding the different biochemical characteristics described in the earlier literature reports. The role of staphylopain as a virulence factor was primarily based on the observation that the enzyme was shown to cleave a number of host proteins. Although casein was

A., (1967), Biochem Biophys Res Commun, 27 (2), 157-162). generally employed as the substrate, haemoglobin (Vesterberg, O., et al, (1967), Biochim Biophys Acta, 133 (3), 435-445; Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667), alcohol dehydrogenase (Bjδrklind, A. and Jornvall, H., (1974), Biochim Biophys Acta, 370 (2), 524-529.) and elastin (Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667) have also been shown to be substrates. Further evidence of its role as a virulence factor was provided by reports that described tissue degradation in diseased chicken due to cysteine peptidase production (Takeuchi, S. and Suto, T., (1976), Jpn J Microbiol, 20 (3), 155-162) and that inhibition of the cysteine peptidase activity resulted in inhibition of S. aureus growth (Takahashi, M., et al, (1994), FEBS Lett, 355 (3), 275-278; Takahashi, M., et al, (1999), Biofactors, 10 (4), 339-345). Further evidence of its role as a virulence factor has been provided by the purification of a staphylopain from the culture supernatant of a S. aureus strain isolated from diseased chickens (Takeuchi, S., et al, (1999), Vet Microbiol, 67 (3), 195-202).

Substrates for monitoring S. aureus cysteine peptidase activity An essential feature of the characterisation of enzymes has been the ability to monitor catalytic activity. This requirement has not only been important in determining the various biochemical roles of enzymes, but more recently it has been essential to the discovery and design of inhibitors. Enzyme activity assays are usually carried out in two formats, direct and indirect. Direct assays are best defined as when substrate conversion is concomitant with signal generation and therefore does not require any form of post-assay sample processing. The indirect methods usually employ the same assay protocol (i.e. buffers, enzyme, additives, etc) but differ in that the substrate depletion or product formation cannot be measured directly. In this latter case cleavage is monitored by post-assay sample processing, which may include, but is not restricted to, differential precipitation (e.g. acid, organic solvent, etc), sample separation (e.g. chromatography, electrophoresis, etc), component partitioning, etc. Direct assays have a number of key advantages over in- direct assays. Firstly, activity, in particular rates, can be determined directly from signal generation. Secondly, as assays do not require post-assay processing, errors due to post-assay sample handling are eliminated. The elimination of post-assay processing also significantly reduces the time between starting the assay and obtaining the final data. Assays can also be made extremely sensitive and are adaptable to multiple assay formats.

A number of protein-based substrates have been used as substrates to monitor S. aureus cysteine peptidase activity. Examples include casein (Arvidson, S., et al, (1971), Acta Pathol Microbiol Scand [BJ Microbiol Immunol, 79 (3), 399-405; Arvidson, S., et al, (1973), Biochim Biophys Acta, 302 (1), 135-148; Arvidson, S., (1973), Biochim Biophys Acta, 302 (1), 149-157; Tirunarayanan, M. O. and Lundblad, G., (1966), Acta Pathol Microbiol Scand, 68 (1), 135-141), gelatin (Tirunarayanan, M. O. and Lundblad, G., (1966), Acta Pathol Microbiol Scand, 68 (1), 135-141), haemoglobin (Vesterberg, O., et al, (1967), Biochim Biophys Acta, 133 (3), 435-445; Tirunarayanan, M. O. and Lundblad, G., (1966), Acta Pathol Microbiol Scand, 68 (1), 135-141), dehydrogenase (yeast and horse liver) (Bjδrklind, A. and Jornvall, H., (1974), Biochim Biophys Acta, 370 (2), 524-529) and elastin (Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667). Proteins were principally used as substrates because they were cheap and relatively easily available in a pure form. Protein-based assays have, in general, been carried out in a stopped- time fashion and principally based on monitoring the increase in absorbance at either 215 nm, 254 nm or 280 nm upon the formation of non-precipitating peptide fragments. After an incubation period the assay mixture is treated with protein precipitants (e.g. strong acid, strong base, detergents, organic solvents, etc) in order to separate the insoluble proteins (i.e. substrates) from the relatively soluble peptide fragments (i.e. products). However these types of assays suffer from a number of drawbacks. Firstly, the assays are less sensitive because of the relatively poor extinction coefficient of the amino acid-based chromophores (peptide bond, or the aromatic aminoacids phenylalanine, tyrosine, tryptophan and histidine). Secondly, post-incubation sample processing, which requires multiple steps, could not guarantee uniformity in sample recovery as substrate was converted to product. One obvious variation has been the use of casein-impregnated polyacrylamide gels (t.e. zymogram gels; (Golonka, E., et al, (2001), 2nd General Meeting of the International Proteolysis Society, Freising, Germany, International Proteolysis Society)), but this assay system is limited in its scope. This was principally because of the limited number of samples that could be processed, the time-consuming assay protocols and an inability of the assay to be adapted for efficient screening of relatively large numbers of compounds. For the conventional protein-based solution assays, the drawbacks discussed have been overcome in a number of ways and variations have principally focused on reducing, eliminating or automating the post- assay processing step(s). The simplest solution has been the automation of the post- sample processing. This was generally achieved by using separation techniques amenable to high sample throughput, automatic sample manipulation and automated data analysis (e.g. high performance liquid chromatography (HPLC)). Another method for overcoming this post-assay processing was to monitor the amount of casein remaining in the assay mixture. Azodye (e.g. Coomasie R250) binding to proteins produces a bathochromic shift and hyperchromic affect in the dye absorption spectra, which can be used to measure protein concentration. With reference to casein hydrolysis, as the protein was degraded less dye was bound resulting in less colour. This assay has been adapted to monitor peptidase activity (Fournier, B. and Hooper, D. C, (2000), J Bacteriol, 182 (14), 3955-3964; Sloot, N., et al, (1992), J Med Microbiol, 37 (3), 201-205). However, this still has the disadvantage that the assay was a stopped-time assay and numerous samples had to be manipulated in order to obtain event the simplest information (e.g. a number of data points have to be collected to obtain a basic rate). Coupled to this, product generation was divorced from quantification. A key requirement has therefore been to couple substrate conversion with signal generation. A much more sensitive alternative to this has been the use of fluorescence-based assays (Potempa, J., et al, (1988), JBiol Chem, 263 (6), 2664-2667; Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Eugene, Molecular Probes, Inc.), where the fluorescent dye was covalently bound to the protein substrate. In its native state the dye was contained within the solvent-inaccessible protein structure and fluorescence was quenched, upon cleavage of the substrate, the dye becomes exposed to solvent and a fluorescence signal was produced. This assay has the advantage that substrate cleavage was concomitant with signal generation and therefore no post-assay sample processing was required. Also, depending on the fluorophore chosen, the assay can be extremely sensitive. However, a major drawback to these large macromolecular protein-based substrates, and even relatively small polypeptides, was their susceptibility to be cleaved at more than one peptide bond, making the nature of the substrate-enzyme interaction more complex. For example, if two cleavage sites were present in the same protein substrate, then they may not necessarily have been cleaved at the same rate. Added to this, the two cleavage sites may not have had the same affinity for the enzyme. Also cleavage of a particular bond in a protein may expose new substrate sites that were previously masked within the protein structure. This would have complicated an interpretation of the nature of the interaction between substrate and enzyme even more. Under these sorts of conditions, the observed rate was usually made up of the sum of a number of minor rates each attributable to different cleavage events. This also meant that in reality more than one substrate was present in the reaction and multiple products were generated. This therefore restricted description of the precise rate of substrate conversion to product and as such these types of substrate are not easily amenable to detailed kinetic and mechanistic characterisation of the interaction of a ligand with the enzyme. Additionally, many dye-labelled proteins were relatively insoluble restricting the upper concentration limits that may be used in an assay.

Based on the limitations discussed already, a necessary goal has been a direct assay for precisely quantifying staphylopain peptidase activity, where substrate cleavage was concomitant with signal generation and where only a single site within the peptide was cleaved. To this end, various semi-empirical approaches describing the elucidation of such substrates have been reported. Arvidson, et al, 1982 described the use of a N-benzoyl-L-Tyr-ethyl ester substrate for monitoring calpain esterase activity (Arvidson, S., et al, (1973), Biochim Biophys Acta, 302 (1), 135-148). However, a major drawback of ester-based substrates was their susceptibility to base- catalysed cleavage. As such, they were extremely sensitive to the pH adopted in the assay and were also susceptible to hydrolysis. This therefore restricted their practicality. Potempa, et al, 1986 identified CBZ-Phe-Leu-Glu-pαrα-nitro-anilide (CBZ-Phe-Leu-Glu-/?ΝA) as a substrate for extracellular S. aureus cysteine peptidase (Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664-2667) and more recently Oleksy, et. al, 2001 reported that staphylopain from S. epidermis also had a Glu substrate preference (Oleksy, A., et al, (2001), 2nd General Meeting of the International Proteolysis Society, Freising, Germany, International Proteolysis Society). Although this substrate did not possess the disadvantages of the para- nitrophenyl ester substrates, the relatively poor kinetic parameters against the cysteine peptidase made the assay insensitive (CBZ-Phe-Leu-Glu-pNA K_M- 0.5 mM; k_cΛ = 0.16 s^"1; k_c K_u = 320 M^"V (Potempa, J., et al, (1988), JBiol Chem, 263 (6), 2664-2667)). Conversely, the kinetic parameters of this substrate for the V8 protease were relatively good (CBZ-Phe-Leu-Glu-pNA .K_M=0.286 mM; k_cat = 6.6 s^"1; k_c K_u = 23,077 M^'V¹ (Potempa, J., et al, (1988), J Biol Chem, 263 (6), 2664- 2667)). On the basis of the second order rate constants, it was possible that even a minor contaminant (i.e. <0.1%) due to the V8 protease could have accounted for the observed activity. However Potempa, 1988 reported that this activity was only observed in the presence of reducing agent. Nevertheless, this did not overcome the lack of sensitivity or specificity for CBZ-Phe-Leu-Glu-pNA as a substrate for monitoring staphylopain peptidase activity. More recently the substrate specificity of SspB cysteine protease has been reported as having a preference for a hydrophobic residue at P2 and a strong preference for arginine at PI (Massimi, I., et al, (2002), J. Biol. Chem., Ill, 41770-41777).

The invention provides substrates for Staphylococcus sp. extracellular cysteine peptidase, which are useful in monitoring enzyme activity. The substrates are particularly useful in the investigation of candidate modulators, particularly inhibitors, of enzyme activity, and as such may be useful in screening for potentially useful therapeutic agents.

According to a first aspect of the invention, there is provided a peptide cleavable by Staphylococcus sp. extracellular cysteine peptidase, the peptide being selected from the following: A. A compound of general formula (I)

Y-A'-A²-A³-A⁴-R-Glu-NH₂ (I) wherein

Y and R are reporter groups;

A¹ is either the D-isomer or the L-isomer form of isoleucine; each of A² and A³ is independently Ala or 2-aminobutyric acid (Abu); A⁴ is Pro, L-3-hydroxyproline (Pro(3-OH)), L-4-hydroxyproline (Pro (4-OH)), L- tetrahydroisoquinoline-1 -carboxylic acid (THIQ), L-tetrahydroisoquinoline-3- carboxylic acid (Tic), L-pipecolic acid (Pip), 1 -amino- 1-cyclopentane carboxylic acid (1-ACP) or D-Pro; and any one of A¹, A², A³ and A⁴ can be replaced by any other amino acid group (Xaa); or

B. one of the following compounds:

Abz-Ile-Ala-Pro-Arg-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Leu-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂;

Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂.

Abz-Ile-Ala-Ser-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ Abz-Tyr-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Phe-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Lys-Ala-Tyr(3-NO₂)-Glu-NH₂ Abz-Tyr-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Glu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂

Abz-Ue-Ala-Thr-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Phe-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Tyr-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂ Abz-Leu-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Ala-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Asp-Val-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Tyr-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Phe-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

wherein

2-Thi represents β-(2-thienyl)-L-alanine

Abz is 2-aminobenzyl;

Tyr(3-NO₂) is 3-nitrotyrosine;

or a variant of one of these in which Abz and Tyr(3-NO₂) are replaced by other groups Y and R as defined for general formula (I); or

C. a compound of general formula (II):

CAP-A'-A²-L (II) wherein:

A¹ and A² are as defined for general formula (I);

CAP is Boc-, N-acetyl-, 4-morpholino-, Benzoyl-, cyclohexoyl-, benzyloxycarbonyl- (CBZ-) or Y, where Y is as defined above for general formula (I); and L is a leaving group.

Optionally, one or more of the following compounds may be excluded from general formula (I): Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Leu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Ala-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Ala-Gln-Pro-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Ala-Thr-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂; Abz-Tyr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂.

However, in that case, these compounds comprise a separate aspect of the present invention.

In general formula (I) described in A above, it is preferred that, independently or in any combination: A¹ is the L-isomer form of isoleucine;

A² is Ala; A³ is Ala; and A⁴ is Pro.

The reporter groups Y and R in general formula (I) and the variants of the compounds of B are preferably groups which can be monitored by a biophysical or chemical method such as fluorimetry, colourimetry, derivatisation (e.g. fluorescamine {Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, Oregan, USA}) or chromatography (for example HPLC).

In one embodiment, Y and R may be a FRET pair. In the intact peptide, the fluorescence is quenched because the absorption spectrum of one member of the FRET pair overlaps with the emission spectrum of the other member, but when the peptide is cleaved by the enzyme, the fluorescence is no longer quenched. This enables product to be distinguished from substrate and the course of the reaction to be monitored.

Preferred Y groups include Abz, 4-aminobenzyl (4-Abz), TFA.NH₂(CH₂)_nCO, TFA.NH₂(CH₂)„CO-Abz, TFA.NH₂(CH₂)_nCO-4-Abz, Abz-Allo, 4-Abz-Allo, Abz- Xaa or 4-Abz-Xaa; n is an integer from 2 to 8;

Xaa is any amino acid residue.

All of these Y groups are fluorogenic, apart from TFA.NH₂(CH₂)_nCO, which can be monitored by a chromatographic method such as HPLC.

Fluorogenic Y groups may, as mentioned above, form a FRET pair with the group R. In the case of the Y groups mentioned above, it is preferred that R is Tyr(3-NO₂).

Abz and Tyr(3-NO₂) can be replaced by any FRET pair described in the prior art (Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, Oregan, USA; Wu, P. and Brand, L., (1994), Anal Biochem, 218(1), 1-13).

When Y and R do not form part of a FRET pair, they may be leaving groups, L (as defined in C above), which are preferably chromogenic or fluorogenic. Examples of such leaving groups include acridines (e.g. acridine orange), coumarins (e.g. 1- amido-4-methyl coumarin (AMC)), dipyrrines, oxazenes (e.g. cresyl violet), xanthenes (e.g. rhodamine 123), naphthylamides, p-nitroanilides, p-nitro- phenylesters, etc.. Other suitable chromogenic and fluorogenic leaving groups are described in the prior art (Haugland, R. P., (2002), Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, Oregan, USA; Bachem (UK), St. Helens, Merseyside, U.K.).

The above peptides are listed in decreasing order of preference. Among the most preferred peptides are:

Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Ala-Pro-Arg-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Leu-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂; Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂.

Preferred compounds are compounds of formula (I), particularly those in which: Y is Abz, 4-Abz, TFA.NH₂(CH₂)_nCO, TFA.NH₂(CH₂)_nCO-Abz, TFA.NH₂(CH₂)_nCO-4-Abz, Abz-Allo, 4-Abz-Allo, Abz-Xaa or 4-Abz-Xaa; n is an integer from 2 to 8;

Xaa is any amino acid residue; and R is Tyr(3-NO₂).

In addition to Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂;

listed above, other preferred compounds of formula I include the compounds specified in Examples 6 to 128 below, i.e.

