Tripeptide rhodamine compound

Abstract

The present invention concerns rhodamine based fluorescent probes which have use in detecting coagulase-producing bacterial strains. In particular, wherein the bacterial strain is MRSA or MSSA.

Claims

1. A composition for use in detection of a coagulase-producing bacterial strain or strains, said composition comprising 50 M to 100 M of a compound of Formula II, 50 M to 100 M prothrombin or prethrombin or both, and a bacterial strain, wherein the bacterial strain is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA), or combinations thereof, and the bacteria is present in a concentration of 10.sup.2 to 10.sup.6, wherein the compound of Formula II has the structure: ##STR00034##

2. The composition of claim 1 wherein said composition comprises 50 M to 100 M prothrombin.

3. A method of detecting a coagulase-producing Staphylococcus aureus bacterial strain comprising: detecting an optical response in the composition of claim 1.

4. The method according to claim 3, wherein the composition comprises 50 M to 100 M prethrombin.

5. The method according to claim 3, wherein the optical response is fluorescence.

6. The method according to claim 3, wherein the bacterial strain is from a sample selected from the group consisting of a biological fluid, a tissue sample, a tissue section, a cell sample and a non-biological fluid or substrate.

7. The method according to claim 3, wherein the bacterial strain is from a human sample or an animal sample.

8. The method according to claim 3, wherein the bacterial strain is from a food sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will now be described, by way of example, with reference to the accompanying figures, in which:

(2) FIG. 1 illustrates the use of a substrate-coupled dye system which is used as a bacterial detecting agent;

(3) FIG. 2A shows MRSA 10.sup.6 Concentration with 50 M LGX;

(4) FIG. 2B shows MRSA 10.sup.5 Concentration with 50 M LGX;

(5) FIG. 2C shows MRSA 10.sup.3 Concentration with 50 M LGX;

(6) FIG. 2D shows MRSA 10.sup.2 Concentration with 50 M LGX;

(7) FIG. 3A shows MRSA 10.sup.4 Concentration with 100 M LGX;

(8) FIG. 3B shows MRSA 10.sup.3 Concentration with 100 M LGX;

(9) FIG. 3C shows MRSA 10.sup.2 Concentration with 100 M LGX;

(10) FIG. 4A shows MSSA 10.sup.6 Concentration with 50 M LGX;

(11) FIG. 4B shows MSSA 10.sup.5 Concentration with 50 M LGX;

(12) FIG. 4C shows MSSA 10.sup.3 Concentration with 50 M LGX;

(13) FIG. 4D shows MSSA 10.sup.2 Concentration with 50 M LGX;

(14) FIG. 5A shows MSSA 10.sup.4 Concentration with 100 M LGX;

(15) FIG. 5B shows MSSA 10.sup.3 Concentration with 100 M LGX;

(16) FIG. 5C shows MSSA 10.sup.2 Concentration with 100 M LGX;

(17) FIG. 6A (1) shows E. coli 10.sup.6 Concentration with 50 M LGX;

(18) FIG. 6A (2) (decreased Y axis value): E. coli 10.sup.5 Concentration with 50 M LGX;

(19) FIG. 6B (1) shows E. coli 10.sup.5 Concentration with 50 M LGX;

(20) FIG. 6B (2) shows (decreased Y axis value): E. coli 10.sup.5 Concentration with 50 M LGX;

(21) FIG. 6C (1) shows E. coli 10.sup.3 Concentration with 50 M LGX;

(22) FIG. 6C (2) shows (decreased Y axis value): E. coli 10.sup.3 Concentration with 50 M LGX;

(23) FIG. 6D (1) shows E. coli 10.sup.2 Concentration with 50 M LGX;

(24) FIG. 6D (2) shows (decreased Y axis value): E. coli 10.sup.2 Concentration with 50 M LGX;

(25) FIG. 7A (1) shows E. coli 10.sup.4 Concentration with 100 M LGX;

(26) FIG. 7A (2) shows (decreased Y axis value): E. coli 10.sup.4 Concentration with 100 M LGX;

(27) FIG. 7B (1) shows E. coli 10.sup.3 Concentration with 100 M LGX;

(28) FIG. 7B (2) shows (decreased Y axis value): E. coli 10.sup.3 Concentration with 100 M LGX;

(29) FIG. 7C (1) shows E. coli 10.sup.2 Concentration with 100 M LGX;

(30) FIG. 7C (2) shows (decreased Y axis value): E. coli 10.sup.2 Concentration with 100 M LGX;

(31) FIG. 8A (1) shows Coagulase Negative Staphylocci 10.sup.6 Concentration with 50 M LGX;

(32) FIG. 8A (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.6 Concentration with 50 M LGX;

(33) FIG. 8B (1) Coagulase Negative Staphylocci 10.sup.5 Concentration with 50 M LG;

(34) FIG. 8B (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.5 Concentration with 50 M LGX;

(35) FIG. 8C (1) shows Coagulase Negative Staphylocci 10.sup.3 Concentration with 50 M LGX;

(36) FIG. 8C (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.3 Concentration with 50 M LGX;

(37) FIG. 8D (1) shows Coagulase Negative Staphylocci 10.sup.2 Concentration with 50 M LGX;

(38) FIG. 8D (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.2 Concentration with 50 M LGX;

(39) FIG. 9A (1) shows Coagulase Negative Staphylocci 10.sup.4 Concentration with 100 M LGX;

(40) FIG. 9A (2) shows (decreased Y axis value) Coagulase Negative Staphylocci 10.sup.4 Concentration with 100 M LGX;

(41) FIG. 9B (1) shows Coagulase Negative Staphylocci 10.sup.3 Concentration with 100 M LGX;

(42) FIG. 9B (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.3 Concentration with 50 M LGX;

(43) FIG. 9C (1) shows Coagulase Negative Staphylocci 10.sup.2 Concentration with 50 M LGX;

(44) FIG. 9C (2) shows (decreased Y axis value): Coagulase Negative Staphylocci 10.sup.2 Concentration with 50 M LGX;

(45) FIG. 10 shows the effects of 500 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin on staphylocoagulase mediated fluorescence in varying bacterial species;

(46) FIG. 11 shows the effects of 100 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin on staphylocoagulase mediated fluorescence in varying bacterial species;

(47) FIG. 12 shows a direct comparison between 100 M and 500 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin on staphylocoagulase mediated fluorescence in varying bacterial species and 100 M LGX on staphylocoagulase mediated fluorescence in varying bacterial species;

(48) FIG. 13 shows high bacterial concentrations of MRSA, MSSA, S. epidermidis and E. coli with 50 M LGX;

(49) FIG. 14 shows low bacterial concentrations of MRSA, MSSA, S. epidermidis and E. coli with 100 M LGX; and

(50) FIG. 15 shows varying bacterial concentrations of MRSA, MSSA, S. epidermidis and E. coli with 100 M coumarin.