TFA.H₂N-(CH₂)nCO-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH2 (n=2-7); the compounds where n=2-5 and 7 are designated (6-10) TFA.H₂N-(CH2)nCO-4Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH2 (n=2-7) ; the compounds where n=2-5 and 7 are designated (11-16) Abz-Gly-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (17) Abz-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (18) Abz-Val-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (19) Abz-Leu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (20) Abz-Ile-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (21) Abz-Phe-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (22) Abz-Tyr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (23) Abz-T -Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (24) Abz-Ser-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (25) Abz-Thr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (26) Abz-Cys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (27) Abz-Met-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (28) Abz-Asn-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (29) Abz-Gln-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (30) Abz-Asp-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (31) Abz-Glu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (32) Abz-Lys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (33) Abz-Arg-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (34) Abz-His-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (35) Abz-β-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (36) Abz-D-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (37) Abz-Ile-Ala-Ala-Thr-Tyr(3-NO₂)-Glu-NH₂ (38) Abz-Ile-Ala-Gly-Pro-Tyr(3-NO₂)-Glu-NH₂ (39) Abz-Ile-Ala-Thr-Pro-Tyr(3-NO₂)-Glu-NH₂ (40) Abz-Ile-Gly-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (41) Abz-Ile-Thr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (42) Abz-Gly-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (43) Abz-Thr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (44) Abz-D-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (45) Abz-Allo-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (46) Abz-Ile-Abu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (47) Abz-Ile-Ala-Abu-Pro-Tyr(3-NO₂)-Glu-NH₂ (48) Abz-Ile-Ala-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (49) Abz-Ile-Ala-Ala-Met-Tyr(3-NO₂)-Glu-NH₂ (50)

Abz-Ile-Ala-Met-Pro-Tyr(3-NO₂)-Glu-NH₂ (51)

Abz-Ile-Met-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (52)

Abz-Ala-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (53) Abz-Met-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (54)

Abz-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (55)

Abz-Allo-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (56)

Abz-Ile-Abu-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (57)

Abz-Ile-Ala-Abu-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (58) Abz-Ile-Ala-Ala-Val-Tyr(3-NO₂)-Glu-NH₂ (59)

Abz-Ile-Ala-Ala-Asn-Tyr(3-NO₂)-Glu-NH₂ (60)

Abz-Ile-Ala-Val-Pro-Tyr(3-NO₂)-Glu-NH₂ (61)

Abz-Ile-Ala-Asn-Pro-Tyr(3-NO₂)-Glu-NH₂ (62)

Abz-Ile-Val-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (63) Abz-Ile-Asn-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (64)

Abz-Val-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (65)

Abz-Asn-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (66)

Abz-Ile-Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (67)

Abz-Allo-Ile-Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (68) Abz-Ile-Abu-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (69)

Abz-Ile-Ala-Abu-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (70)

Abz-Ile-Ala-Ala-Leu-Tyr(3-NO₂)-Glu-NH₂ (71)

Abz-Ile-Ala-Ala-Gln-Tyr(3-NO₂)-Glu-NH₂ (72)

Abz-Ile-Ala-Leu-Pro-Tyr(3-NO₂)-Glu-NH₂ (73) Abz-Ile-Ala-Gln-Pro-Tyr(3-NO₂)-Glu-NH₂ (74)

Abz-Ile-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (75)

Abz-Ile-Gln-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (76)

Abz-Leu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (77)

Abz-Gln-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (78) Abz-Ile-Ala-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (79)

Abz-Allo-Ile-Ala-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (80)

Abz-Ile-Abu-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (81) Abz-Ile-Ala-Abu-THIQ-Tyr(3-NO₂)-Glu-NH₂ (82) Abz-Ile-Ala-Ala-Ile-Tyr(3-NO2)-Glu-NH₂ (83) Abz-Ile-Ala-Ala-Asp-Tyr(3-NO₂)-Glu-NH₂ (84) Abz-Ile-Ala-Ile-Pro-Tyr(3-NO₂)-Glu-NH₂ (85) Abz-Ile-Ala-Asp-Pro-Tyr(3-NO₂)-Glu-NH₂ (86) Abz-Ile-Ile-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (87) Abz-Ile-Asp-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (88) Abz-Asp-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (89) Abz-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (90) Abz-Allo-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (91) Abz-Ile-Abu-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (92) Abz-Ile-Ala-Abu-Tic-Tyr(3-NO₂)-Glu-NH₂ (93) Abz-Ile-Ala-Ala-Phe-Tyr(3-NO₂)-Glu-NH₂ (94) Abz-Ile-Ala-Ala-Glu-Tyr(3-NO₂)-Glu-NH₂ (95) Abz-Ile-Ala-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂ (96) Abz-Ile-Ala-Glu-Pro-Tyr(3-NO₂)-Glu-NH₂ (97) Abz-Ile-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (98) Abz-Ile-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (99) Abz-Phe-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (100) Abz-Glu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (101) Abz-Ile-Ala-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (102) Abz-Allo-Ile-Ala-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (103) Abz-Ile-Abu-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (104) Abz-Ile-Ala-Abu-Pip-Tyr(3-NO₂)-Glu-NH₂ (105) Abz-Ile-Ala-Ala-Tyr-Tyr(3-NO₂)-Glu-NH₂ (106) Abz-fle-Ala-Ala-Lys-Tyr(3-NO₂)-Glu-NH₂ (107) Abz-Ile-Ala-Tyr-Pro-Tyr(3-NO₂)-Glu-NH₂ (108) Abz-Ile-Ala-Lys-Pro-Tyr(3-NO₂)-Glu-NH₂ (109) Abz-Ile-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (110) Abz-Ile-Lys-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (111) Abz-Tyr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (112) Abz-Lys-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (113) Abz-Ile-Ala-Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (114)

Abz-Allo-Ile-Ala-Ala-l-ACP-Tyr(3-NO₂)-Glu-NH₂ (115)

Abz-Ile-Abu-Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (116)

Abz-Ile-Ala-Abu-l-ACP-Tyr(3-NO₂)-Glu-NH₂ (117) Abz-Ile-Ala-Ala-Ser-Tyr(3-NO₂)-Glu-NH₂ (118)

Abz-Ile-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (119)

Abz-Ile-Ala-Pro-Pro-Tyr(3-NO₂)-Glu-NH₂ (120)

Abz-Ile-Ser-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (121)

Abz-Ile-Pro-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (122) Abz-Ser-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (123)

Abz-Pro-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (124)

Abz-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (125)

Abz-Allo-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (126)

Abz-Ile-Abu-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (127) Abz-Ile-Ala-Abu-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (128)

Abz-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (156)

Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂ (171)

where "Allo" (for example in Allo-Ile) indicates an amino acid in which the C-alpha carbon stereochemistry remains the same as in the naturally occuring form but the beta-carbon stereochemistry is inverted.

Other preferred compounds are peptide derivatives of general formula (II), particularly those in which, independently or in combination A¹ is the L-isomer form of isoleucine and A² is Ala, Cys or Tyr, with Ala being most preferred.

The following compounds of general formula (II) are especially preferred: Boc-Ile-Ala-AMC (129) HCl.Ile-Ala-AMC (130) N-acetyl-Ile-Ala-AMC (131) 4-morpholino-Ile-Ala-AMC (132)

Benzoyl-Ile-Ala-AMC (133) cyclohexoyl-Ile-Ala-AMC (134) CBZ-Ile-Ala-AMC (135) Boc-Ile-Cys(Bzl)-AMC (136) CBZ-Ile-Cys(Bzl)-AMC (137) Boc-Ile-Tyr-AMC (138) CBZ-Ile-Tyr-AMC (139).

wherein "CBZ" represents benzyloxycarbonyl and "AMC" represents 7- amido-4-methyl-coumarin;

or a variant of one of the above in which AMC is replaced by an alternative leaving group L.

The peptides can by synthesised by those skilled in the art using solid phase techniques, as detailed in the examples.

According to a second aspect of the invention, there is provided a method of assaying Staphylococcus sp. extracellular cysteine peptidase activity, the method comprising allowing the enzyme to catalyse the cleavage of a peptide as described above.

Another feature of the present invention is the use of such substrates for monitoring cysteine peptidase activity during protein purification. It is known to those skilled in the art that protein purification usually involves selective partitioning of protein samples using biophysical techniques. In order to differentiate between fractions that contain the target activity and those that do not, enzyme activity is the most convenient method for selecting cysteine peptidase-containing fractions.

In another feature of the invention, the peptidase activity may be assayed in the presence of a candidate modulator (enhancer or inhibitor) of peptidase activity, in which case the assay functions as a screen of such candidate compounds. Inhibitors of Staphylococcus sp. extracellular cysteine peptidase may have valuable pharmacological properties. Such a screening method itself forms an aspect of the invention. In another aspect of the invention, the peptide substrates identified for Staphylococcus sp. extracellular cysteine peptidase may be elaborated using methods described in the prior art to produce mechanism-based inhibitors (Otto, H-H and Schirmeister, T., (1997, Chem. Rev., 97, 133-171 ; Veber, D.F. and Thompson, S.K., (2000), Curr. Opinion in Drug Discovery & Development, 3(4), 362-369; Leung, D, et al, (2000), J. Med. Chem., 43(3), 305-332). Such compounds may be used as biological tools or may in themselves form the basis of pharmaceutically active compounds.

Therefore, in a further aspect of the invention there is provided a compound of general formula (III):

CAP-A'-A^G (III)

wherein:

1

A and A are as defined for general formula (I);

CAP is as defined for general formula (II); and

G is a protease inhibitor cap, for example an aldehyde, a Michael acceptor (e.g. vinyl sulfone) or an epoxide.

As with general formula (II), preferred compounds of general formula (III) are those in which A¹ is the L-isomer form of isoleucine and/or A² is Ala, Cys or Tyr, with Ala being most preferred.

Compounds of general formula (III) may be used in medicine, in particular for inhibiting Staphylococcus sp. extracellular cysteine peptidases and in the treatment of staphylococcal infection. Therefore, the compounds are of use in the preparation of an agent for the treatment of staphylococcal infection.

Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis. Preferred embodiments and examples of the invention will now be described with reference to the accompanying drawings, in which:

Figure 1 shows a plot of initial rate (VJ) versus substrate concentration for enzyme- catalysed reactions following Michaelis-Menten kinetics (Michaelis, L. and Menten, M. L., (1913), Biochem. Z., 49 333).

Figure 2 shows an example of a fluorescence progress curve for the novel FRET substrate Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (2).

Figure 3 shows the Michaelis-Menten plot for an example of the novel substrates of the present invention, Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (2).

Figure 4 shows the Michaelis-Menten plot for casein-BODIPY FL.

Figure 5 shows the Michaelis-Menten plot for elastin-BODIPY FL.

Figure 6 shows monitoring peptidase inactivation by an irreversible inhibitor (E-64) using novel substrate Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (2).

Figure 7 shows determination of the second order inactivation rate constant for the irreversible inhibitor E-64.

Figure 8 shows monitoring peptidase inactivation by a reversible inhibitor (leupeptin) using the novel substrates Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (2).

Figure 9 shows the K\ determination for the reversible inhibitor leupeptin using the novel substrate Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (2).

Background to enzyme kinetics and substrate specificity

The enzyme-catalysed conversion of substrate to product has been the subject of extensive study (Cannon, W. R., et al, (1996), Nat Struct Biol, 3 (10), 821-833; Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company, ; Comish-Bowden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd., ; Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). Crucial to many of these studies has been the identification and classification of natural and synthetic substrates, together with the determination of whether substrates may be considered to be general or specific for a given enzyme. The availability of a substrate has been crucial to the characterisation of an enzyme and as such, the lack of substrate can seriously hamper progress in the study of an enzyme. This requirement for a substrate becomes crucial in any drug discovery programme since substrate turnover is the means by which compound inhibition is measured and quantified. In its simplest interpretation, a substrate molecule is said to be specific when it is the only substrate available for conversion to a product by a particular enzyme. Although this may be the case for some enzyme-catalysed reactions, it is rarely the case. Practically, many enzymes and in particular peptidases, exhibit a spectrum of activity against a wide range of substrates. This feature, plus the requirement to categorise substrates, led to the development of various biochemical methods for the classification of substrate preferences.

It is found experimentally that at low substrate concentrations, the initial rate of product formation is linear with increasing substrate concentration (Figure 1). However as the substrate concentration increases this relationship breaks down and a saturating rate is observed. This relationship is described by Equations 1 and 2 (Figure 1).

V_mm = [E₀].k_cat (Eq. 2)

In Equation 1, 'V_J' is the observed initial rate, 'V_mΛ is the observed maximum activity at saturating substrate concentration, ^lK_M' in this case is defined as substrate concentration at ^lΛVmax (Fig. 1), '[S„]' is the initial substrate concentration. In Equation 2 '[E„]' is the active enzyme concentration and ' r_cat' is the turnover number.

Interpretation of kinetic parameters for single substrate reactions The Michaelis-Menten mechanism of enzyme-catalysed reactions is the foundation of enzyme kinetics and is described by the general Scheme 1. The reaction is made up of two steps, the first is the binding phase between enzyme ('E') and substrate ('S') leading to the formation of the enzyme-substrate complex ('ES'). The second step is conversion of the enzyme-substrate complex to enzyme plus product ('P').

E + S ES—^→E + P (Scheme 1)

Under Scheme 1 , the dissociation constant of the enzyme-substrate complex (K_s) is assumed to be rapid and reversible and in this case is equal to K_M- In cases where free enzyme and substrate are not in rapid equilibrium with the enzyme-substrate complex, then the Michaelis-Menten mechanism no longer holds true. This second situation is best described by Scheme 2, where k₂« _\ (Briggs, G. Ε. and Haldane, J. B. S., (1925), Biochem. J, 19 338).

- E + S ES— ^- E + (Scheme 2)

Under the general Scheme 2, the rate equation is identical to Εq. 1, but in this case K >K and reduces to the definition given in Equation 3 (Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company).

These simplified enzyme mechanisms become more complicated as the number of steps in the conversion of substrate to product increases. Additionally, for many enzymes, multiple substrates may be converted into multiple products. This further complicates derivation of the various kinetic parameters and an example of a comprehensive treatise on enzyme mechanisms has been published elsewhere (Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). Specifically in relation to the peptidase-catalysed conversion of peptide substrate to products, the general Scheme 3 applies. In Scheme 3, 'E' is the enzyme, 'S' is the substrate, 'PI' is the product generated from the molecular fragment C-terminal of the scissile bond, 'EAc' is the acyl-enzyme intermediate and 'P2' is the molecular fragment N- terminal of the scissile bond. For peptide amide substrates &₂ is rate limiting and as such is equivalent to k_cat, for peptide ester substrates &₃ is rate limiting and as such is equivalent to k_cat. Depending on the mechanism that applies, K can be defined as a function of the individual microscopic rate constants.

-

E + S ES EAc + P\ E + P2 (Scheme 3) t_, *_ k_,

Because of the time-consuming and practical difficulties in accurately determining the individual rate constants (e.g. Scheme 3), certain experimental shortcuts and assumptions are made in the determination of A_M and k_cat- In many cases, K_M is calculated from the Michaelis-Menten plot, monitoring initial rate as a function of substrate concentration (Figure 1). Therefore in these cases, K actually describes the macroscopic binding constant for the overall reaction and as such should be more accurately be referred to as K_M ^3PP- Similarly, the turnover number is also determined from the relationship described in Εq. 2, again from the Michaelis-Menten plot monitoring initial rate as a function of substrate concentration. In this case the amount of enzyme is converted into moles of protein as determined from the protein molecular mass. However, in order to accurately determine the value of k_cat, the precise mole amount of active enzyme must be determined. It is known, to those skilled in that art, that processing of proteins samples in order to obtain purified material usually results in the inactivation of a variable amount of the protein. Therefore, enzyme preparations may appear pure, for example by chromatographic or electrophoretic analysis, but may not necessarily be totally active. In the case of peptidases for example, if a protein preparation were to be fifty percent active, then the calculation of the turnover number would under-estimate the value of k_cat. Values based only on the amount of protein provide a lower limit for calculating the turnover number, however since the amount of active protein can vary dramatically from preparation to preparation and protein to protein, it is important to determine this value experimentally in order to obtain an accurate value for k_cat. Added to this, protein concentrations are usually determined by dye-binding methods rather than by amino acid analysis. This tends to result in an error in the determination of protein concentration, which translates into an error in £_Cat- The amount of active enzyme is best determined by active-site titration using an irreversible mechanism-based molecular probe whose chemical composition is either accurately determined or known (Silverman, R. B., (1996), Contemporary enzyme kinetics and mechanism, London, Academic Press, 291-334).

Substrate specificity

The term 'specificity' is best encapsulated by the value of the second order rate constant for the conversion of substrate to product. This value is generally calculated from the value

where k_cΛ is the unimolecular first-order rate constant for the conversion of enzyme-substrate complex to enzyme-product complex and K_M is the macroscopic binding constant, or Michaelis constant, for the substrate (Cannon, W. R., et al, (1996), Nat Struct Biol, 3 (10), 821-833; Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company, ; Cornish- Bowden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd., ; Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). The turnover number k_cat is expressed in units of per second (i.e. s^"1) and is the number of moles of substrate converted to product by each mole of active enzyme per second (Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company, ; Cornish-Bowden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd., ; Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). The value of K_M is expressed as a concentration (i.e. molar (M)) and is equivalent to the substrate concentration required to exhibit half-maximal activity (Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company, ; Cornish-Bowden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd., ; Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). In its simplest interpretation, the maximal observed activity (K_max ^app) is, under saturation kinetics (either hyperbolic or sigmoidal kinetics), the rate observed at infinite substrate concentration as calculated from the Michaelis-Menten equation 1. More accurately, the true maximal velocity (i.e. V_max) can be calculated from the relationship in equation 2, where at saturating substrate concentration V_max equals [E]₀.&_cat; where '[E]₀' is the active enzyme concentration. This however requires that the accurate concentration of active enzyme and the precise value of λr_cat are known. The parameter k_c K_M is therefore the bimolecular second-order rate constant for the conversion of substrate to product and is expressed in units of 'M^'V¹'. Its value is restricted by the rate of diffusion to less than or equal to 10⁸ M^'V¹ (Cannon, W. R., et al, (1996), Nat Struct Biol, 3 (10), 821-833; Fersht, A., (1985), Enzyme Structure and Mechanism, New York, W.H. Freeman and Company, ; Cornish-Bo wden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd., ; Segal, I. H., (1993), Enzyme Kinetics, John Wiley & Sons Inc). The kinetic parameters K_M and k_cat are mutual terms that define affinity of a substrate for an enzyme and the rate at which substrate is converted to product, respectively. For example, compounds 'A' and 'B' may have the same affinity (i.e. K_M) for an enzyme, but compound A may be converted at a faster rate (i.e. it has a higher k_cat value) than compound B. In this case, because compound A would have the higher second order rate constant (i.e. K_M), it would be considered to be more specific for the enzyme than compound B. Similarly, where two molecules have the same £_Cat value, the one that binds tighter (i.e. lower K_M value) would be considered to be more specific, again because it would have the higher second order rate constant (i.e. k_cat/K_M). Substrates are therefore categorised by the value of the second order rate constant and those with the higher value may be considered to be more specific for an enzyme than those with lower values.

Because of the time-consuming and practical difficulties in accurately determining the individual rates constants (e.g. Scheme 3), certain experimental shortcuts and assumptions are made in the determination of K_M and k_cat. Therefore, K_M is re¬

calculated from the Michaelis-Menten plot, monitoring initial rate as a function of substrate concentration. Practically, K_M is calculated empirically from the relationship described in Eq. 1 (Figure 1) and should more accurately be referred to as A_M ^app. The turnover number is calculated from an active-site titration. In this case the second order rate constant should more accurately be described as '&_cat/A^'M^app'.

EXAMPLES

General Experimental Methods All solvents were purchased from ROMIL Ltd. (Waterbeach, Cambridge, UK) at SpS or 'Hi-Dry' grade unless otherwise stated. General peptide synthesis reagents were obtained from Chem-Impex International Inc. (Wood Dale, IL 60191, USA). Amino acids and/or derivatives were purchased from Chem-Impex International Inc. (Wood Dale, IL 60191, USA), Bachem UK (Bachem UK, St. Helens, Merseyside, U.K.), CN Biosciences (CN Biosciences, Beeston, U.K.) or Neosystems (SNPE England, Croydon, Surrey, U.K.) are of the L-configuration unless otherwise stated. All 7-amido-4-methylcoumarin (AMC) based substrates and O-succinimidyl esters were purchased from Bachem UK. Analytical HPLC was carried out on a Jupiter C4 column (5 μ, 300 A; 4.6 x 250 mm; Phenomenex) at 1.5 ml/min. using the following gradient: 0-2 min., 10% buffer B; 2- 27 min, 10-90% buffer B; 27-32 min., 90% buffer B; 32-35 min., 90-10% buffer B; 35-45 min., 10% buffer B. Buffer A consisted of 0.1% TFA in water and buffer B consisted of 90% acetonitrile containing 0.1% TFA plus 10% buffer A. HPLC-MS analysis was performed on Agilent 1100 series LC/MSD, using an automated HPLC system (HP 1100 system; Agilent Technologies, Bracknell, UK). Column elution was carried out at 0.4 ml/min. on a Columbus C8 column (5μ, 30θA, 50 x 2.0 mm; Phenomenex) using the following gradient: 0-7 min., 10-90% buffer B; 7-8.5 min, 90% buffer B; 8.5-8.7 min., 90-10% buffer B; 8.7-11 min., 10% buffer B. The mass spectrometer was set to API-ES ionisation mode, positive polarity; scanning in the 100-1500 Da mass range with a gas temperature was set to 350°C. Semi-preparative HPLC purification of crude samples was performed on Phenomenex Jupiter C4 (5μ, 300 A; 10 x 250 mm) using a linear increasing gradient of solvent B in solvent A using the gradient indicated, at 4.0 ml/min. The eluant absorbance was monitored at 215 nm and fractions collected manually, analysed and then lyophilised.

Example 1 - Synthesis of Compounds (1) to (36) General Solid Phase Peptide Synthesis Methods

Substrates, utilizing fluorescence resonance energy transfer methodology (FRET- based substrates), were synthesized at Incenta Limited using general solid phase protocols (Atherton & Sheppard, (1989), Solid Phase Peptide Synthesis, IRL Press, Oxford, U.K; Meldal, M. and Breddam, K., (1991), Anal. Biochem., 195, 141-147).

Peptide synthesis was carried out in 5 ml polypropylene plastic syringes fitted with an end-cap, a teflon scinter and stopper. Resin (NOVASYN TGR resin; 0.2 mmol/g; CN Biosciences) was added to the syringe as required. Resin was solvated in dimethylformamide (DMF) (3-5ml) on a rotating bed (SRT1 , Stuart Scientific; Fisher Scientific, Loughborough, Leicestershire, U.K) for approximately 10 min. Peptide synthesis was carried out in repetitive cycles consisting of a coupling step, a reagent wash step, an 9-fluorenylmethyloxycarbonyl (Fmoc) de-protection step, a wash step followed by the next coupling round. Between each step, excess reagent and solvent were removed by application of a vacuum. Each coupling step was commenced by activating a three-mole excess of Fmoc-amino acid (with respect to total resin loading capacity) via 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexofluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N- methylmorpholine (NMM). The activation mixture was pre-mixed in DMF (2-3 ml) for 30 s and the coupling step initiated by addition to the drained resin. The syringe was capped and agitated on the rotating bed for 1 h. The excess reagents were then drained (by application of the vacuum) and the bed washed with 6 x DMF (5 ml per wash). Fmoc deprotection then commenced by continuously washing the resin bed, under gravity flow, for 10 min. with piperidine:DMF (20:80, vol./vol.). The excess reagents were again drained (by application of the vacuum) and the bed washed with 10 x DMF (5 ml per wash). The resin was drained as before and ready for the next round of coupling. Upon completion of the sequence, substrates were cleaved with one of two cleavage cocktails for 75 min. For peptides containing Arg, His, Trp or Cys residues, a cocktail of 92.5% TFA:2.5% triisopropylsilane:2.5% water:2.5% ethanedithiol (40 ml/g resin) was used. For peptides not containing these residues, a cocktail of 95% TFA:2.5% triisopropylsilane:2.5% water was used. The resin was removed by filtration and the filtrate concentrated by sparging with nitrogen gas. The crude products were precipitated by addition of 50 ml cold methyl tert-butyl ether (MTBE), the sample shaken vigorously and the precipitate collected by centrifugation (5500 r.p.m. for 5 min). The supernatant was discarded and the process repeated. The final crude products were re-dissolved in 50:50 (vol./vol.) acetonitrile: water and analysed by RP-HPLC-MS. Crude products at >97% purity by UV analysis, were subsequently lyophilised. When required, poorer quality crude products were purified by semi-preparative HPLC and desired fractions pooled then lyophilised.

Synthesis of Abz-Ile- Ala-Pro- Arg-Tyr(3-NO₂)-Glu-NH₂ (1)

Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.1 g, 20 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (25.5 mg, 60 μmol), Fmoc-Tyr(3-NO₂)-OH (26.9 mg, 60 μmol), Fmoc-Arg(Pmc)-OH (39.8 mg, 60 μmol), Fmoc-Pro-OH (20.2 mg, 60 μmol), Fmoc-Ala-OH (18.7 mg, 60 μmol), Fmoc-Ile-OH (21.2 mg, 60 μmol), and Boc-2-Abz-OH (14.2 mg, 60 μmol), each coupling step utilising HBTU (22.8 mg, 60 μmol), HOBt (9.2 mg, 60 μmol) and NMM (13.2 μl, 120 μmol).

Substrate (1) was cleaved and lyophilised, yield 9.01 mg (9.9 μmol, 49%), ESI-MS 456.1 [M + 2H]²⁺ , 911.2 [M + H]⁺ (calc. Mw 909.98) with Rt 11.32 min (>97 %).

Synthesis of Abz-Ile- Ala- Ala-Pro-Tyr(3-NO₂)-Glu-NH2 (2)

Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.5 g, 100 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (127.6 mg, 300 μmol), Fmoc-Tyr(3-NO₂)-OH (134.5 mg, 300 μmol), Fmoc-Pro-OH (101.2 mg, 300 μmol), Fmoc-Ala-OH (93.3 mg, 300 μmol), Fmoc-Ala-OH (93.3 mg, 300 μmol), Fmoc-Ile-OH (106.0 mg, 300 μmol) and Boc-2-Abz-OH (71.1 mg, 300 μmol), each coupling step utilising HBTU (113.7 mg, 300 μmol), HOBt (45.9mg, 300 μmol) and NMM (66 μl, 600 μmol).

Substrate (2) was cleaved with 95% TFA:2.5% triisopropylsilane:2.5% water for 75 mins. and worked up as detailed above. The crude product was lyophilised, yield 31.86 mg (38.6μmol, 12.9%), ESI-MS 413.6 [M + 2H]²⁺ , 826.2 [M + H]⁺ (calc. Mw 824.87) with Rt 11.89 min (>97%).

Synthesis of Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂ (3) Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.1 g, 20 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (25.5 mg, 60 μmol), Fmoc-Tyr(3-NO₂)-OH (26.9 mg, 60 μmol), Fmoc-Pro-OH (20.2 mg, 60 μmol), Fmoc-Phe-OH (23.2 mg, 60 μmol), Fmoc-Phe-OH (23.2 mg, 60 μmol), Fmoc-Ile-OH (21.2 mg, 60 μmol) and Boc-2-Abz-OH (14.2 mg, 60 μmol), each coupling step utilising HBTU (22.8 mg, 60 μmol), HOBt (9.2 mg, 60 μmol) and NMM (13.2 μl, 120 μmol).

Substrate (3) was cleaved and lyophilised, yield 11.51 mg (11.78μmol, 59%), ESI- MS 489.6 [M + 2H]²⁺ , 978.2 [M + H]⁺ (calc. Mw 977.02) with Rt 17.12 min (>97%).

Synthesis of Abz-Ile-Leu-Thi-Glv-Tyr(3-NO₂)-Glu-NH₂ (4)

Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.1 g, 20 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (25.5 mg, 60 μmol), Fmoc-Tyr(3-NO₂)-OH (26.9 mg, 60 μmol), Fmoc-Gly-OH (17.8 mg, 60 μmol), Fmoc-2-Thi-OH (Fmoc-2-thienylalanine, ex Chem-Impex, 23.6 mg, 60 μmol), Fmoc-Leu-OH (21.2 mg, 60 μmol), Fmoc-Ile-OH (21.2 mg, 60 μmol) and Boc-2-Abz-OH (14.2 mg, 60 μmol), each coupling step utilising HBTU (22.8 mg, 60 μmol), HOBt (9.2 mg, 60 μmol) and NMM (13.2 μl, 120 μmol).

Substrate (4) was cleaved and lyophilised, yield 6.5 mg (7.1 μmol, 36%), ESI-MS 455.6 [M + 2H]²⁺ , 910.1 [M + H]⁺ (calc. Mw 909.0) with Rt 16.26 min (>97%). Synthesis of Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂ (5)

Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (0.1 g, 20 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (25.5 mg, 60 μmol), Fmoc-Tyr(3-NO₂)-OH (26.9 mg, 60 μmol), Fmoc-Arg(Pmc)-OH (39.8 mg, 60 μmol), Fmoc-Phe-OH (23.2 mg, 60 μmol), Fmoc-Tyr(tBu)-OH (27.6 mg, 60 μmol), Fmoc-Leu-OH (21.2 mg, 60 μmol) and Boc-2-Abz-OH (14.2 mg, 60 μmol), each coupling step utilising HBTU (22.8 mg, 60 μmol), HOBt (9.2 mg, 60 μmol) and NMM (13.2 μl, 120 μmol).

Substrate (5) was cleaved and lyophilised, yield 10.7 mg (10.17μmol, 50.8%), ESI- MS 527.2 [M + 2H]²⁺ , 1053.2 [M + H]⁺ (calc. Mw 1052.14) with Rt 15.25 min (>97%).

Synthesis of N-terminal extended analogues of Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu- NH₂ (6-10)

Following the general solid phase techniques detailed earlier, NOVASYN TGR resin (2.0 g, 400 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (510.6 mg, 1.2 mmol), Fmoc-Tyr(3-NO₂)-OH (538. lmg, 1.2 mmol), Fmoc-Pro-OH (404.9 mg, 1.2 mmol), Fmoc-Ala-OH (373.6 mg, 1.2 mmol), Fmoc-Ala-OH and (373.6 mg, 1.2 mmol), Fmoc-Ile-OH (424.1 mg, 1.2 mmol) each coupling step utilising HBTU (455.1 mg, 1.2 mmol), HOBt (183.7 mg, 1.2 mmol) and NMM (264.8 μl, 2.4 mmol).

The resin was de-protected, washed with methanol followed by MTBE and then allowed to dry by evaporation for 72 h at room temperature and stored until required.

A test cleavage of the sample yielded ESI-MS 707.3 [M + H]⁺, 354.2 [M + H]²⁺,

1413.6 [2M + H]⁺ (calc. Mw 707.3) with Rt 5.0 min (>97%).

Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3-

NO₂)-Glu-NOVASYN TGR resin (0.2 g, 40 μmol) was stepwise N-terminally capped with a range of straight chain Fmoc-NH-(CH₂)_n-COOH reagents (where n = 2

- 8), each individual coupling step utilising HBTU (45.5 mg, 120 μmol), HOBt (18.4 mg, 120 μmol) and NMM (26.5 μl, 240 μmol). Resins were the Fmoc de-protected and cleaved with the appropriate cleavage mixture, described above, to yield:

Where n = 2 (coupling with Fmoc-β-Ala-OH; 37.4 mg; 120 μmol), compound (6) was cleaved and lyophilised, yield 16.1 mg (20.7 μmol, 52%), ESI-MS 778.4 [M + H]⁺, 389.7 [M + 2H]²⁺ (calc. Mw 777.82) with Rt 6.27 min (>97%).