(51) FIG. 16A shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1 prothrombin at 15 minutes.

(52) FIG. 16B shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1 prothrombin at 30 minutes.

(53) FIG. 16C shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1 prothrombin at 60 minutes.

(54) FIG. 17A shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1prethrombin at 15 minutes.

(55) FIG. 17B shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1prethrombin at 30 minutes.

(56) FIG. 17C shows the detection of S. aureus in samples by standard techniques and the relative fluorescence of the samples with 100 M LGX with 1prethrombin at 60 minutes.

(57) FIG. 18 shows the mean values for S. aureus positive and S. aureus negative samples

DETAILED DESCRIPTION OF THE INVENTION

(58) The present invention involves the use of rhodamine coupled to the tripeptide Val-Pro-Arg. This generates a bacterial detecting agent for detecting coagulase-producing bacterial strains. In particular, the present invention is concerned with Staphylococcus aureus coagulase-specific dye systems which are used as a point of care tool to detect MSSA and MRSA within any healthcare based environment (see FIG. 1).

(59) The key advantages that substrate-coupled dye system according to the present invention has over the highly specific and reliable ELISA and western blotting techniques for protein identification are; no need for use of expensive monoclonal antibodies; no need for reagents for plate development; no protracting incubation time for cell culture or pre-enrichment; rapid detection (parts per billion quantities of, for example, rhodamine are visible to the naked eye); it can be used to detect enzymes in multiple matrices e.g. including directly on surfaces potential for equally high sensitivity to PCR/ELISA techniques

(60) The existing molecular method relies upon on MRSA/MSSA coagulase detection. Boc-Val-Pro-Arg-7-AMC (see below) was first reported in 1977 (Ford et al).

(61) ##STR00010##

(62) The tripeptide had been identified as a substrate that mimics fibrogen and cleavage of the tripeptide from the aminomethylcoumarin (AMC) by MRSA coagulase complex results in a shift in the fluorescence and UV spectrum of the compound. This shift can be used to identify the coagulase and consequently the MRSA. One of the drawbacks is sensitivity, requiring cell culture techniques (24 hours), for sufficient quantities of coagulase to be externally expressed for detection to be possible. The other drawback is that there is no colour change observable to the human eye. A further issue is high cost of the BOC-Val-Pro-Arg-7-AMC (Sigma-Aldrich), sufficient for the testing of only small numbers (<5) of samples.

(63) The choice of rhodamine as the chromophore is based on its ability to co-exist as a red zwitterion or colourless lactone. This switch is exploited by attaching a peptide bond to the pendant rhodamine amine groups, thus converting them into electron-deficient amide groups, rendering the coloured zwitterionic structure unable to form. Upon cleavage of the amide bond/s, the zwitterionic form, which is preferred in aqueous solution, will automatically form, granting a substantial change in colour, from colourless to fluorescent yellow.

(64) ##STR00011##

(65) The Val-Pro-Arg tripeptide sequence is known to be a recognisable substrate for staphylothrombin, a complex formed by SA expression of staphylocoagulase and prothrombin. The staphylothrombin complex mistakes the tripeptide for part of the fibrogen protein, which it converts to fibrin. The amidase activity of the staphylothrombin complex is specific to this tripeptide sequence as indicated in previous research and the use of this compound and is the basis of the current commercial tube coagulase test.

(66) LGX is a compound which functions in much the same way, but works as a staphylothrombin assay which is much more sensitive to a given concentration of staphylothrombin, and therefore the number of SA colony forming units.

(67) The estimate of enhanced sensitivity is predicated on the presence of two substrate tripeptides on one dye molecule, essentially doubling the concentration and the fact that coumarin dyes have an extinction coefficient of 20,000 (although not within the visible region), compared to rhodamine extinction coefficient of 110,000.

(68) The current commercial dyes absorb in the UV region at 372 nm and therefore no visible colour change results and fluorescence spectrophotometry must be used to detect the loss of the tripeptide. In the case of the novel compound LGX, it is possible to detect a change within the visible region (488 nm) and therefore, cheaper colorimetery spectrophotometers can be used.

(69) For enhanced sensitivity, however, LGX also has a distinct fluorescence profile, which can also be exploited. The enhanced sensitivity of LGX means that the same quantity of staphylothrombin (and therefore SA colony forming units) can be determined over a much shorter timescale. An alternative view is that smaller quantities of SA bacteria could be identified over the same time period.

(70) LGX works as a staphylothrombin assay to be used in the detection of MRSA and MSSA and is 10 times as sensitive as the existing commercial dye system.

(71) It is to be appreciated that the compound, method and kit of the invention may be used in to detect MSSA and MRSA within any healthcare based environment or agricultural environment. The compound, method and kit of the invention may also be used in to detect MSSA and MRSA in any food or beverage product.

Embodiment 1

(72) In a preferred embodiment of the present invention R.sup.7 of formula I is represented by

(73) ##STR00012##

(74) This embodiment is Embodiment 1 of the present invention.

(75) In a preferred embodiment of Embodiment 1, X is C(O).

(76) In a preferred embodiment of Embodiment 1, Y is O, S or NH, preferably O.

(77) In a preferred embodiment of Embodiment 1, R.sup.1a and R.sup.1b are each independently methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably ethyl, propyl, isopropyl, most preferably propyl or isopropyl. In an embodiment of Embodiment 1, R.sup.1a and R.sup.1b are identical.

(78) In a preferred embodiment of Embodiment 1, R.sup.2a and R.sup.2b are each independently H, OR.sup.a, C(O)R.sup.a, C(O)OR.sup.b, preferably C(O)R.sup.a or C(O)OR.sup.b, preferably where R.sup.a and R.sup.b are each independently a methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, most preferably tert-butyl. In an embodiment of Embodiment 1, R.sup.2a and R.sup.2b are identical.

(79) In a preferred embodiment of Embodiment 1, R.sup.3a and R.sup.3b are each independently a H, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably H, methyl or ethyl, most preferably H. In an embodiment of Embodiment 1, R.sup.3a and R.sup.3b are identical.

(80) In a preferred embodiment of Embodiment 1, R.sup.4a and R.sup.4b are each independently a H, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably H, methyl or ethyl, most preferably H. In an embodiment of Embodiment 1, R.sup.4a and R.sup.4b are identical.

(81) In embodiments where the any of the substituent groups are aryl or C.sub.1-6 alkyl, or contain an aryl or C.sub.1-6 alkyl group, the aryl or C.sub.1-6 alkyl group may be substituted with one or more halo substituents. In preferred embodiments, they are substituted with 1 to 10 halo substituents, more preferably 1 to 7 and most preferably 1 to 4.

(82) In a preferred embodiment of Embodiment 1, n is 0 or 1, preferably 0.

(83) In a preferred embodiment of Embodiment 1, m is 0 or 1 for both R.sup.6a and R.sup.6b, preferably 0.

(84) When R.sup.5 is present, it may occupy any vacant position on the aryl ring. In preferred embodiments R.sup.5 occupies the -meta and/or -para position of the aryl ring.

(85) In a preferred embodiment of Embodiment 1:

(86) X is C(O);

(87) Y is O, S or NH, most preferably O;

(88) R.sup.1a and R.sup.1b are each independently ethyl, propyl, or isopropyl, most preferably isopropyl;

(89) R.sup.2a and R.sup.2b are each independently C(O)R.sup.a or C(O)OR.sup.b, where R.sup.a and R.sup.b are each preferably isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, most preferably tert-butyl;

(90) R.sup.3a and R.sup.3b are each independently H, methyl or ethyl, most preferably H;

(91) R.sup.4a and R.sup.4b are each independently H, methyl or ethyl, most preferably, H;

(92) n is 0 or 1, most preferably 0; and

(93) m is 0 or 1 for both R.sup.6a and R.sup.6b, most preferably 0.

(94) In a preferred embodiment of Embodiment 1 there is provided,

(95) ##STR00013##

(96) wherein

(97) X represents C(O) or S(O).sub.t;

(98) t represents 1 or 2;

(99) Y represents O, S, NH or CH.sub.2;

(100) R.sup.1a and R.sup.1b each independently represent H or C.sub.1-6 alkyl, wherein the latter group is optionally substituted with one or more halo;

(101) R.sup.2a and R.sup.2b each independently represent H, C(O)R.sup.a, C(O)OR.sup.b, -L-aryl or a G group;

(102) R.sup.3a, R.sup.3b, R.sup.4a and R.sup.4b each independently represent H, NO.sub.2, OH, C(O)R.sup.c or C.sub.1-6 alkyl, wherein the latter group is optionally substituted with one or more halo;

(103) each R.sup.5 independently represents halo, OR.sup.d, C(O)R.sup.e, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo;

(104) and/or any two adjacent R.sup.5 groups may be joined together to form, together with the carbon atoms to which they are necessarily attached, a 5- or 6-membered aryl or cycloalkyl group, wherein the latter two groups may be optionally substituted with one or more halo;

(105) n represents 0 to 4;

(106) each R.sup.6 independently represents N(H)R.sup.f, N(R.sup.g)R.sup.h, C(O)R.sup.h, OR.sup.i, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo;

(107) each m independently represents 0 to 3;

(108) each R.sup.a to R.sup.i independently represents C.sub.1-6 alkyl, optionally substituted with one or more halo;

(109) L represents a direct bond or CH.sub.2;

(110) G represents any amino acid which may be further substituted.

(111) In an embodiment of the present invention when:

(112) X=C(O);

(113) Y=O;

(114) R.sup.1a and R.sup.1b are each isopropyl;

(115) R.sup.3a, R.sup.3b, R.sup.4a and R.sup.4b are each H; and

(116) n is 0 for R.sup.5; and

(117) m is 0 for R.sup.6a and R.sup.6b;

(118) R.sup.2a and R.sup.2b may not each be represented by Cbz.

(119) In an embodiment of the present invention when:

(120) X=C(O);

(121) Y=O;

(122) R.sup.2a and R.sup.2b are each represented by Cbz;

(123) R.sup.3a, R.sup.3b, R.sup.4a and R.sup.4b are each H;

(124) n is 0 for R.sup.5; and

(125) m is 0 for R.sup.6a and R.sup.6b;

(126) R.sup.1a and R.sup.1b may not be each be sec-butyl.

(127) In a more preferred embodiment of Embodiment 1 there is provided a compound according to formula III:

(128) ##STR00014##

(129) In another embodiment of Embodiment 1 there is provided a compound according to formula IIIa:

(130) ##STR00015##

(131) In a further embodiment of Embodiment 1 there is provided a compound according to formula IIIb:

(132) ##STR00016##

(133) In an aspect of the present invention, there is provided a mixture of the compounds depicted by formula III, formula IIIa and formula IIIb optionally wherein:

(134) the compound of formula III is present in an amount of about 95.0% to about 100% by weight of the mixture;

(135) the compound of formula IIIa is present in an amount of about 0.1% to about 5.0% by weight of the mixture; and

(136) the compound of formula IIIb is present in an amount from about 0.001% to about 0.1% by weight of the mixture.

(137) For example, the compound of formula III is present in an amount of about 97.0% to about 99.5% by weight of the mixture;

(138) the compound of formula IIIa is present in an amount of about 1.0% to about 3.0% by weight of the mixture; and

(139) the compound of formula IIIb is present in an amount from about 0.01% to about 0.05% by weight of the mixture.

(140) In a particularly preferred embodiment of Embodiment 1, there is provided a compound according to formula II:

(141) ##STR00017##

(142) In another embodiment of Embodiment 1 there is provided a compound according to formula IIa and formula IIb:

(143) ##STR00018##

(144) In another aspect of the present invention, there is provided a mixture of the compounds depicted by formula II, formula IIa and formula IIb optionally wherein:

(145) the compound of formula II is present in an amount of about 95.0% to about 100% by weight of the mixture;

(146) the compound of formula IIa is present in an amount of about 0.1% to about 5.0% by weight of the mixture; and

(147) the compound of formula IIb is present in an amount from about 0.001% to about 0.1% by weight of the mixture.