Where n - 3 (coupling with Fmoc-4-aminobutyric acid-OH; 39.0 mg; 120 μmol), compound (7) was cleaved and lyophilised, yield 16.1 mg (20.4 μmol, 51%), ESI- MS 792.4 [M + H]⁺, 396.7 [M + 2H]²⁺ (calc. Mw 791.85) with Rt 11.89 min (>97%).

Where n = 4 (coupling with Fmoc-5-aminopentanoic acid-OH; 39 mg; 120 μmol), compound (8) was cleaved and lyophilised, yield 14.88 mg (18.4 μmol, 46%), ESI- MS 806.4 [M + H]⁺, 403.7 [M + 2H]²⁺ (calc. Mw 805.88) with Rt 5.28 min. (>97%).

Where n = 5 (coupling with Fmoc-6-aminohexanoic acid-OH; 42.4 mg; 120 μmol), compound (9) was cleaved and lyophilised, yield 11.97 mg (14.6 μmol, 36.5%), ESI- MS 820.4 [M + H]⁺, 410.7 [M + 2H]²⁺ (calc. Mw 819.9) with Rt 5.01 min. (>97%). Where n=7 (coupling with Fmoc-8-aminooctanoic acid-OH; 45.8 mg; 120 μmol), compound (10) was cleaved and lyophilised, yield 16.26 mg (19.1 μmol, 48%), ESI- MS 848.4 [M + H]⁺, 424.8 [M + 2H]²⁺ (calc. Mw 847.96) with Rt 5.59 min. (>97%).

Synthesis of 4- Abz-Ile- Ala- Ala-Pro-Tyr(3-NO₂)-Glu-NOVASYN TGR Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (2.0 g, 400 μmol) was elaborated with Fmoc-4- Abz (285 mg; 1200 μmol), each coupling step utilising HBTU (455 mg, 1200 μmol), HOBt (184 mg, 1200 μmol) and NMM (265 μl, 2400 μmol).

A test sample of the resin was Fmoc de-protected and cleaved with the appropriate cleavage mixture, described above, to yield ESI-MS 826.4 [M + H]⁺, 413.7 [M + H]²⁺, 848.4 [M + Na]⁺ (calc. Mw 825.8) with Rt 5.48 min. (>97%).

Synthesis of N-terminal extended analogues of 4- Abz-Ile- Ala- Ala-Pro-Tyr(3-NO₂)- Glu-NH₂ (11-16)

Following the general solid phase techniques detailed earlier, 4-Abz-Ile-Ala-Ala-Pro- Tyr(3-NO₂)-Glu-NOVASYN TGR resin (0.2 g, 40 μmol) was stepwise N-terminally capped with a range of straight chain Fmoc-NH-(CH₂)_n-COOH reagents (where n = 2 - 8), each individual coupling step utilising N-[(dimethylamino)-lH- 1,2,3- triazolo[4,5-b]pyridin- 1 -ylmethylene]-Ν-methylmethanaminium hexafluorophosphate N-oxide [ΗATU; PE Biosystems, Warrington, U.K.] (152.1

mg, 400 μmol), 3H-l,2,3-triazolo-[4,5-b]pyridine-3-ol [ΗOAt; Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan] (54.4 mg, 400 μmol) and 2,4,6-collidine (105.9 μl, 800 μmol). Resins were the Fmoc de-protected and cleaved with the appropriate cleavage mixture, described above, to yield: Where n = 2 (coupling with Fmoc-β-Ala-OH; 124.5 mg; 400 μmol), compound (11) was cleaved and lyophilised, yield 9.66 mg (10.7 μmol, 27%), ESI-MS 897.4 [M + H]⁺, 449.2 [M + 2H]²⁺ (calc. Mw 896.9) with Rt 6.05 min. (>97%).

Where n = 3 (coupling with Fmoc-4-aminobutyric acid-OH; 130.2 mg; 400 μmol), compound (12) was cleaved and lyophilised, yield 12.5 mg (13.7 μmol, 34%), ESI- MS 911.4 [M + H]⁺, 456.2 [M + 2H]²⁺ (calc. Mw 910.9) with Rt 6.60 min (>95%).

Where n = 4 (coupling with Fmoc-5-aminopentanoic acid-OH; 135.8 mg; 400 μmol), compound (13) was cleaved and lyophilised, yield 15.2 mg (16.5 μmol, 41%), ESI- MS 925.4 [M + H]⁺, 463.3 [M + 2H]²⁺ (calc. Mw 925.0) with Rt 5.92 min (>95%).

Where n = 5 (coupling with Fmoc-6-aminohexanoic acid-OH; 141.4 mg; 400 μmol), compound (14) was cleaved and lyophilised, yield 16.2 mg (17.3 μmol, 43%), ESI- MS 939.4 [M + H]⁺, 470.3 [M + 2H]²⁺ (calc. Mw 939.0) with Rt 6.25 min (>95%).

Where n= 7 (coupling with Fmoc-8-aminooctanoic acid-OH; 152.6 mg; 400 μmol), compound (15) was cleaved and lyophilised, yield 8.5 mg (8.8 μmol, 22%), ESI-MS 967.5 [M + H]⁺, 484.3 [M + 2H]²⁺ (calc. Mw 967.0) with Rt 6.29 min (>95%).

Synthesis of Abz-Gly-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (17) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Gly-OH (11.1 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (17) was cleaved and lyophilised, yield 2.86 mg (3.2 μmol, 26%), ESI-MS 883.4 [M + H]⁺, 442.3 [M + 2H]²⁺, 905.3 [M+Na]⁺ (calc. Mw 882.8) with Rt 5.77 min (>97%).

Synthesis of Abz-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (18) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Ala-OH (11.7 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (18) was cleaved and lyophilised, yield 4.19 mg (4.7 μmol, 37%), ESI-MS 897.4 [M + H]⁺, 449.3 [M + 2H]²⁺, 919.4 [M + Na]⁺ (calc. Mw 896.8) with Rt 6.24 min (>97%).

Synthesis of Abz-Val-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (19) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Val-OH (12.7 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (19) was cleaved and lyophilised, yield 3.1 mg (3.4 μmol, 27%), ESI-MS 925.4 [M + H]⁺, 463.3 [M + 2H]²⁺, 947.4 [M + Na]⁺ (calc. Mw 924.8) with Rt 6.30 min (>97%).

Synthesis of Abz-Leu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (20) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Leu-OH (13.3 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (20) was cleaved and lyophilised, yield 5.3 mg (5.7 μmol, 45%), ESI-MS 939.4 [M + H]⁺, 470.2 [M + 2H]²⁺, 961.4 [M + Na (calc. Mw 938.8) with Rt 7.58 min (>97%).

Synthesis of Abz-Ile-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (21) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Ile-OH (13.3 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (21) was cleaved and lyophilised, yield 3.76 mg (4.0 μmol, 32%), ESI-MS 939.4 [M + H]⁺, 470.3 [M + 2H]²⁺, 961.4 [M + Na]⁺ (calc. Mw 938.8) with Rt 6.79 min (>97%).

Synthesis of Abz-Phe-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (22) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Phe-OH (14.5 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (22) was cleaved and lyophilised, yield 4.6 mg (4.7 μmol, 38%), ESI-MS 973.4 [M + H]⁺, 487.2 [M + 2H]²⁺, 995.4 [M + Na]⁺ (calc. Mw 972.8) with Rt 9.14 min (>97%).

Synthesis of Abz-Tyr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (23) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Tyr(tBu)-OH (17.2 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol). Substrate (23) was cleaved and lyophilised, yield 5.0 mg (5.1 μmol, 41%), ESI-MS 989.4 [M + H]⁺, 495.3 [M + 2H]²⁺, 1011.4 [M + Naf (calc. Mw 988.8) with Rt 8.47 min (>97%).

Synthesis of Abz-Trp-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (24)

Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Trp(Boc)-OH (19.7 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (24) was cleaved and lyophilised, yield 4.4 mg (4.4 μmol, 35%), ESI-MS 1012.4 [M + H]⁺, 506.8 [M + 2H]²⁺, 1034.4 [M + Naf (calc. Mw 1011.9) with Rt 7.99 min (>97%).

Synthesis of Abz-Ser-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (25) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Ser(tBu)-OH (14.4 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (25) was cleaved and lyophilised, yield 4.27 mg (4.7 μmol, 37%), ESI-MS 913.4 [M + H]⁺, 457.2 [M + 2H]²⁺, 935.3 [M + Na]⁺ (calc. Mw 912.8) with Rt 5.51 min (>97%).

Synthesis of Abz-Thr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (26) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Thr-OH (12.8 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol). Substrate (26) was cleaved and lyophilised, yield 4.78 mg (5.2 μmol, 41%), ESI-MS 927.4 [M + Hf , 464.2 [M + 2H]²⁺, 949.3 [M + Naf (calc. Mw 926.8) with Rt 5.56 min (>97%).

Synthesis of Abz-Cys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (27) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Cys(Trt)-OH (14.4 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (27) was cleaved and lyophilised, yield 4.64 mg (5.0 μmol, 40%), ESI-MS 929.4 [M + Hf , 465.2 [M + 2H]²⁺, 951.3 [M + Naf (calc. Mw 928.8) with Rt 6.15 min (>95%).

Synthesis of Abz-Met-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (28) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Met-OH (13.9 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (28) was cleaved and lyophilised, yield 4.35 mg (4.5 μmol, 36%), ESI-MS 957.3 [M + H]⁺, 479.3 [M + 2H]²⁺, 979.3 [M + Naf (calc. Mw 956.8) with Rt 8.34 min (>97%).

Synthesis of Abz- Asn-Ile- Ala- Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (29) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Asn(Trt)-OH (22.4 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (29) was cleaved and lyophilised, yield 3.39 mg (3.6 μmol, 29%), ESI-MS 940.4 [M + Hf , 470.8 [M + 2H]²⁺, 962.3 [M + Naf (calc. Mw 940.8) with Rt 5.61 min (>97%).

Synthesis of Abz-Gln-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (30) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Gln(Trt)-OH (22.9 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (30) was cleaved and lyophilised, yield 4.5 mg (4.7 μmol, 38%), ESI-MS 954.4 [M + Hf , 477.8 [M + 2H]²⁺, 976.3 [M + Naf (calc. Mw 953.8) with Rt 5.84 min (>97%).

Synthesis of Abz-Asp-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (31)

Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Asp(OtBu)-OH (15.4 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (31) was cleaved and lyophilised, yield 3.3 mg (3.5 μmol, 28%), ESI-MS 941.3 [M + Hf , 471.3 [M + 2H]²⁺, 963.3 [M + Naf (calc. Mw 939.8) with Rt 6.06 min (>97%).

Synthesis of Abz-Glu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (32) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Glu(OtBu)-OH (16.0 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (32) was cleaved and lyophilised, yield 3.72 mg (3.9 μmol, 31%), ESI-MS 955.4 [M + Hf , 478.4 [M + 2H]²⁺, 977.2 [M + Naf (calc. Mw 954.8) with Rt 5.71 min (>97%).

Synthesis of Abz-Lys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (33) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Lys(Boc)-OH (17.6 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (33) was cleaved and lyophilised, yield 3.96 mg (4.2 μmol, 33%), ESI-MS

954.4 [M + Hf , 477.8 [M + 2H]²⁺ (calc. Mw 953.8) with Rt 7.73 min (>97%).

Synthesis of Abz-Arg-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (34) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-Arg(Pmc)-OH (24.9 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (34) was cleaved and lyophilised, yield 4.38 mg (4.5 μmol, 36%), ESI-MS

982.5 [M + Hf , 491.8 [M + 2H]²⁺ (calc. Mw 981.8) with Rt 6.65 min (>97%).

Synthesis of Abz-His-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (35) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-His(Trt)-OH (23.2 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (35) was cleaved and lyophilised, yield 5.3 mg (5.5 μmol, 44%), ESI-MS 963.4 [M + Hf , 482.3 [M + 2H]²⁺, 987.4 [M + Naf (calc. Mw 962.8) with Rt 5.99 min (>97%).

Synthesis of Abz-β-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (36) Following the general solid phase techniques detailed earlier, Ile-Ala-Ala-Pro-Tyr(3- NO₂)-Glu-NOVASYN TGR resin (0.05 g, 12.5 μmol) was stepwise elaborated with Fmoc-β-Ala-OH (11.7 mg; 37.5 μmol) and Boc-2-Abz (8.9 mg; 37.5 μmol), each coupling step utilising HBTU (14.2 mg, 37.5 μmol), HOBt (5.7 mg, 37.5 μmol) and NMM (8.3 μl, 75 μmol).

Substrate (36) was cleaved and lyophilised, yield 3.84 mg (4.3 μmol, 34%), ESI-MS 897.4 [M + Hf , 449.3 [M + 2H]²⁺, 919.4 [M + Naf (calc. Mw 896.8) with Rt 5.77 min (>95%).

Example 2 - Synthesis of Compounds(37) to (128)

Combinatorial solid-phase peptide synthesis using SynPhase™ gears

Substrates were prepared using gear assembly consisting of SynPhase™ solid-phase gears (1.9 μmole loading; Mimo topes, Heswall, Cheshire, U.K.) attached to the stems, which were inserted in a stemholder rack. Standard Fmoc-based chemistries were employed for solid-phase peptide synthesis (see 'Solid Phase Peptide Synthesis', Atherton, E. and Sheppard, R. C, IRL Press Ltd, Oxford, UK, 1989. for a general description). Peptide synthesis was carried out in rounds consisting of a coupling step, a short wash step, a de-protection step, a long wash step and a drying step. Each round elaborated the growing peptide chain with a single building block and upon completion substrates were then released (cleaved) from the solid phase, dissolved, analysed and assayed for activity against S. aureus extracellular cysteine peptidase.

Coupling cycles

The coupling of standard Fmoc-aminoacids/peptidomimetics (10 mole equivalence) was performed via carboxyl activated with 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU, 10 mole equivalence), 1- hydroxybenzotriazole (HOBT, 10 mole equivalence) and N-methylmorpholine (NMM, 20 mole equivalence) in dimethylformamide (DMF), with pre-activation for 5 min. Activated species were dispensed into the appropriate wells of a polypropylene 96-well plate (Beckman, 1 ml wells, 250 μl solution per well) in the pattern required for product synthesis.

Short wash step

The short wash step consisted of immersing the gear assembly twice into ~ 250 ml DMF for 3 min. each. In between each immersion, the assembly was removed, dried by shaking and re-immersed into fresh DMF. The assembly was removed, excess solvent removed by shaking and carried onto the next step.

De-protection step

The assembly was immersed into ~ 250 ml 80%DMF:20% piperidine for 30 min. The assembly was then removed, excess solvent removed by shaking and the gears washed thoroughly using the long wash step.

Long wash step

The long wash step consisted of immersing the gear assembly four times into ~ 250 ml DMF for 3 min. each followed by immersing the gear assembly four times into ~ 250 ml acetonitrile for 3 min. each. In between each immersion, the assembly was removed, dried by shaking and re-immersed into fresh solvent. Finally the assembly was removed, excess solvent removed by shaking and the gear dried in vacuo for 30 min. Acidolytic Cleavage Cycle

A mixture of 95% TFA / 5% triethylsilane was pre-dispensed into two polystyrene 96-well plates (Beckman, 1ml wells, 300 μl solution per well) in a pattern corresponding to that of the synthesis. The completed multipin assembly was added to the first plate ('mother' plate), the block covered in tin foil and cleavage reaction allowed to proceed for 2 hours. The cleaved multipin assembly was then removed from the first plate and added to the second plate ('daugther' plate) for 10 min. The spent multipin assembly was then discarded and the 'mother' and 'daughter' plates evaporated to dryness in a HT-4 Gene Vac plate evaporator (GeneVac Limited, Ipswich, U.K.).

Cleavage and analysis of substrates

The dried 'mother' and 'daughter' plates were recovered and 160 μl dimethylsulphoxide (DMSO) was added to each of the appropriate wells of the daughter plate, thoroughly mixed, transferred to the corresponding post cleaved and dried mother plate well. Again this was thoroughly mixed. An aliquot (10 μl) of this DMSO solution was diluted to 100 μl with a 90% acetonitrile / 10% 0.1 %aq TFA mixture and 20 μl aliquots were analysed by HPLC-MS and full analytical HPLC. HPLC-MS was carried out on a Phenomenex Columbus C8 (5μ, 30θA, 50 x 2.0 mm; Phenomenex) at 0.4 ml/min. using the following isocratic gradient: 0-3 min., 65% buffer B. Analytical HPLC was carried out as described previously. In each case the crude example molecules gave the expected [M + H]⁺ ion and product purity estimated from the analytical HPLC chromatogram (215 nm). The samples were dissolved to approximately 10 mM DMSO stock solutions and appropriately diluted for substrate screening assays against S. aureus extracellular cysteine peptidase. The following figures relate to the Fmoc-protected version of the compound structures and the relevant abbreviations, where applicable, used for compounds 37- 128.

Fmoc- 1 -amino- 1-cyclopentane carboxylic acid (Fmoc-l-ACP)

Fmoc-nipecotic acid (Fmoc-Nip)

Fmoc-L-4-hydroxy-prolιne (Fmoc-Pro(4-θH))

Fmoc-L-3-hydroxy-prolιne (Fmoc-Pro(3-OH))

Fmoc-L-pipecolic acid (Fmoc-Pip)

Fmoc-L-tetrahydroisoquinoline-1 -carboxylic acid (Fmoc-THIQ)

Fmoc- 1 ,2,3,4-L-tetrahydroisoquinoline-3-carboxylic acid (Fmoc-Tic)

Fmoc-L-2-aminobutyric acid (Fmoc-Abu) Abz-D-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (37)

ESI-MS 786.3 [M + H]⁺ (calc. Mw 785.4) with Rt 11.65 min. (>95%).

Abz-Ile-Ala-Ala-Thr-Tyr(3-NO₂)-Glu-NH₂ (38) ESI-MS 830.4 [M + Hf (calc. Mw 829.4) with Rt 12.08 min. (>95%).

Abz-Ile-Ala-Gly-Pro-Tyr(3-NO₂)-Glu-NH₂ (39)

ESI-MS 812.3 [M + Hf (calc. Mw 811.4) with Rt 12.00 min. (>95%).

Abz-Ile-Ala-Thr-Pro-Tyr(3-NO₂)-Glu-NH₂ (40)

ESI-MS 856.4 [M + H]⁺ (calc. Mw 855.4) with Rt 11.98 min. (>90%).

Abz-Ile-Gly-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (41) ESI-MS 812.4 [M + H]⁺ (calc. Mw 811.4) with Rt 11.91 min. (>95%).

Abz-Ile-Thr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (42)

ESI-MS 856.4 [M + Hf (calc. Mw 855.4) with Rt 11.97 min. (>90%).

Abz-Gly-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (43) ESI-MS 770.3 [M + H]⁺ (calc. Mw 769.4) with Rt 8.79 min. (>95%).

Abz-Thr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (44) ESI-MS 814.3 [M + Hf (calc. Mw 813.4) with Rt 9.14 min. (>90%).

Abz-D-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (45)

ESI-MS 826.4 [M + H]⁺ (calc. Mw 825.9) with Rt 12.68 min. (>95%).

Abz-AUo-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (46) ESI-MS 826.4 [M + H]⁺ (calc. Mw 825.9) with Rt 11.67 min. (>95%).

Abz-Ile-Abu-Ala-Pro-Tyr(3-NO_?)-Glu-NH_? (47) ESI-MS 840.4 [M + Hf (calc. Mw 840) with Rt 12.47 min. (>95%).

Abz-Ile-Ala-Abu-Pro-Tyr(3-NO )-Glu-NH₂ (48) ESI-MS 840.4 [M + Hf (calc. Mw 840) with Rt 11.86 min. (>97%).

Abz-Ile-Ala-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (49)

ESI-MS 800.4 [M + H]⁺ (calc. Mw 799.9) with Rt 12.94 min. (>95%).

Abz-Ile-Ala-Ala-Met-Tyr(3-NO₂)-Glu-NH₂ (50) ESI-MS 860.4 [M + H]⁺ (calc. Mw 859.9) with Rt 14.99 min. (>95%).

Abz-Ile-Ala-Met-Pro-Tyr(3-NO₂)-Glu-NH₂ (51)

ESI-MS 886.4 [M + H]⁺ (calc. Mw 885.9) with Rt 13.69 min. (>90%).

Abz-Ile-Met-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (52)

ESI-MS 886.4 [M + H]⁺ (calc. Mw 885.9) with Rt 13.80 min. (>90%).

Abz-Ala-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (53) ESI-MS 784.3 [M + Hf (calc. Mw 783.9) with Rt 9.24 min. (>95%).

Abz-Met-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (54)

ESI-MS 844.3 [M + H]⁺ (calc. Mw 843.9) with Rt 11.40 min. (>95%).

Abz-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (55) ESI-MS 842.4 [M + Hf (calc. Mw 842.9) with Rt 9.29 min. (>90%).

Abz-AUo-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (56) ESI-MS 842.4 [M + H]⁺ (calc. Mw 842.9) with Rt 9.99 min. (>95%).

Abz-Ile-Abu-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (57)

ESI-MS 856.4 [M + H]⁺ (calc. Mw 857) with Rt 11.67 min. (>95%). Abz-Ile-Ala-Abu-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (58)

ESI-MS 856.4 [M + H]⁺ (calc. Mw 857) with Rt 11.72 min. (>95%).

Abz-Ile-Ala-Ala-Val-Tyr(3-NO₂)-Glu-NH₂ (59) ESI-MS 828.4 [M + H]⁺ (calc. Mw 827.9) with Rt 14.44 min. (>97%).

Abz-Ile-Ala-Ala-Asn-Tyr(3-NO₂)-Glu-NH₂ (60)

ESI-MS 843.4 [M + H]⁺ (calc. Mw 843.9) with Rt 11.49 min. (>75%).

Abz-Ile-Ala-Val-Pro-Tyr(3-NO₂)-Glu-NH₂ (61)

ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 13.22 min. (>97%).

Abz-Ile-Ala-Asn-Pro-Tyr(3-NO₂)-Glu-NH₂ (62) ESI-MS 869.4 [M + Hf (calc. Mw 869.9) with Rt 12.34 min. (>90%).

Abz-Ile- Val-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (63)

ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 13.16 min. (>97%).

Abz-Ile-Asn-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (64) ESI-MS 869.4 [M + Hf (calc. Mw 869.9) with Rt 11.30 min. (>90%).

Abz-Val-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (65)

ESI-MS 812.4 [M + H]⁺ (calc. Mw 811.9) with Rt 10.90 min. (>95%).

Abz-Asn-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (66)

ESI-MS 827.3 [M + H]⁺ (calc. Mw 827.9) with Rt 8.62 min. (>70%).

Abz-Ile- Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (67)

ESI-MS 842.4 [M + H]⁺ (calc. Mw 842.9) with Rt 10.11 min. (>95%).

Abz-Allo-Ile-Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (68) ESI-MS 842.4 [M + H]⁺ (calc. Mw 842.9) with Rt 10.83 min. (>95%). Abz-Ile-Abu-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (69)

ESI-MS 856.4 [M + H]+ (calc. Mw 857) with Rt 9.85 min. (>95%).

Abz-Ile-Ala-Abu-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (70)

ESI-MS 856.4 [M + H]⁺ (calc. Mw 857) with Rt 11.52 min. (>95%).

Abz-Ile- Ala-Ala-Leu-Tyr(3-NO₂)-Glu-NH₂ (71) ESI-MS 842.4 [M + H]⁺ (calc. Mw 841.9) with Rt 16.19 min. (>95%).

Abz-Ile-Ala-Ala-Gln-Tyr(3-NO₂)-Glu-NH₂ (72)

ESI-MS 857.4 [M + Hf (calc. Mw 856.9) with Rt 11.40 min. (>80%).

Abz-Ile-Ala-Leu-Pro-Tyr(3-NO₂)-Glu-NH₂ (73) ESI-MS 868.4 [M + H]⁺ (calc. Mw 867.9) with Rt 14.47 min. (>95%).

Abz-Ile- Ala-Gln-Pro-Tyr(3-NO₂)-Glu-NH₂ (74) ESI-MS 883.4 [M + Hf (calc. Mw 882.9) with Rt 11.20 min. (>80%).

Abz-Ile-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (75)

ESI-MS 868.4 [M + H]⁺ (calc. Mw 867.9) with Rt 14.65 min. (>95%).

Abz-Ile-Gln-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (76) ESI-MS 883.4 [M + Hf (calc. Mw 882.9) with Rt 11.47 min. (>80%).

Abz-Leu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (77)

ESI-MS 826.4 [M + H]⁺ (calc. Mw 825.9) with Rt 12.37 min. (>95%).

Abz-Gln-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (78)

ESI-MS 841.4 [M + H]⁺ (calc. Mw 840.9) with Rt 8.58 min. (>75%). Abz-Ile-Ala-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (79)

ESI-MS 888.4 [M + H]⁺ (calc. Mw 887.9) with Rt 14.45 min. (>70%).

Abz-AUo-Ile-Ala-Ala-THIQ-Tyr(3-NO_?)-Glu-NH₂ (80) ESI-MS 888.4 [M + H]⁺ (calc. Mw 887.9) with Rt 14.5 min. (>70%).

Abz-Ile-Abu-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (81)

ESI-MS 902.5 [M + Hf (calc. Mw 901.4) with Rt 14.99 min. (>70%).

Abz-Ile-Ala-Abu-THIQ-Tyr(3-NO₂)-Glu-NH₂ (82)

ESI-MS 902.4 [M + H]⁺ (calc. Mw 901.4) with Rt 14.15 min. (>97%).

Abz-Ile-Ala-Ala-Ile-Tyr(3-NO₂)-Glu-NH₂ (83)

ESI-MS 842.4 [M + H]⁺ (calc. Mw 841.9) with Rt 15.68 min. (>97%).

Abz-Ile-Ala-Ala-Asp-Tyr(3-NO₂)-Glu-NH₂ (84)

ESI-MS 844.4 [M + H ^■f,+ (calc. Mw 843.8) with Rt 11.95 min. (>97%).

Abz-Ile-Ala-Ile-Pro-Tyr(3-NO₂)-Glu-NH₂ (85) ESI-MS 868.4 [M + Hf (calc. Mw 867.9) with Rt 14.08 min. (>95%).

Abz-Ile-Ala-Asp-Pro-Tyr(3-NO₂)-Glu-NH₂ (86)

ESI-MS 870.4 [M + Hf (calc. Mw 868.9) with Rt 12.45 min. (>95%).

Abz-Ile-Ile-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (87)

ESI-MS 868.4 [M + H]⁺ (calc. Mw 867.9) with Rt 14.05 min. (>95%).

Abz-Ile-Asp-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (88)

ESI-MS 870.4 [M + Hf (calc. Mw 868.9) with Rt 1 1.77 min. (>95%).

Abz-Asp-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH2 (89)

ESI-MS 828.3 [M + Hf (calc. Mw 826.9) with Rt 9.15 min. (>90%). Abz-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (90)

ESI-MS 888.4 [M + H]⁺ (calc. Mw 887.4) with Rt 14.60 min. (>95%).

Abz-Allo-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (91)

ESI-MS 888.4 [M + H]⁺ (calc. Mw 887.4) with Rt 14.3 min. (>97%).

Abz-Ile-Abu-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (92) ESI-MS 902.4 [M + Hf (calc. Mw 901.1) with Rt 15.11 min. (>95%).

Abz-Ile-Ala-Abu-Tic-Tyr(3-NO₂)-Glu-NH₂ (93)

ESI-MS 902.5 [M + Hf (calc. Mw 901.1) with Rt 12.81 min. (>95%).

Abz-Ile-Ala-Ala-Phe-Tyr(3-NO₂)-Glu-NH₂ (94) ESI-MS 876.4 [M + H]⁺ (calc. Mw 875.9) with Rt 16.23 min. (>94%).

Abz-Ile-Ala-Ala-Glu-Tyr(3-NO₂)-Glu-NH₂ (95)

ESI-MS 858.4 [M + Hf (calc. Mw 857.9) with Rt 12.15 min. (>95%).

Abz-Ile-Ala-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂ (96)

ESI-MS 902.4 [M + H]⁺ (calc. Mw 901.9) with Rt 14.94 min. (>95%).

Abz-Ile-Ala-Glu-Pro-Tyr(3-NO₂)-Glu-NH₂ (97) ESI-MS 884.4 [M + H]⁺ (calc. Mw 883.9) with Rt 11.73 min. (>94%).

Abz-Ile-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (98)

ESI-MS 902.4 [M + H]⁺ (calc. Mw 901.9) with Rt 15.34 min. (>95%).

Abz-Ile-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (99) ESI-MS 884.4 [M + H]⁺ (calc. Mw 883.9) with Rt 12.00 min. (>95%).

Abz-Phe-Ala-Ala-Pro-Tyr(3-NO_?)-Glu-NH₂ (100) ESI-MS 860.4 [M + H]⁺ (calc. Mw 859.9) with Rt 13.19 min. (>95%).

Abz-Glu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (101) ESI-MS 842.3 [M + H]⁺ (calc. Mw 841.9) with Rt 9.28 min. (>95%).

Abz-Ile-Ala-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (102)

ESI-MS 840.4 [M + H]⁺ (calc. Mw 839.9) with Rt 12.72 min. (>79%).

Abz-Allo-Ile-Ala-Ala-Pip-Tyr(3-NO2)-Glu-NH₂ (103) ESI-MS 840.4 [M + H]⁺ (calc. Mw 839.9) with Rt 12.82 min. (>78%).

Abz-Ile-Abu-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (104)

ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 13.31 min. (>82%).

Abz-Ile- Ala-Abu-Pip-Tyr(3-NO₂)-Glu-NH₂ (105)

ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 13.34 min. (>95%).

Abz-Ile-Ala-Ala-Tyr-Tyr(3-NO₂)-Glu-NH₂ (106) ESI-MS 892.4 [M + Hf (calc. Mw 891.9) with Rt 14.15 min. (>95%).

Abz-Ile- Ala-Ala-Lys-Tyr(3-NO₂)-Glu-NH₂ (107)

ESI-MS 857.4 [M + H]⁺ (calc. Mw 856.9) with Rt 11.44 min. (>95%).

Abz-Ile- Ala-Tyr-Pro-Tyr(3-NO₂)-Glu-NH₂ (108) ESI-MS 918.4 [M + H]⁺ (calc. Mw 917.9) with Rt 13.10 min. (>95%).

Abz-Ile-Ala-Lys-Pro-Tyr(3-NO_z)-Glu-NH₂ (109)

ESI-MS 883.5 [M + Hf (calc. Mw 882.9) with Rt 11.04 min. (>95%).

Abz-Ile-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (110)

ESI-MS 918.2 [M + Hf (calc. Mw 917.9) with Rt 13.19 min. (>95%). Abz-Ile-Lys-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (111)

ESI-MS 883.5 [M + Hf (calc. Mw 882.9) with Rt 11.61 min. (>95%).

Abz-Tyr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (112) ESI-MS 876.4 [M + H]⁺ (calc. Mw 875.9) with Rt 10.86 min. (>95%).

Abz-Lys-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (113)

ESI-MS 841.4 [M + H]⁺ (calc. Mw 840.9) with Rt 8.57 min. (>95%).

Abz-Ile- Ala- Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (114)

ESI-MS 840.4 [M + Hf (calc. Mw 839.9) with Rt 15.18 min. (>95%).

Abz-Allo-Ile-Ala-Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (115) ESI-MS 840.4 [M + H]⁺ (calc. Mw 839.9) with Rt 15.14 min. (>95%).

Abz-Ile- Abu- Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (116)

ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 15.74 min. (>95%).

Abz-Ile- Ala- Abu- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (117) ESI-MS 854.4 [M + H]⁺ (calc. Mw 853.9) with Rt 15.76 min. (>95%).