Embodiment 2

(148) In a preferred embodiment of the present invention m of formula I is 0 for both R.sup.6a and R.sup.6b.

(149) This embodiment is Embodiment 2 of the present invention.

(150) In a preferred embodiment of Embodiment 2, X is C(O).

(151) In a preferred embodiment of Embodiment 2, Y is O, S or NH, preferably O.

(152) In a preferred embodiment of Embodiment 2, R.sup.1a is a methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably ethyl, propyl, isopropyl, more preferably propyl or isopropyl, most preferably isopropyl.

(153) In a preferred embodiment of Embodiment 2, R.sup.2a is H, OR.sup.a, C(O)R.sup.a, C(O)OR.sup.b, preferably C(O)R.sup.a or C(O)OR.sup.b, preferably where R.sup.a and R.sup.b are each independently a methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, most preferably tert-butyl.

(154) In a preferred embodiment of Embodiment 2, R.sup.3a and R.sup.4a each independently represent H, NO.sub.2, OH, C(O)R.sup.c or C.sub.1-6 alkyl, wherein the latter group is optionally substituted with one or more halo, preferably H, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, preferably H, methyl or ethyl, most preferably H. In an embodiment of Embodiment 1, R.sup.3a and R.sup.4a are identical.

(155) In a preferred embodiment of Embodiment 2, R.sup.5 and R.sup.7 are each independently NO.sub.2, or OR.sup.i, wherein each R.sup.i is independently H, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo, and R.sup.7 may be H. In a preferred embodiment R.sup.i is a C.sub.1-3 alkyl, preferably methyl or ethyl, most preferably methyl.

(156) In embodiments where the any of the substituent groups are aryl or C.sub.1-6 alkyl, or contain an aryl or C.sub.1-6 alkyl group, the aryl or C.sub.1-6 alkyl group may be substituted with one or more halo substituents. In preferred embodiments, they are substituted with 1 to 10 halo substituents, more preferably 1 to 7 and most preferably 1 to 4.

(157) In a preferred embodiment of Embodiment 2, n is 0 or 1.

(158) In Embodiment 2, preferred combinations of R.sup.5 and R.sup.7 are:

(159) R.sup.5 is not present (i.e. n=0), and R.sup.7 is H, OMe or NO.sub.2; and

(160) R.sup.5 (n=1) is OMe or NO.sub.2 and R.sup.7 is H, OMe or NO.sub.2.

(161) When R.sup.5 is present, it may occupy any vacant position on the aryl ring. In preferred embodiments R.sup.5 occupies the -meta and/or -para position of the aryl ring.

(162) In a preferred embodiment of Embodiment 2:

(163) X is C(O);

(164) Y is O, S or NH;

(165) R.sup.1a is ethyl, propyl, or isopropyl, most preferably isopropyl;

(166) R.sup.2a is C(O)R.sup.a or C(O)OR.sup.b, where R.sup.a and R.sup.b are each preferably isopropyl, butyl, iso-butyl, sec-butyl or tert-butyl, most preferably tert-butyl;

(167) R.sup.3a and R.sup.4a are independently H, methyl or ethyl, most preferably H;

(168) R.sup.5 and R.sup.6b, if present, are each independently NO.sub.2, or OR.sup.i where R.sup.i is a C.sub.1-3 alkyl, most preferably methyl; and

(169) n is 0 or 1.

(170) In very preferred embodiments of Embodiment 2 the following structures are provided:

(171) ##STR00019## ##STR00020## ##STR00021##

(172) According to another aspect of the present invention there is provided the rhodamine derivatives RD1 and RD2:

(173) ##STR00022##

(174) wherein independently in RD1 and RD2;

(175) Y represents O, S, NH or CH.sub.2;

(176) R.sup.7 may represent H, NO.sub.2, N(H)R.sup.f, N(R.sup.g)R.sup.h, OR.sup.i, SR.sup.i, C(O)R.sup.h, C(O)OR.sup.h, C(O)N(H)R.sup.h, C(O)NR.sub.2.sup.h, S(O).sub.3R.sup.h, S(O).sub.2N(H)R.sup.h, S(O).sub.2NR.sub.2.sup.h, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo;

(177) each R.sup.5 independently represents NO.sub.2, OR.sup.d, NR.sup.d.sub.2, SR.sup.d, C(O)R.sup.e, C(O)OR.sup.e, C(O)N(H)R.sup.e, C(O)NR.sub.2.sup.e, S(O).sub.3R.sup.e, S(O).sub.2N(H)R.sup.e, S(O).sub.2NR.sub.2.sup.e, halo, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo;

(178) and/or any two adjacent R.sup.5 groups may be joined together to form, together with the carbon atoms to which they are necessarily attached, a 5- or 6-membered aryl or cycloalkyl group, wherein the latter two groups may be optionally substituted with one or more halo;

(179) n represents 0 to 4;

(180) R.sup.6a and R.sup.6b independently represents NO.sub.2, N(H)R.sup.f, N(R.sup.g)R.sup.h, OR.sup.i, SR.sup.i, C(O)R.sup.h, C(O)OR.sup.h, C(O)N(H)R.sup.h, C(O)NR.sub.2.sup.h, S(O).sub.3R.sup.h, S(O).sub.2N(H)R.sup.h, S(O).sub.2NR.sub.2.sup.h, aryl or C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo;

(181) each m independently represents 0 to 3;

(182) each R.sup.d to R.sup.i independently represents H, aryl, C.sub.1-6 alkyl, wherein the latter two groups may be optionally substituted with one or more halo.

(183) In an embodiment, when the compounds of Embodiment 2 are cleaved by coagulase enzymes from a coagulase-producing bacterial strain, the rhodamine derivative RD1 is produced. In aqueous solutions the rhodamine derivative RD1 will exist predominantly as the fluorescent rhodamine derivative RD2. The fluorescence profiles of the fluorescent rhodamine derivatives RD2 are dependent on the electron donating/electron withdrawing properties of the substituent groups present on the aromatic rings. Accordingly, the detection wavelength and minimum detection concentration of the rhodamine derivatives can be modulated by altering the number and positioning of electron donating/electron withdrawing substituents on the aromatic moieties.

(184) ##STR00023##

(185) Upon enzymatic cleavage of the amide bond linking the tripeptide sidechain to the rhodamine core in the very preferred compounds of Embodiment 2 (SFG1 to SFG9), the following RD1 rhodamine derivatives are produced.

(186) ##STR00024## ##STR00025##

Experimental

(187) To ensure that the effect of staphylocoagulase on fluorescence potential of the compounds of the present invention was not a strain specific phenomenon and was indeed a species specific reaction, various clinical isolates of MRSA (sample name 1-3-12, 13, 15-17, 19-20 inclusive), MSSA (sample name 58-59, 61-64, 66-69, 71, 74 and 65-78 inclusive), and 68-76), E-coli (sample name 1-11, 12, 15, and 17, and 1 NTCT strain), and coagulase negative Staphylococal species (S. epidermidis 1-4, Coagulase negative S. epidermidis (CNS), S. hominus, S. warneri, M. Luteus and 2 laboratory strains of S. epidermidis were cultured on nutrient agar prior to being grown overnight in nutrient broth under shaking conditions at 37 C. Each sample was rinsed liberally in sterile 1PBS and varying concentrations of bacterial suspension from 10.sup.0-10.sup.6 were generated by serial dilution in 1PBS. Additionally, bacterial cultures were furthermore maintained on nutrient agar to assess the coagulase status of the isolate.

(188) To determine the effects of varying bacterial concentrations with respect to bacterial strains and concentrations of the compounds according to the present invention on staphylocoagulase activity, 100 and 50 M solutions containing LGX were prepared in 1PBS with the addition of 0.05 M Tris Buffer and 0.1 M NaCl to maintain a pH of 8.5 using LGX dissolved in MeOH so that final concentration of methanol was no more than 2.5%. Furthermore, an appropriate concentration (100 M and 50 M, respectively) of human prothrombin was added to the solution.

(189) 90 l of 50 M or 100 M of prepared LGX solution was added to varying concentrations of bacteria (10.sup.6, 10.sup.5, 10.sup.3, 10.sup.2 and 10.sup.4, 10.sup.3, 10.sup.2, 10.sup.0, respectively) in a microtitre plate and staphylocoagulase activity determined by fluorescence spectrophotometry exhibiting excitation at 488 nm and emission at 525 nm at 15 minute intervals over a 6 hour time period. The plate reader used was a Fluostar Optima, and the plates used were Nunclon Surface 96 well plates. Positive controls for each experiment was a strain of MRSA, and the negative control for each experiment was a strain of E. coli, both of which were picked at random to ensure there was no bias within the data. A 1:1 ratio of the LGX solution with PBS was also assayed as LGX alone sample, which has been designated as a black line on the graphs below. n=3 cultures were assayed in each case.

(190) In tandem with all fluorometric assays using LGX, the positive/negative/false positive status of coagulase present in each respective bacterial isolates was determined, using a staphylase text kit (Oxoid), under manufacturer's instructions. Loopfuls of bacteria, which had been cultured overnight on agar, were assayed based on coagulase mediated clumping of fibrinogen-sensitised ovine blood cells. As opposed to being a colourometric or fluorometric assay, providing instant visible coagulation of the reagents following exposure to bacteria. n=3 cultures were assayed in each case.

(191) To ensure that the effect of staphylocoagulase on fluorescence potential of the LGX compound was not a strain specific phenomenon and was indeed a species specific reaction, 20 clinical isolates of MRSA were assayed under the same conditions as detailed above, and have been designated the nomenclature MRSA 1-20. Using the staphylase test, all isolates were confirmed to be coagulase positive. As can be observed in FIG. 2A-2D, whereby LGX concentration was at 50 M and bacterial concentration was 10.sup.6, 10.sup.5, 10.sup.3 and 10.sup.2, there is a considerable degree of variation in the efficacy of the compound with regard to fluorescence reaching its maximum over time in varying bacterial strains, however, at a 10.sup.6 bacterial concentration, the maximum level was reached by 2 hours for all samples, whereas at 10.sup.5, 10.sup.3 and 10.sup.2, fluorescence tended to reach maximum at varying times.

(192) It is evident from these results that at high bacterial concentrations of some strains of MRSA (10.sup.6), there was an almost instantaneous reaction, with fluorescence reaching the maximum reading of 65,000 RFU within 2 hours in the majority (90%). 10% of the samples failed to reach maximum, however, these were significantly higher than the LGX alone and negative control by several orders of magnitude. At a lower concentration (10.sup.5) of both MRSA, a more gradual increase in fluorescence was observed, with fluorescence reaching the maximum reading of 65,000 RFU within 2 hours in the majority (60%). However, 20% of the samples failed to reach maximum fluorescence, within 5 hours, yet, there is still a highly significant discrimination between these slower strains than the negative control even at 6 hours.

(193) At even lower concentration (10.sup.3 and 10.sup.2) of MRSA samples, a more gradual increase in fluorescence was observed, with fluorescence reaching the maximum reading of 65,000 RFU within 4 hours in the majority (60% and 40%, respectively). However, 40% and 60% of the samples, respectively, failed to reach maximum fluorescence, within the 6 hour time frame of the experiment, however again there is still a highly significant discrimination between these samples at 6 hours. It is important to note that, in spite of the vast variance in efficacy across the bacterial strains, fluorescence is still significantly higher in all samples when compared to the negative control, where fluorescence remains consistently low across the 6 hour time period.

(194) Further to this, using higher concentrations (100 M) LGX and lower (10.sup.4, 10.sup.3 and 10.sup.3) bacterial concentrations, as demonstrated in FIG. 3 A-D, results were again consistent with what was observed in FIGS. 2A-D, whereby at higher bacterial concentrations, fluorescence reached its upper limits within 2 hours in 50% of the samples (10.sup.4), in 40% of the samples within 3 hours (10.sup.3) or within 4 hours in 40% of the samples (10.sup.2). Over the 6 hours time period, 90% of MRSA at 10.sup.4 concentration reached maximum fluorescence, whereas at lower concentrations of 10.sup.3 and 10.sup.4, 40% of isolates failed to reach maximum fluorescence, however, a distinction between MRSA strains and the negative control and LGX alone can be observed in all cases, whereby the negative control remained low and failed to demonstrate any increase over time.