Abz-Ile-Ala-Ala-Ser-Tyr(3-NO₂)-Glu-NH₂ (118)

ESI-MS 816.4 [M + H]⁺ (calc. Mw 815.9) with Rt 11.14 min. (>95%).

Abz-Ile- Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (119)

ESI-MS 842.4 [M + H]⁺ (calc. Mw 841.9) with Rt 11.77 min. (>95%).

Abz-Ile- Ala-Pro-Pro-Tyr(3-NO₂)-Glu-NH₂ (120)

ESI-MS 852.4 [M + H]⁺ (calc. Mw 851.9) with Rt 1 1.68 min. (>95%).

Abz-Ile-Ser-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (121)

ESI-MS 842.4 [M + H -f,+ (calc. Mw 841.9) with Rt 11.55 min. (>95%). Abz-Ile-Pro-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (122)

ESI-MS 852.4 [M + Hf (calc. Mw 851.9) with Rt 12.36 min. (>95%).

Abz-Ser-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (123)

ESI-MS 800.3 [M + H]⁺ (calc. Mw 799.9) with Rt 11.96 min. (>95%).

Abz-Pro-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (124) ESI-MS 810.4 [M + H]⁺ (calc. Mw 809.9) with Rt 7.03 min. (>50%).

Abz-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (125)

ESI-MS 826.4 [M + H]⁺ (calc. Mw 825.9) with Rt 12.35 min. (>95%).

Abz-Allo-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (126) ESI-MS 826.4 [M + H]⁺ (calc. Mw 825.9) with Rt 12.38 min. (>95%).

Abz-Ile-Abu-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (127)

ESI-MS 840.4 [M + Hf (calc. Mw 839.9) with Rt 12.79 min. (>95%).

Abz-Ile- Ala-Abu-D-Pro-Tyr(3-NO₂)-Glu-NH (128)

ESI-MS 840.4 [M + H]⁺ (calc. Mw 839.9) with Rt 13.11 min. (>95%).

Example 3 - Synthesis of Compounds (129) to (139) by Solution Phase Synthesis

Peptidyl 7-amino-4-methylcoumarin-based (AMC-based) substrates (129-139) were prepared in solution using traditional peptide coupling and de-protection techniques (see Bodanszky, M and Bodanszky, A, (1994), 77ιe Practice of Peptide Synthesis, 2^nd Ed, Springer- Verlag Berlin, Heidelberg).

Synthesis of Boc-Ile-Ala-AMC (129)

H-Ala-AMC (10 mg, 39 μmol in 0.3 ml tetrahydrofuran (THF)) was mixed with Boc-Ile-O-succinimidyl ester (12.8 mg, 39 μmol in 0.3 ml THF). After 16 h at room temperature, the mixture was evaporated in vacuo then transferred with ethyl acetate (EtOAc) (10 ml) to a separating funnel. The reaction vial was washed out with a second volume of EtOAc (10 ml), then two aliquots of 0.1 M hydrochloric acid and the washings added to the separating funnel. The organic layer was extracted and then washed with saturated sodium hydrogen carbonate (lOmL) and brine (lOmL). The organic layer was dried (Na₂SO₄), filtered and evaporated in vacuo to a white solid.

The solid was dissolved in acetonitrile and lyophilised to give substrate (129), yield 15.38 mg (33.5μmol, 86%), ESI-MS 360.2 [M + H - Bocf , 819.4 [2M + H - Bocf , 941.4 [2M + Naf (calc. Mw 458.24) with Rt 16.94 min (>95%).

Synthesis of HClIle-Ala-AMC (130)

4 M hydrochloric acid in dioxan was added to Boc-Ile-Ala-AMC (12 mg, 26.1 μmol), stirred on ice for 2 h, evaporated in vacuo. The residual solid was dissolved acetonitrile:water (50:50, vol./vol.) (lmL), then lyophilised to give substrate (130), yield: 15.38 mg (25μmol, 96 %), ESI-MS 360.1 [M + H]⁺, 719.2 [2M + H]⁺, 742.2 [2M + Naf (calc. Mw 358.24) with Rt 10.48 min (>95%).

Synthesis of N-acetyl-Ile-Ala-AMC (131)

Acetic anhydride (6.38 μl, 67.7 μmol; Chemlmpex) and NMM (1.5 μl, 13.5 μmol) were mixed with HCIH-Ile-Ala-AMC (1.3 mg, 6.7 μmol) in 0.25 ml DMF and the sample stirred overnight at room temperature. The product was purified by semi- preparative HPLC using the following column elution profile: 0-1 min., 10% buffer B; 1-15 min., 10-90% buffer B; 15-19 min., 90% buffer B; 19-21 min., 90-10% buffer B; 21-25 min., 10% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually.

Appropriate fractions were lyophilised to give (131), yield: 15.38 mg (25 μmol, 96 %), ESI-MS 403.2 [M + H]⁺, 803.2 [2M + H]⁺ , 825.2 [2M + Naf, (calc. Mw 401.46) with Rt 12.40 min. (>97%). Synthesis of 4-morpholino-Ile-Ala-AMC (132)

4-morpholine carbonyl chloride (10.1 mg, 67.7 μmol; Aldrich) and NMM (1.5 μl, 13.5 μmol) were mixed with a solution of HCIH-Ile-Ala-AMC (1.3 mg, 6.7 μmol) in 0.25 ml DMF and the sample stirred overnight at room temperature. The product was purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 10% buffer B; 1-15 min., 10-90% buffer B; 15-19 min., 09% buffer B; 19- 21 min., 90-10% buffer B; 21-25 min., 10%_> buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually.

Appropriate fractions were lyophilised to give (132), yield: 15.38 mg (25 μmol, 96 %), ESI-MS 473.2 [M + H]⁺, 945.5 [2M + H]⁺ (calc. Mw 472.53), with Rt 6.3 min. (>97%)

Synthesis of Benzoyl-Ile-Ala-AMC (133) Benzoic acid (8.2 mg, 67.7 μmol; Aldrich), HBTU (25 mg, 67.7 μmol), HOBt (10 mg, 67.7 μmol) and NMM (15 μl, 135 μmol) were mixed in 0.25 ml DMF and stirred at room temperature for 5 min. HCIH-Ile-Ala-AMC (1.3 mg, 6.7 μmol) and NMM (7.5 μl, 67.7 μmol) were added to the mixture and the sample stirred overnight at room temperature. The product was purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 10% buffer B; 1-15 min., 10-90% buffer B; 15-19 min., 90% buffer B; 19-21 min., 90-10% buffer B; 21-25 min., 10% buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually.

Appropriate fractions were lyophilised to give (133), yield: 15.38 mg (25 μmol, 96 %), ESI-MS 464.2 [M + H⁺], 927.2 [2M + H - Bocf, 949.2 [2M + H - Boc + Naf (calc. Mw 463.53) with Rt 7.7 min. (>97%).

Synthesis of cyclohexoyl-Ile-Ala-AMC (134) Cyclohexane carboxylic acid (8.7 mg, 67.7 μmol; Aldrich), HBTU (25 mg, 67.7 μmol), HOBt (10 mg, 67.7 μmol) and NMM (15 μl, 135 μmol) were mixed in 0.25 ml DMF and the reaction incubated at room temperature for 5 min. HCIH-Ile-Ala- AMC (1.3 mg, 6.7 μmol) and NMM (7.5 μl, 67.7 μmol) were added to the reaction and the sample stirred overnight at room temperature. The product was purified by semi-preparative HPLC using the following column elution profile: 0-1 min., 10% buffer B; 1-15 min., 10-90% buffer B; 15-19 min., 90% buffer B; 19-21 min., 90- 10%) buffer B; 21-25 min., 10%) buffer B. The eluant absorbance was monitored at 230 nm and fractions were collected manually.

Appropriate fractions were lyophilised to give (134), yield: 15.38 mg (25 μmol, 96 %), ESI-MS 470.2 [M + H]⁺, 961.4 [2M + H]⁺ (calc. Mw 469.57) with Rt 8.2 min. (>97%).

Synthesis of CBZ-Ile-Ala-AMC (135)

H-Ala-AMC (10 mg, 39 μmol in 0.3 ml THF) was mixed with CBZ-Ile-O- succinimidyl ester (14.1 mg, 39 μmol in 0.3 ml of THF). After overnight incubation at room temperature, the mixture was evaporated in vacuo then transferred with ethyl acetate (EtOAc) (10 ml) to a separating funnel. The reaction vial was washed out with a second volume of EtOAc (10 ml), then two aliquots of 0.1 M hydrochloric acid and the washings added to the separating funnel. The organic layer was extracted and then washed with saturated sodium hydrogen carbonate (lOmL) and brine (lOmL). The organic layer was dried (Na₂SO₄), filter and evaporated in vacuo to a white solid.

The solid was dissolved in acetonitrile and lyophilised to give substrate (135), yield 2.11 mg (4.3 μmol, 11 %), ESI-MS 494.3 [M +H⁺], 987.5 [2M + H]⁺ (calc. Mw 493.22) with Rt 17.80 min. (>95%).

Example 4 - Synthesis of Compunds (136) to (139)

General technique for solution phase synthesis of peptidyl 7-amino-4- methylcoumarin-based (AMC-based) substrates

For compounds 136-139, AMC-based substrate in 0.1 ml dichloromethane (DCM) was mixed with aliquots of a 100 mM solution (in DCM) of O-succinimidyl ester (OSu). The reaction was incubated at room temperature and product formation was monitored by RP-HPLC-MS. The product was purified by precipitation upon addition of water to the reaction mixture. The precipitate was recovered by centrifugation at 5500 r.p.m. for 5 min. and the supernatant discarded. The residual solid was dissolved in acetonitrile (1 ml) and lyophilised to give product.

Synthesis of Boc-Ile-Cys(Bzl)-AMC (136)

Following the general technique for solution phase synthesis of AMC-based substrates detailed earlier, H-Cys(Bzl)-AMC (10 mg, 27.1 μl) was mixed with Boc- Ile-OSu (27.1 μl, 27.1 μmol).

Compound (136), yield: 7.24 mg (12.4 μmol, 46 %), ESI-MS 482.1 [M + H - Bocf, 1063.2 [2M + H - Bocf, 1185.2 (calc. Mw 581.72) with Rt 21.05 min. (>95%).

Synthesis of CBZ-Ile-Cys(Bzl)-AMC (137)

Following the general technique for solution phase synthesis of AMC-based substrates detailed earlier, H-Cys(Bzl)-AMC (10 mg, 27.1 μl) was mixed with CBZ- Ile-OSu (27.1 μl, 27.1 μmol).

Compound (137), yield: 2.73 mg (4.4 μmol, 16.4 %), ESI-MS 616.1 [M + H⁺], 1231.2 [2M + Hf (calc. Mw 615.74) with Rt 21.34 min. (>97%).

Synthesis of Boc-Ile-Tyr-AMC (138)

Following the general technique for solution phase synthesis of AMC-based substrates detailed earlier, H-Tyr-AMC (10 mg, 29.5 μmol) was mixed with Boc-Ile- OSu (29.5 μl, 29.5 μmol).

Compound (138), yield: 2.36 mg (4.27 μmol, 14.5 %), ESI-MS 452.1 [M + H - Bocf (calc. Mw 551.63) with Rt 17.95 min. (>95%). Synthesis of CBZ-Ile-Tyr-AMC (139)

Following the general technique for solution phase synthesis of AMC-based substrates detailed earlier, H-Tyr-AMC (10 mg, 29.5 μmol) was mixed with CBZ- Ile-OSu (29.5 μl, 29.5 μmol).

Compound (139), yield: 12.98 mg (22.1 μmol, 75 %) ESI-MS 586.1 [M + Hf, 1171.2 [2M + Hf (calc. Mw 585.65) with Rt 18.21 min. (>97%).

The following compounds were also prepared using the methods outlined in Examples 1 to 4 above

Abz-Ile-Ala-Ser-Gly-Tyr(3-NO₂)-Glu-NH₂ (140) Abz-Ile-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (141) Abz-Leu-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (142) Abz-Tyr-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (143)

Abz-Leu-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (144) Abz-Ile-Phe-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂ (145) Abz-Leu-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (146) Abz-Ile-Leu-Lys-Ala-Tyr(3-NO₂)-Glu-NH₂ (147) Abz-Tyr-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂ (148)

Abz-Ile-Glu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (149) Abz-Ile-Leu-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂ (150) Abz-Ile-Ala-Thr-Gly-Tyr(3-NO₂)-Glu-NH₂ (151) Abz-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (152) Abz-Leu-Phe-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂ (153)

Abz-Ile-Tyr-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂ (154) Abz-Leu-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂ (155) Abz-Ile-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂ (156) Abz-Leu-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (157) Abz-Ile-Ala-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂ (158)

Abz-Tyr-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (159) Abz-Ile-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (160) Abz-Leu-Tyr-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (161)

Abz-Ile-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (162)

Abz-Asp-Val-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂ (163)

Abz-Leu-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (164) Abz-Tyr-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂ (165)

Abz-Leu-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (166)

Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂ (167)

Abz-Leu-Leu-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂ (168)

Abz-Leu-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (169) Abz-Ile-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂ (170)

Abz-Leu-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (171)

Abz-Ile-Tyr-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂ (172)

Abz-Leu-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂ (173)

Abz-Tyr-Phe-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂ (174) Abz-Leu-Tyr-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (175) wherein "2-Thi" represents β-(2-thienyl)-L-alanine.

Assays for Cysteine Protease Activity

General materials and methods

Unless otherwise stated, all general chemicals and biochemicals were purchased from either the Sigma Chemical Company, Poole, Dorset, U.K. or from Fisher Scientific UK, Loughborough, Leicestershire, U.K. Absorbance assays were carried out in flat-bottomed 96-well plates (Spectra; Greiner Bio-One Ltd., Stonehouse, Gloucestershire, U.K) using a SpecfraMax PLUS384 plate reader (Molecular Devices, Crawley, U.K). Fluorescence high throughput assays were carried out in either 384-well microtitre plates (Corning Costar 3705 plates, Fisher Scientific) or 96-well 'U' bottomed Microfluor WI microtitre plates (Thermo Labsystems, Ashford, Middlesex, U.K). Fluorescence assays were monitored using a SpecfraMax Gemini fluorescence plate reader (Molecular Devices). As the substrates employed a 3-amino-benzoyl (Abz) fluorophore, assays were monitored at an excitation wavelength of 310 nm and an emission wavelength of 445 nm; the fluorescence plate reader calibrated with 3-amino-benzamide (Fluka). Unless otherwise indicated, all the peptidase substrates were purchased from Bachem UK, St. Helens, Merseyside, UK. Hydroxyethylpiperazine ethanesulfonate (HEPES), tris-hydroxylmethylamino- methane (tris) base, bis-tris-propane (BTP) and all the biological detergents (e.g. Triton X-100, Tween 20, CHAPS, β-octyl-gluopyranoside; zwittergents, etc) were purchased from CNBiosciences UK, Beeston, Nottinghamshire, U.K. Glycerol was purchased from Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K. Stock solutions of substrate or inhibitor were made up to 10 mM in 100 % dimethylsulfoxide (DMSO) (Rathburns, Glasgow, U.K) and diluted as appropriately required. In all cases the DMSO concentration in the assays was maintained at less than l% (vol./vol).

Measurement of the inhibition constants

The inhibition constant (K) for each compound was determined on the basis that inhibition was reversible and occurred by a pure-competitive mechanism. The K values were calculated, from the dependence of enzyme activity as a function of inhibitor concentration, by direct regression analysis (Prism v 3.02) using Equation 4 (Cornish-Bowden, A., 1995).

In Equation 4 'vj' is the observed residual activity, ' V_max ^app, is the observed maximum activity (i.e. in the absence of inhibitor), Α_M ^app' is the apparent macroscopic binding (Michaelis) constant for the substrate, '[S]' is the initial substrate concentration, 'Ay is the apparent dissociation constant and '[I]' is the inhibitor concentration.