(195) 20 clinical isolates of MSSA were assayed under the same conditions as detailed above, and have been designated the nomenclature MSSA 58-65, and 68-76. Using the staphylase test, all isolates were confirmed to be coagulase positive. As can be observed in FIG. 4A-D, whereby LGX concentration was at 50 M and bacterial concentration was 10.sup.6, 10.sup.5, 10.sup.3 and 10.sup.2, there is a considerable degree of variation in the efficacy of the compound with regard to fluorescence reaching its maximum over time in varying bacterial strains, however, at a 10.sup.6 bacterial concentration, the maximum level was reached by 2 hours, whereas at 10.sup.5, 10.sup.3 and 10.sup.2, fluorescence tended to reach maximum at varying times. It is evident from these results that at high bacterial concentrations of some strains MSSA (10.sup.6), there was an almost instantaneous reaction, with fluorescence reaching the maximum reading of 65,000 RFU in all samples within 1 hour and 45 minutes.

(196) At a lower concentration (10.sup.5) of both MSSA, a more gradual increase in fluorescence was observed, with fluorescence reaching the maximum reading of 65,000 RFU within 2 hours in the majority (75%). Only 5% of the samples failed to reach maximum fluorescence, within 6 hours, however again, this was significantly higher than the negative control and LGX alone. At even lower concentration (10.sup.3) of MSSA samples, a more gradual increase in fluorescence was observed, with fluorescence reaching the maximum reading of 65,000 RFU in 25% of the samples within 2.5 hours, and within 6 hours 60% of samples had reached maximum fluorescence in the 10.sup.3 isolates. In the 10.sup.2 concentration samples, no samples reached maximum fluorescence, yet again, discrimination between MSSA at this concentration and the negative control was observed.

(197) Further to this, using higher concentrations (100 M) LGX and lower (10.sup.4, 10.sup.3 and 10.sup.3) bacterial concentrations, as demonstrated in FIGS. 5A-C, results were again consistent with what was observed in FIG. 2A-D, whereby at higher bacterial concentrations, fluorescence reached its upper limits within 1 hours in 70% of the samples (10.sup.4), yet, at this concentration, 20% of samples remained below maximum fluorescence, yet results still maintained a differentiation between MSSA and the negative control. 45% of the samples reached maximum fluorescence within 4 hours in the 10.sup.3 samples, or within 2 hours in 5% of the samples and 25% of the samples over a 6 hour time period (10.sup.2). Again, all samples at both 10.sup.3 and 10.sup.2 demonstrated a significant discrimination between the presence of MRSA at any concentration and the negative control, again whereby the negative control remained low and failed to demonstrate any increase over time.

(198) To further demonstrate the discriminatory potential of LGX, between coagulase positive MRSA and MSSA and coagulase negative bacterial species, various strains of E. coli and proven coagulase negative staphylocci were assayed in conjunction with a positive control (MRSA). FIGS. 6, A-D (1 & 2), demonstrates the effect of the presence of high concentrations (10.sup.6, 10.sup.5, 10.sup.3 and 10.sup.2) of E. coli strains on amide cleavage induced fluorescence of LGX, designated the nomenclature E. coli 1-20. It is important to note that graphs have been separated into 1 and 2, 1 being reaching 80000 units on the Y axis and 2 demonstrating a Y axis reaching maximum of 30000, to illustrate the differences in fluorescence units within E. coli samples alone.

(199) At 10.sup.6, 10.sup.5 and 10.sup.3 concentrations, fluorescence readings reach a maximum almost immediately or within an hour for the positive control, or at 6 hours for the lower concentrations, whereas the coagulase negative strains of staphylococci remain consistently low and tend to not deviate from a baseline level throughout the duration of the experiment. On occasion, there are instances at which fluorescence falls below baseline level, which may be suggestive of hydrolysis of the compound or an alternative mechanism which may affect the fluorescence potential of LGX. It important to note that there is no discrimination between the coagulase negative staphylococci at any concentration or at any time point, and results remain consistent throughout the experiment.

(200) Again, as demonstrated by the results depicted in FIGS. 7A-C (1 & 2), and consistent with the data presented in FIG. 4, lower concentrations of the respective bacterial strains (10.sup.4, 10.sup.3 and 10.sup.3) were assayed in the presence of 100 M LGX solution and results tended to follow the same pattern, whereby fluorescence, which is relative to staphylocoagulase activity, had a tendency to reach a maximum within 3 hours at all concentrations in MRSA, whereas coagulase negative bacterial strains remained consistently low across the 6 hour time period.

(201) FIGS. 8, A-D (1 & 2), demonstrates the effect of the presence of high concentrations (10.sup.6, 10.sup.5, 10.sup.3 and 10.sup.2) of S. Epidermidis species, CNS strains, laboratory strains, S. Hominus, S. Warneri and M. luteus, all of which are coagulase negative, on amide cleavage induced fluorescence of LGX, in comparison to coagulase positive MRSA. It is important to note that graphs have been separated into 1 and 2, 1 being reaching 80000 units on the Y axis and 2 demonstrating a Y axis reaching maximum of 30000, to illustrate the differences in fluorescence units within staphyloccal samples alone. Using the staphylase test, all isolates were confirmed to be coagulase negative, with the exception being M. luteus, which proved to demonstrate a false positive result. At 10.sup.6, 10.sup.6 and 10.sup.3 concentrations, fluorescence readings reach a maximum almost immediately or within an hour for the positive control, whereas the coagulase negative strains of staphylococci remain consistently low and tend to not deviate from a baseline level throughout the duration of the experiment. On occasion, there are instances at which fluorescence falls below baseline level, which may be suggestive of hydrolysis of the compound or an alternative mechanism which may affect the fluorescence potential of LGX.