In situations where the apparent dissociation constant (A,^app) approached the enzyme concentrations, the ^ values were calculated using a quadratic solution in the form described by Equation 3 (Morrison, J. F., (1969), Biochim Biophys Acta, 185 (2), 269-286; Stone, S. R. and Hofsteenge, J., (1986), Biochemistry, 25 (16), 4622-4628). F{E₀ -I₀ -Kr + l(E₀ -I₀ -KD² + .Kr.E₀ } v. =- (5)

A,^ = A, (1 + [S /A_M ^W) (6)

In Equation 5 'V_J' is the observed residual activity, 'F' is the difference between the maximum activity (i.e. in the absence of inhibitor) and minimum enzyme activity, 'E₀' is the total enzyme concentration, 'AY ^P' is the apparent dissociation constant and 'I₀' is the inhibitor concentration. Curves were fitted by non-linear regression analysis (Prism) using a fixed value for the enzyme concentration. Equation 6 was used to account for the substrate kinetics, where 'A7 is the inhibition constant, '[S₀]' is the initial substrate concentration and Α_M ^app is the apparent macroscopic binding (Michaelis) constant for the substrate (Morrison, 1982).

The second-order rate of reaction of inhibitor with enzyme Where applicable, the concentration dependence of the observed rate of reaction ( _0bs) o each compound with enzyme was analysed by determining the rate of enzyme inactivation under pseudo-first order conditions in the presence of substrate (Morrison, J. F., (1969), Biochim Biophys Acta, 185 (2), 269-286; Morrison, J. F. and Walsh, C. T., (1988), Adv Enzymol Relat Areas Mol Biol, 61, 201-301; Tsou, C. L., (1988), Advances in Enzymology, 61, 382-436; Tian, W. X. and Tsou, C. L., (1982), Biochemistry, 21 (5), 1028-1032). Assays were carried out by addition of various concentrations of inhibitor to assay buffer containing substrate. Assays were initiated by the addition of enzyme to the reaction mixture and the change in fluorescence monitored over time. During the course of the assay less than 10% of the substrate was consumed.

The activity fluorescence progress curves were fitted by non-linear regression analysis (Prism) using Equation 7 (Morrison, 1969; Morrison, 1982); where 'F' is the fluorescence response, 't' is time, 'v₀' is the initial velocity, 'v_s' is the equilibrium steady-state velocity, 'A₀bs' is the observed pseudo first-order rate constant and 'D' is the intercept at time zero (i.e. the ordinate displacement of the curve). The second order rate constant was obtained from the slope of the line of a plot of _0bs versus the inhibitor concentration (i.e. k₀,_s/[l]). To correct for substrate kinetics, Eq. 8 was used, where '[S₀]' is the initial substrate concentration and ΑM^app' is the apparent macroscopic binding (Michaelis) constant for the substrate.

Staphylococcus aureus culture supernatant

S. aureus strain V8 was a generous gift from Prof. Steffan O. Arvidson, Karolinska Institute, Stockholm, Sweden. Culture supernatant was produced as a service by Dr. Peter Lambert, Aston University, Birmingham, U.K., according to the method previously described (Drapeau, G. R., et al, (1972), J Biol Chem, 247 (20), 6720- 6726). Culture supernatant was freated with cetrimide (cetyltrimethylammonium bromide) and frozen as aliquots (50 ml) at minus 20°C until required. Routinely samples were thawed and spun at 5500 r.p.m. for 5 min at 15°C to remove any precipitate. The supernatant was recovered and this was freated as the crude extract.

Partial purification of the extracellular cysteine peptidase activity

To a stirred aliquot (50 ml) of the S. aureus crude extract solid ammonium sulphate was slowly added to a final concentration of 600 g/1. The sample was stirred on ice for 30 min and subsequently centrifuged at 5500 rpm for 30 min at 15°C. The supernatant was discarded and the tubes were inverted over a towel to remove excess liquid. An equivalent volume to the original sample (i.e. 50 ml) of 10 mM glycine, pH 10.5 was added to each tube and the protein solubilised by gentle inversion. The sample was loaded onto a Q-Sepharose (Amersham Pharmacia Biotech, Little Chalfont, U.K) column (XK series; 1.6 x 20 cm; Amersham Pharmacia Biotech) previously equilibrated in 10 mM glycine, pH 10.5. The column was washed with four volumes of 10 mM glycine, pH 10.5 and developed with a total of 150 ml of an increasing linear gradient of 0-1 M NaCl in 10 mM glycine, pH 10.5 at 1 ml min^"1. Column elution was monitored by absorbance at 280 nm (A_{2 0 nm}) and fractions (2.5 ml) containing peptidase activity were pooled. The sample concentrated to -20-30 ml using an ultrafiltration cell (Amicon 8200; Millipore (UK) Limited, Watford, U.K) fitted with a YM10 membrane (Millipore) by ultrafiltration. Upon concentration the sample was subjected to four rounds of diafiltration by diluting the concentrated sample with 50 mM sodium acetate, pH 5.0 to 200 ml and re- concentrating the sample to 20-30 ml. Upon concentration the sample was subjected to four rounds of diafiltration, as before, using ultrafiltration. The concentrated sample was loaded onto a SP-Sepharose (Amersham Pharmacia Biotech) column (XK series; 1.6 x 20 cm; Amersham Pharmacia Biotech) previously equilibrated in 50 mM sodium acetate, pH 5.0. The column was washed with four volumes of 50 mM sodium acetate, pH 5.0 and developed with a total of 150 ml of an increasing linear gradient of 0-0.5 M sodium chloride in 50 mM sodium acetate, pH 5.0 at 1 ml min^"1. Column elution was monitored as before and fractions (2.5 ml) containing peptidase activity were pooled and the sample concentrated as before.

Polyacrylamide, zymogram and isoelectric focusing (IEF) gel electrophoresis Protein purity was routinely assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) using 4-20% NuPAGE gels (Invitrogen, Paisley, U.K) employing the 2-(N-morpholino)ethanesulfonic acid (MES) buffer system (Invitrogen) according the manufacturers instructions. Protein visualisation was carried out using the SilverExpress stain kit (Invitrogen). Zymogram gels were run using 20% beta-casein gels (Invitrogen); sample preparation, gel renaturation and staining were carried out according to the manufacturers instructions. The zymogram gels were developed by incubation in 50 mM bis-tris-propane, pH 6.8 containing 1 mM EDTA, 100 μM Tween 20 and 10 mM L-cysteine for 1 h at room temperature. Gels were subsequently stained with SimplyBlue Safestain (Invitrogen). Isolelectric focussing (IEF) gels were run on a pH 3-10 gradient using a complete system consisting of gels, electrode buffers, sample buffer and IEF markers according to the manufacturers instructions (Invitrogen). Gels were stained according to the manufacturers protocol supplied with the IEF gels. In all cases the gels were dried using the gel drying kit (Invitrogen) and for presentation purposes, gels were scanned at 300 dpi resolution using grey scale false colour (OfficeJet Pro 1175c; Hewlett Packard).

Assay protocols were based on literature precedent (Barrett, A.J., Rawlings, N.D. and Woessner, J.F., 1998, Handbook of Proteolytic Enzymes, Academic Press, London and references therein) and modified as required to suit local assay protocols. Enzyme was added as required to initiate the reaction and the activity, as judged by the change in fluorescence upon conversion of substrate to product, was monitored over time. All assays were carried out at 25±1°C.

Extracellular S. aureus V8 cysteine peptidase (cysteine peptidase) peptidase activity assays S. aureus V8 was obtained from Prof. S. Arvidson, Karolinska Institute, Stockholm, Sweden. Extracellular S. aureus V8 cysteine peptidase (cysteine peptidase) activity assays were carried out using partially purified S. aureus V8 culture supernatant (obtained from Dr. Peter Lambert, Aston University, Birmingham, U.K). Activity assays were carried out in 10 mM BTP, pH 6.5 containing 1 mM EDTA, 5 mM 2- mercaptoethanol and ImM calcium chloride using two-times diluted partially purified extract. For the inhibition assays, Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu- NH₂ (A_M ^app - 117 μM; Incenta Limited) was used as the substrate at a concentration equivalent to A_M ^app. The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time.

EnzChek assays for monitoring peptidase activity

Unless otherwise indicated all assays were carried out at 25±0.5°C monitoring the steady-state rates. Casein EnzChek (Molecular Probes, Leiden, Netherlands) and elastin EnzChek (Molecular Probes) were solubilised with water to a concentration of 1 mg ml^"1. The substrates were diluted as required and the cleavage reaction monitored by changes in fluorescence using 485 nm as the excitation wavelength (Ex_{4 5 nm}) and a 513 nm as the emission wavelength (Em_{513 nm}). The apparent Michaelis constant (A_M ^app) and the apparent maximal rate of conversion of substrate to product ( _max ^app) were calculated by measuring the rate of product formation as a function of substrate concentration (Cornish-Bowden, A., (1995), Fundementals of Enzyme Kinetics, London, Portland Press Ltd.). The pH-activity profile was carried out in a number of buffers including buffer mixtures that maintained constant ionic strength over the pH range investigated (Ellis, K. J. and Morrison, J. F., (1982), Methods Enzymol, 87 405-426).

Example 5 - Peptidase Activity Assays Using Novel Substrates

It is known to those skilled in the art that labelling of protein substrate with dyes, especially fluorogenic dyes, can make the assay amenable to continuous real-time monitoring. Casein and elastin have been used as the traditional substrates for monitoring S. aureus extracellular cysteine peptidase activity. Although other proteins have been used, the literature indicates that cysteine peptidase activity was not specific in its substrate preference (Bjδrklind, A. and Jornvall, H., (1974), Biochim Biophys Acta, 370 (2), 524-529.). Surprisingly we have found that the small peptide-based substrates of the invention can be used for monitoring S. aureus extracellular cysteine peptidase activity and exhibit substrate specificity as indicated by the preponderance of certain amino acids in the preferred substrates. For example, a plot for the substrate Abz-Ile-Ala-Ala-Pro-Tyr(3NO2)-Glu-NH2 (2) is shown in Figure 2. These substrates also demonstrate favourable kinetic parameters, and a Michaelis-Menten plot for S. aureus extracellular cysteine peptidase activity monitored with compound (2) is shown in Figure 3. For comparison the S. aureus extracellular cysteine peptidase activity monitored with dye-labelled casein and elastin are also shown (Figures 4 and 5 respectively).

Table 1 below shows the kinetic parameters derived for other substrates of the invention. Table 1

Example 6 - Cleavage Sites in Substrates

A number of substrates were discovered and they exhibit a similar pattern of amino acid preference. The analysis of the post-cleavage reaction mixture also indicated that the majority (>90% after 2 h) cleavage only occurred at a single site. Table 2 shows the pattern of substrates and cleavage sites (J.).

Table 2

Example 7 - E-64 inactivation

Another aspect of the present invention is the use of such substrates for monitoring peptidase inactivation. Figure 6 shows the time course for inactivation of the cysteine peptidase by the irreversible inhibitor E-64 using the novel substrate compund (2). This enables mechanism-based active site inhibitors to be categorised in order of activity as determined by the value of the second order rate constant for inactivation (Figure 7). Example 8 - Leupeptin inhibition

Another aspect of the present invention is the use of such substrates for monitoring peptidase inhibition. Figure 8 shows the inhibition of the cysteine peptidase activity by leupeptin using novel substrate compound (2). The results show that the novel substrates of the present invention enable compounds to be assayed as potential inhibitors of cysteine peptidase activity. Figure 9 shows A, determinations using casein and FRET peptide (2) of the invention as substrates.

Example 9 - Enzyme class-specific inhibitors using EnzChek and New Substrate Compound (2)

It is known to those skilled in the art that various class-specific peptidase inhibitors can be used to differentiate the mechanistic class of an un-classified peptidase activity (North, M. J., (1989), Proteolytic enzymes a practical approach, Oxford, IRL Press, 105-124). Table 3 gives the results of an EnzChek assay using casein as substrate, while Table 4 gives the results of a comparable assay in which Compound (2) was used as substrate. The results show that the novel substrates of the present invention indicate that the activity being monitored was a cysteine peptidase analogous to the data obtained with casein, the prior art S. aureus cysteine peptidase substrate.

TABLE 3. Inhibitors and activators of S. aureus extracellular peptidase activity⁸

Relative activity Relative activity

Reactant (%ofcontrol)^b (% of control)⁰

- 10 mM L-cysteine + 10 mM L-cysteine control (no additives) 100 ±40 100 ±10

10 mM L-cysteine 2800 ±10 - lOmMDTT 1720 ±16 -

10 mM 2-mercaptoethanol 630 ± 25 -

10 μM E-64 - 13 ± 2

1 mM mercury chloride - 80 ±2

1 mM iodoacetic acid - 0±3

1 mM iodoacetamide - 20 ±2

1.6μMHEWC - 88 ±7

1 mM CaCl₂ - 88 ±7

100 μM EDTA - 98 ±6

10 μM phosphoramidon - 81 ± 9

1 mM 1,10-phenanthroline - 90 ±6

1 mM ZnCl₂ -

10 μM elastatinal - 74 ±3

5 μM SBTI - 80 ±10

200 μM PMSF - 90 ±2

1 mM benzamidine - 98 ±4

100 μM leupeptin - 4±1

10 μMpepstatin - 89 ±7

10 μMbestatin - 89 ±3 steady-state activity measured using 1.6 μg ml^" casein EnzChek in 10 mM bis-tris propane, pH 6.5 containing 100 μM Tween 20 over 30 min without pre-incubation of enzyme with inhibitor ^bsteady-state rate compared to control rate ^c steady-state rate compared to control assay rate containing 10 mM L-cysteine without inhibitor TABLE 4. Inhibitors and activators of S. aureus extracellular peptidase activity³

Relative activity Relative activity

Reactant (%ofcontrol)^b (% of control)⁰

+ 10 mM L-cysteine + 10 mM L-cysteine control (no additives) 0 100 ±10

10 mM L-cysteine 100 ±1 - lOmMDTT 37 ± 0.4 -

10 mM 2-mercaptoethanol 4 ± 0.03 -

10 μM E-64 - 2±1

1 mM mercury chloride - 35 ±4

1 mM iodoacetic acid - 0

1 mM iodoacetamide - 8±4

1.6μMHEWC - 54 ±6

1 mM CaCl₂ - 76 ±17

100 μM EDTA - 83 ±19

10 μM phosphoramidon - 64 ±9

1 mM 1,10-phenanthroline - 65 ±13

1 mM ZnCl₂ -

10 μM elastatinal - 75 ±15

5 μM SBTI - 43 ±14

200 μM PMSF - 82 ±12

1 mM benzamidine - 100 ±10

100 μM leupeptin - l±l

10 μM pepstatin - 92 ±10

10 μM bestatin - 47 ±6 ^a steady-state activity measured using 35μM Abz-Ile-Ala-Ala-Pro-Tyr(3-N0₂)-Asp-NH₂ in 10 mM bis-tris propane, pH 6.5 containing 100 μM Tween 20 over 30 min without pre-incubation of enzyme with inhibitor ^bsteady-state rate compared to control rate containing lOmM L-cysteine

⁰ steady-state rate compared to control assay rate containing 10 mM L-cysteine without inhibitor TABLE 5. Second order inactivation rates and inhibition constants for inhibitors

^~! 64 ΪJ OAxW - iodoacetic acid 3.6 ± 0.6 x 10 iodoacetamide 3.9±0.7xlθ' leupeptin 1.1 ± 0.2 x 10³ 2.7 ± 2.4 x IO^"6

Claims

1. A peptide cleavable by Staphylococcus sp. extracellular cysteine peptidase, the peptide being selected from the following:

A. A compound of general formula (I)

Y-A^A^A^A^R-Glu-NHz (I) wherein Y and R are reporter groups;

A¹ is either the D-isomer or the L-isomer form of isoleucine; each of A and A is independently Ala or 2-aminobutyric acid (Abu);

A⁴ is Pro, L-3-hydroxyproline (Pro(3-OH)), L-4-hydroxyproline (Pro (4-OH)), L- tetrahydroisoquinoline-1 -carboxylic acid (THIQ), L-tetrahydroisoquinoline-3- carboxylic acid (Tic), L-pipecolic acid (Pip), 1 -amino- 1-cyclopentane carboxylic acid (1-ACP) or D-Pro; and any one of A¹, A², A³ and A⁴ can be replaced by any other amino acid group (Xaa); or

B. one of the following compounds:

Abz-Ile-Ala-Pro-Arg-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂;

Abz-Ile-Leu-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂; Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂.

Abz-Ile-Ala-Ser-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ Abz-Leu-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Phe-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Leu-Lys-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Glu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Ala-Thr-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Phe-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Tyr-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile- Ala-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Tyr- Ala-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Asp-Val-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

Abz-Ile-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ Abz-Ile-Tyr-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂

Abz-Tyr-Phe-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂

Abz-Leu-Tyr-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

wherein

2-Thi represents β-(2-thienyl)-L-alanine Abz is 2-aminobenzyl; Tyr(3-NO₂) is 3-nitrotyrosine;

C. a compound of general formula (II):

CAP-A¹ -A²-L (II)

wherein:

A¹ and A² are as defined for general formula (I);

CAP is Boc-, N-acetyl-, 4-morpholino-, Benzoyl-, cyclohexoyl-, benzyloxycarbonyl-

(CBZ-) or Y, where Y is as defined above for general formula (I); and

L is a leaving group.

2. A compound as claimed in claim 1 wherein, in general formula (I), independently or in any combination:

A¹ is the L-isomer form of isoleucine; A² is Ala; A³ is Ala; and

A⁴ is Pro.

3. A compound as claimed in claim 1 or claim 2 wherein reporter groups Y and R are groups which can be monitored by a biophysical or chemical method such as fluorimetry, colourimetry, derivatisation or chromatography.

4. A compound as claimed in claim 3 wherein Y is Abz, 4-aminobenzyl (4-Abz), TFA.NH₂(CH₂)_nCO, TFA.NH₂(CH₂)_nCO-Abz, TFA.NH₂(CH₂)_nCO-4-Abz, Abz- Allo, 4-Abz-Allo, Abz-Xaa or 4-Abz-Xaa; n is an integer from 2 to 8; Xaa is any amino acid residue.

5. A compound as claimed in claim 3 wherein Y and R are a FRET pair.

6. A compound as claimed in claim 5 wherein Y is Abz, 4-aminobenzyl (4-Abz), TFA.NH₂(CH₂)_nCO-Abz, TFA.NH₂(CH₂)_nCO-4-Abz, Abz-Allo, 4-Abz-Allo, Abz- Xaa or 4-Abz-Xaa; n is an integer from 2 to 8;

Xaa is any amino acid residue; and

R is Tyr(3-NO₂).

7. A compound as claimed in claim 3 wherein either or both of Y and R is a fluorogenic or chromogenic leaving group L.

8. A compound as claimed in any one of claims 1 to 3 or claim 7 wherein the group L (in general formula II or Y and R) is selected from acridines (e.g. acridine orange), coumarins (e.g. 7-amido-4-methyl coumarin (AMC)), dipyrrines, oxazenes (e.g. cresyl violet), xanthenes (e.g. rhodamine 123), naphthylamides, p-nitroanilides and p-nitro-phenylesters.

A peptide as claimed in claim 1 which is selected from:

Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile- Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Ala-Pro-Arg-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂; Abz-Ile-Leu-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂;

Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂.

10. A compound of general formula (I) as defined in claim 1 wherein: Y is Abz, 4- Abz, TFA.NH₂(CH₂)_nCO, TFA.NH₂(CH₂)_nCO-Abz, TFA.NH₂(CH₂)_nCO-4-Abz, Abz-Allo, 4- Abz- Allo, Abz-Xaa or 4-Abz-Xaa; n is an integer from 2 to 8; Xaa is any amino acid residue; and R is Tyr(3-NO₂);

11. A compound as claimed in claim 10 comprising TFA.H₂N-(CH₂)_nCO-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (n=2-5, 7) (6-10)

TFA.H₂N-(CH₂)_nCO-4Abz-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (n=2-5, 7) (11-16)

Abz-Gly-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (17)

Abz-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (18)

Abz-Val-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (19) Abz-Leu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (20)

Abz-Ile-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (21)

Abz-Phe-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (22)

Abz-Tyr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (23)

Abz-Trp-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (24) Abz-Ser-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (25)

Abz-Thr-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (26)

Abz-Cys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (27)

Abz-Met-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (28)

Abz-Asn-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (29) Abz-Gln-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (30)

Abz-Asp-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (31)

Abz-Glu-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (32)

Abz-Lys-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (33)

Abz-Arg-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (34) Abz-His-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (35)

Abz-β-Ala-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (36)

Abz-D-Ile-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (37)

Abz-Ile-Ala-Ala-Thr-Tyr(3-NO₂)-Glu-NH₂ (38)

Abz-Ile-Ala-Gly-Pro-Tyr(3-NO₂)-Glu-NH₂ (39) Abz-Ile-Ala-Thr-Pro-Tyr(3-NO₂)-Glu-NH₂ (40)

Abz-Ile-Gly-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (41)

Abz-Ile-Thr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (42) Abz-Gly-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (43) Abz-Thr-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (44) Abz-D-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (45) Abz-Allo-Ile-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (46) Abz-Ile-Abu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (47) Abz-Ile-Ala-Abu-Pro-Tyr(3-NO₂)-Glu-NH₂ (48) Abz-Ile-Ala-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂ (49) Abz-Ile-Ala-Ala-Met-Tyr(3-NO₂)-Glu-NH₂ (50) Abz-Ile-Ala-Met-Pro-Tyr(3-NO₂)-Glu-NH₂ (51) Abz-Ile-Met-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (52) Abz-Ala-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (53) Abz-Met-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (54) Abz-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (55) Abz-Allo-Ile-Ala-Ala-Pro(3-OH)-Tyr(3-NO2)-Glu-NH₂ (56) Abz-Ile-Abu-Ala-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (57) Abz-Ile- Ala-Abu-Pro(3-OH)-Tyr(3-NO₂)-Glu-NH₂ (58) Abz-Ile-Ala-Ala-Val-Tyr(3-NO₂)-Glu-NH₂ (59) Abz-Ile-Ala-Ala-Asn-Tyr(3-NO₂)-Glu-NH₂ (60) Abz-Ile-Ala-Val-Pro-Tyr(3-NO₂)-Glu-NH₂ (61) Abz-Ile-Ala-Asn-Pro-Tyr(3-NO₂)-Glu-NH₂ (62) Abz-Ile- Val-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (63) Abz-Ile-Asn-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (64) Abz-Val-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (65) Abz-Asn-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (66) Abz-Ile-Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (67)

Abz-Allo-Ile-Ala-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (68) Abz-Ile-Abu-Ala-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (69) Abz-Ile-Ala-Abu-Pro(4-OH)-Tyr(3-NO₂)-Glu-NH₂ (70) Abz-Ile-Ala-Ala-Leu-Tyr(3-NO₂)-Glu-NH₂ (71) Abz-Ile-Ala-Ala-Gln-Tyr(3-NO₂)-Glu-NH₂ (72) Abz-Ile-Ala-Leu-Pro-Tyr(3-NO₂)-Glu-NH₂ (73) Abz-Ile-Ala-Gln-Pro-Tyr(3-NO₂)-Glu-NH₂ (74) Abz-Ile-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (75)

Abz-Ile-Gln-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (76)

Abz-Leu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (77)

Abz-Allo-Ile-Ala-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (80)

Abz-Ile-Abu-Ala-THIQ-Tyr(3-NO₂)-Glu-NH₂ (81)

Abz-Ile-Ala-Abu-THIQ-Tyr(3-NO₂)-Glu-NH₂ (82)

Abz-Ile-Ala-Ala-Ile-Tyr(3-NO₂)-Glu-NH₂ (83) Abz-Ile- Ala- Ala- Asp-Tyr(3-NO₂)-Glu-NH₂ (84)

Abz-Ile-Ala-Ile-Pro-Tyr(3-NO₂)-Glu-NH₂ (85)

Abz-Ile-Ala-Asp-Pro-Tyr(3-NO₂)-Glu-NH₂ (86)

Abz-Ile-Ile-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (87)

Abz-Ile-Asp-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (88) Abz-Asp-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (89)

Abz-Ile- Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (90)

Abz-Allo-Ile-Ala-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (91)

Abz-Ile-Abu-Ala-Tic-Tyr(3-NO₂)-Glu-NH₂ (92)

Abz-Ile-Ala-Abu-Tic-Tyr(3-NO₂)-Glu-NH₂ (93) Abz-Ile-Ala-Ala-Phe-Tyr(3-NO₂)-Glu-NH₂ (94)

Abz-Ile-Ala-Ala-Glu-Tyr(3-NO₂)-Glu-NH₂ (95)

Abz-Ile-Ala-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂ (96)

Abz-Ile-Ala-Glu-Pro-Tyr(3-NO₂)-Glu-NH₂ (97)

Abz-Ue-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (98) Abz-Ile-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (99)

Abz-Phe-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (100)

Abz-Glu-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (101)

Abz-Ile-Ala-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (102)

Abz-Allo-Ile-Ala-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (103) Abz-Ue-Abu-Ala-Pip-Tyr(3-NO₂)-Glu-NH₂ (104)

Abz-Ile-Ala-Abu-Piρ-Tyr(3-NO₂)-Glu-NH₂ (105)

Abz-Ile-Ala-Ala-Tyr-Tyr(3-NO₂)-Glu-NH₂ (106) Abz-Ile-Ala-Ala-Lys-Tyr(3-NO₂)-Glu-NH₂ (107) Abz-Ile-Ala-Tyr-Pro-Tyr(3-NO₂)-Glu-NH₂ (108) Abz-Ile-Ala-Lys-Pro-Tyr(3-NO₂)-Glu-NH₂ (109) Abz-Ile-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (110) Abz-Ile-Lys-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (111) Abz-Tyr- Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (112) Abz-Lys-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (113) Abz-Ile- Ala- Ala- 1 -ACP-Tyr(3-NO₂)-Glu-NH₂ (114) Abz-Allo-Ile-Ala-Ala-l-ACP-Tyr(3-NO₂)-Glu-NH₂ (115) Abz-Ile- Abu- Ala- 1 - ACP-Tyr(3-NO₂)-Glu-NH₂ (116) Abz-Ile-Ala-Abu-l-ACP-Tyr(3-NO₂)-Glu-NH₂ (117) Abz-Ile-Ala-Ala-Ser-Tyr(3-NO₂)-Glu-NH₂ (118) Abz-Ile- Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂ (119) Abz-Ile-Ala-Pro-Pro-Tyr(3-NO₂)-Glu-NH₂ (120) Abz-Ile-Ser-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (121) Abz-Ile-Pro-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (122) Abz-Ser-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (123) Abz-Pro-Ala-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂ (124) Abz-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (125) Abz-Allo-Ile-Ala-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (126) Abz-Ile-Abu-Ala-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (127) Abz-Ile- Ala-Abu-D-Pro-Tyr(3-NO₂)-Glu-NH₂ (128) Abz-Ile- Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂ (156) Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂ (171)

12. Abz-Ile-Phe-Phe-Pro-Tyr(3-NO₂)-Glu-NH₂.

13. Abz-Ile-Leu-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂.

14. Abz-Leu-Tyr-Phe-Arg-Tyr(3-NO₂)-Glu-NH₂.

15. Abz-Ile-Ala-Ser-Gly-Tyr(3-NO₂)-Glu-NH_2.

16. Abz-Ile-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂.

17. Abz-Leu-Tyr-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂.

18. Abz-Tyr-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂.

19. Abz-Leu-Glu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂.

20. Abz-Ile-Phe-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂.

21. Abz-Leu-Leu-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂

22. Abz-Ile-Leu-Lys-Ala-Tyr(3-NO₂)-Glu-NH₂.

23. Abz-Tyr-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂.

24. Abz-Ile-Glu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂.

25. Abz-Ile-Leu-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂.

26. Abz-Ile-Ala-Thr-Gly-Tyr(3-NO₂)-Glu-NH₂.

27. Abz-Leu-Phe-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂.

28. Abz-Ile-Tyr-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂.

29. Abz-Leu-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂.

30. Abz-Ile-Leu-Gln-Ala-Tyr(3-NO₂)-Glu-NH₂.

31. Abz-Leu-Leu-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂.

32. Abz-Ile-Ala-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂.

33. Abz-Tyr-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂.

34. Abz-Ile-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂.

35. Abz-Leu-Tyr-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂.

36. Abz-Ile-Tyr-Ala-Ala-Tyr(3-NO₂)-Glu-NH₂.

37. Abz-Asp-Val-2-Thi-Gly-Tyr(3-NO₂)-Glu-NH₂.

38. Abz-Leu-Phe-Ala-Pro-Tyr(3-NO₂)-Glu-NH₂.

39. Abz-Tyr-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂.

40. Abz-Leu-Glu-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂

41. Abz-Ile-Ala-Ala-Arg-Tyr(3-NO₂)-Glu-NH₂.

42. Abz-Leu-Leu-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂

43. Abz-Leu-Ala-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂.

44. Abz-Ile-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH2.

45. Abz-Leu-Ala-Ser-Pro-Tyr(3-NO₂)-Glu-NH₂.

46. Abz-Ile-Tyr-Ser-Ala-Tyr(3-NO₂)-Glu-NH₂.

47. Abz-Leu-Leu-Thr-Ala-Tyr(3-NO₂)-Glu-NH₂.

48. Abz-Tyr-Phe-Thr-Asp-Tyr(3-NO₂)-Glu-NH₂.

49. Abz-Leu-Tyr-Ala-Gly-Tyr(3-NO₂)-Glu-NH₂.

50. A compound of general formula (II) as defined in claim 1 wherein, independently or in combination, A¹ is the L-isomer form of isoleucine and A² is Ala, Cys or Tyr.

51. A compound as claimed in claim 50 wherein A is Ala.

52. One of the following compounds:

Boc-Ile-Ala-AMC (129)

HCl.Ile-Ala-AMC (130) N-acetyl-Ile-Ala-AMC (131)

4-morpholino-Ile-Ala-AMC (132)

Benzoyl-Ile-Ala-AMC (133) cyclohexoyl-Ile-Ala-AMC (134)

CBZ-Ile-Ala-AMC (135) Boc-Ile-Cys(Bzl)-AMC (136)

CBZ-Ile-Cys(Bzl)-AMC (137)

Boc-Ile-Tyr-AMC (138)

CBZ-Ile-Tyr-AMC (139).

53. A method of assaying Staphylococcus sp. extracellular cysteine peptidase activity, the method comprising allowing the enzyme to catalyse the cleavage of a peptide as claimed in any one of claims 1 to 52.

54. A method as claimed in claim 53 wherein the assay is carried out during protein purification.

55. A method as claimed in claim 53, wherein a candidate modulator of enzyme activity is present.

56. A method as claimed in claim 55, where the candidate modulator is a candidate inhibitor.

57. A method of screening for candidate inhibitors of Staphylococcus sp. exfracellular cysteine peptidase, the method comprising allowing the enzyme to catalyse the cleavage of a peptide as claimed in any one of claims 1 to 52 in the presence of a candidate inhibitor of enzyme activity.

58. A compound of general formula (III):

CAP-A'-A²-G (III)

wherein:

A¹ and A² are as defined for general formula (I); CAP is as defined for general formula (II); and

59. A compound as claimed in claim 66 wherein, independently or in combination A¹ is the L-isomer form of isoleucine and A² is Ala, Cys or Tyr.

60. A compound as claimed in claim 59 wherein A 2 i ^•s Ala

61. A compound as claimed in any one of claims 58 to 60 for inhibiting Staphylococcus sp. extracellular cysteine peptidases and treating staphylococcal infection.

62. The use of a compound as claimed in any one of claims 58 to 60 in the preparation of an agent for the treatment of staphylococcal infection.