(202) It important to note that there is no discrimination between the coagulase negative staphylococci and the negative control at any concentration or at any time point, and results remain consistent throughout the experiment. Again, as demonstrated by the results depicted in FIGS. 9A-C (1 & 2), and consistent with the data presented in FIG. 3, lower concentrations of the respective bacterial strains (10.sup.4, 10.sup.3 and 10.sup.3) were assayed in the presence of 100 M LGX solution and results tended to follow the same pattern, whereby fluorescence, which is relative to staphylocoagulase activity had a tendency to reach a maximum within 3 hours at all concentrations in MRSA, whereas coagulase negative bacterial strains remained consistently low across the 6 hour time period.

(203) The data shown within this study have amply demonstrated the use of LGX to determine the presence of MRSA and MSSA within a sample, when compared to coagulase negative bacterial strains. Using a large number of isolates, the ability of LGX to rapidly detect the presence of staphylocoagulase in a sample, which is culture independent, abolishes the potential to show a false positive result, and can detect a low concentration of bacteria within a sample, which is based on time. More traditional methods of bacterial detection and diagnosis of infection requires an overnight culture, followed by lab based tests, and potentially more confirmatory tests such as ELISA and PCR, whereas this system may provide a potential for the rapid detection of a bacterium without the need for such a lengthy time period, a point of care tool, facilitating prompt diagnosis and treatment of infection.

Conclusions

(204) Across a broad spectrum of bacterial isolates, the use of this system in the identification of MRSA and MSSA in clinical samples or within a laboratory setting requires no culturing and can detect the presence of bacteria as low as 10.sup.2 concentrations.

(205) Using 50 M of LGX and 10.sup.4 and above cell concentrations of both MRSA and MSSA, a fluorescence reading in excess of 20,000 within 30 minutes is considered to be a positive result, however, at 10.sup.3 and 10.sup.2 cell concentrations of MRSA and MSSA, 5% and 20%, of isolates, respectively, failed to reach this level of fluorescence, within this 30 minute time limit.

(206) Increasing the concentration of LGX to 100 M bears a significant effect on the sensitivity and selectivity at lower bacterial concentrations. At 10.sup.3 cell concentration and above, 100% of MRSA and MSSA samples tested to date gave a fluorescence reading greater than 20,000 within 30 minutes, indicating a positive result. At 10.sup.2 concentrations of bacteria, a fluorescence reading of 20,000 is achieved within 1 hour in 95% and 90% of MRSA and MSSA samples, respectively.

(207) No response above 5000 fluorescence units was observed in E. coli and coagulase negative staphylococci at either 50 M or 100 M concentrations of LGX within this 1 hour time frame.

(208) At a 50 M concentration of LGX and using all concentrations of bacteria, more than 80% MRSA and MSSA show a significant absorbance of above 20,000 fluorescence units after 1 hour, whereas 0% of E. coli and coagulase negative staphylocci show the same response over the same time period.

The Potential of N-t-BOC-Val-Pro-Arg-7-Amido-4-Methylcoumarin to Detect Varying Bacterial Species at Varying Bacterial Concentrations

(209) In order to compare and contrast the efficacy of the LGX system, with a similar, well established means of detecting staphylocoagulase (N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin), it was necessary to assess the same spectra of bacterial species and concentrations under the same parameters as used in the LGX system, and also using the same methods which have already been characterised (Ford et al, 1999). As we have already demonstrated, the LGX system effectively detects low bacterial concentrations at a relatively low concentration of compound, and also potently distinguishes between coagulase positive MRSA and MSSA, and coagulase negative E. coli and S. epidermidis.

(210) As described for the LGX system, clinical isolates of Methicillin Resistant Staphylococcus Aureus (MRSA), Methicillin Sensitive Staphylococcus Aureus (MSSA), Staphylococcus Epidermidis and Eschericia coli were cultured on nutrient agar prior to being grown overnight in nutrient broth under shaking conditions at 37 C. To determine the effects of varying bacterial concentrations with respect to bacterial strains and N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin concentrations on staphylocoagulase activity, a direct comparison between the LGX system and the existing use of N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin as a means of detecting staphylocoagulase was necessary (Ford et al, 1999).

(211) 100 M and 500 M solutions of coumarin solution were prepared in 1PBS with the addition of 0.05 M Tris Buffer and 0.1 M NaCl to maintain a pH of 8.5 using N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin dissolved in MeOH. Furthermore, 100 M and 500 M of human prothrombin was added to the solution. 90 l of 50 M or 100 M of N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin solution was added to varying concentrations of bacteria (10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2 and 10.sup.0, respectively) in a microliter plate and staphylocoagulase activity determined by fluorescence spectrophotometry (Excitation of 355 nm, Emmission of 460 nm) at 15 minute intervals over a 6 hour time period. N=3 cultures were assayed in each case. As with the LXG system, the plate reader used was a Fluostar Optima, and the plates used were Nunclon Surface 96 well plates.

(212) FIG. 10 demonstrates the effect of the presence of varying concentrations of MRSA, MSSA, S. epidermidis and E. coli on the fluorescence of 500 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin, using the same parameters as used in the LGX system. It is evident from these results that at high bacterial concentrations of all bacterial species (10.sup.6), there was little discrimination between the fluorescence of all samples, which is certainly the opposite of what is observed in the LGX system, with the exception being MSSA at 10.sup.6, whereby a slight increase was observed, but was significantly less than what one would observe using LGX. Further to this, at both high and low concentrations of MRSA, MSSA, S. epidermidis and E. coli, no increase in fluorescence was observed and results remained consistently low for all bacterial species over the 6 hour time period, thus demonstrating that the use of N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin does not harbour the potential to discriminate between bacterial species at a low concentration.

(213) FIG. 11 demonstrates the effect of the presence of varying concentrations of MRSA, MSSA, S. epidermidis and E. coli on the fluorescence potential of 100 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin. Again, it can be seen that at high bacterial concentrations of MRSA and MSSA (10.sup.6 and 10.sup.5), there is no discrimination between bacterial samples, and this pattern prevails at lower bacterial concentrations in all bacterial species. Again, unlike the LGX system, and consistent with the data presented in FIG. 10, exceptionally high concentrations of N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin were not capable of distinguishing between bacterial species at low concentrations over a 6 hour time period.

(214) FIG. 12 demonstrates the comparison between 100 and 500 m N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin and 100 m LGX in MRSA, MSSA, E. coli and S. Epidermidis at a 10.sup.2 concentration, which would be considered to be a normal level of bacteria. It is vastly evident that in MRSA and MSSA samples at this concentration, there is no comparison between the LGX system and -t-BOC-val-pro-arg-7-amido-4-methylcoumarin- the use of LGX by far produces a superior response in terms of selectivity and speed at a lower concentration than that of the coumarin system.

(215) The LGX system facilitates the production of an instantaneous and increased fluorescent response, even at very low bacterial concentrations, and bears the potential to determine the presence of MRSA and MSSA far more significantly than N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin. The speed and selectivity of the LGX system, as demonstrated in these data, shows that this new system may prove to be a vital tool in the detection of coagulase positive bacteria, more so than the systems which are already in place.

(216) The first generation synthesis of the prototype active molecule (LGX) has been accomplished utilising liquid phase peptide synthesis, taking advantage of the highly reactive coupling reagents Oxyma, COMU and EDCI. Firstly, the rhodamine is coupled to a suitably protected arginine residue (Boc-(Z).sub.2-Arg) utilising EDCI and Oxyma in pyridine/DMF. The two Boc protecting groups are them removed using TFA, before the Rhod(Arg(Z).sub.2NH.sub.2).sub.2 is further coupled to the boo protected peptide dimer (Boc-Val-Pro). Finally deprotection under hydrogenation conditions remove the Cbz protecting groups yielding LGX.

(217) Clinical isolates of Methicillin Resistant Staphylococcus Aureus (MRSA), Methicillin Sensitive Staphylococcus Aureus (MSSA), Staphylococcus Epidermidis, Eschericia coli and other staphyloccal species were cultured on nutrient agar prior to being grown overnight in nutrient broth under shaking conditions at 37 C. Each sample was rinsed liberally in sterile 1PBS and varying concentrations of bacterial suspension from 10.sup.20-10.sup.6 were generated by serial dilution in 1PBS. Additionally, bacterial cultures were furthermore maintained on nutrient agar to assess the coagulase status of the isolate.

(218) To determine the effects of varying bacterial concentrations with respect to bacterial strains and LGX concentrations on staphylocoagulase activity, 100 and 50 M solutions of LGX were prepared in 1PBS with the addition of 0.05 M Tris Buffer and 0.1 M NaCl to maintain a pH of 8.5 using LGX dissolved in MeOH so that final concentration of methanol was no more than 2.5%. Furthermore, an appropriate concentration (100 M and 50 M, respectively) of human prothrombin was added to the solution. 90 l of LGX solution in addition to each respective bacterial isolate at varying concentrations was added to a microtitre plate. Staphylocoagulase activity resulting from cleavage of amide bonds within the compound was determined using fluorescence spectrophotometry exhibiting excitation at 488 nm and emission at 525 nm at 15 minute intervals over a 6 hour time period. n=3 cultures were assayed in each case. The plate reader used was a Fluostar Optima, and the plates used were Nunclon Surface 96 well plates.

(219) Tests were also run to validate that the methanol had no adverse effects on the growth of a number of bacterial strains up to a 5% methanol solution. Varying concentrations of methanol (0.25%, 0.5%, 1%, 2.5 5, 4% and 5%) were incubated with each bacterial species also at varying concentrations (10.sup.2-10.sup.6) for 2 hours prior to plating on nutrient agar to assess the effects of the solvent on bacterial viability. Plates were incubated overnight at 37 C. and the number of colonies was counted and it was found that up to 4% methanol, bacterial viability was not compromised at all bacteria concentrations, thus providing the optimum concentration to both maintain compound solubility and ensure bacterial cell viability to be 2.5%.

(220) To compare the efficacy of this staphylocoagulase assay with current, well established gold standard methods of coagulase detection such as a coumarin assay, coagulase test and latex agglutination test, bacterial isolates were prepared in suspension as above and clinical isolates of each respective strain was cultured overnight on nutrient agar. 90 l of bacterial suspension was added to a solution consisting of 100 M solutions of N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin prepared in 1PBS with the addition of 0.05 M Tris Buffer and 0.1 M NaCl to maintain a pH of 8.5. Staphylocoagulase activity was determined by measuring the absorbance at 385 nm at 15 minute intervals over a 6 hour time period. It should be noted that a concentration of 795 M of the coumarin dye is usually required post-culture (10.sup.6-10.sup.8 bacteria) in order to observe a significant chromogenic response.

(221) Furthermore, to determine the positive/negative/false positive status of coagulase present in bacterial isolates, loopfuls of each respective culture was assessed using a staphylase text kit (Oxoid), under manufacturer's instructions. This particular kit is commonly used in hospital and research laboratories and functions through coagulase mediated clumping of fibrinogen-sensitised ovine blood cells. As opposed to being a colourometric or fluorometric assay, the staphylase kit provides an instant visible coagulation of the reagents following exposure to bacteria following a 24 hour culture. n=3 cultures were assayed in each case.

(222) FIG. 13 demonstrates the effect of the presence of high concentrations of MRSA, MSSA, S. epidermidis and E. coli on the fluorescence potential of 50 M LGX following staphylocoagulase mediated cleavage of amide bonds within the compound. It is evident from these results that at high bacterial concentrations of MRSA and MSSA (10.sup.6), there was an almost instantaneous reaction, with fluorescence reaching the maximum reading of 65,000 RFU for each sample. However, at a lower concentration (10.sup.5) of both MRSA and MSSA, a more gradual increase in fluorescence was observed, with fluorescence reaching a maximum after approximately 3 hours. Further to this, at both high and low concentrations of S. epidermidis and E. coli, no increase in fluorescence was observed and results remained consistently low for both bacterial species over the 6 hour time period.

(223) FIG. 14 demonstrates the effect of the presence of lower concentrations of MRSA, MSSA, S. epidermidis and E. coli on the fluorescence potential of 100 M LGX following staphylocoagulase mediated cleavage of amide bonds within the compound. Again, it can be seen that at high bacterial concentrations of MRSA and MSSA (10.sup.4 and 10.sup.3), and 10.sup.2 concentrations of MSSA, there was an almost instantaneous reaction, with fluorescence reaching the maximum reading of 65,000 RFU for each sample. However, at a lower concentration (10.sup.2) of MRSA, a more gradual increase in fluorescence was observed, with fluorescence reaching a maximum after approximately 3 hours. Consistent with the data presented in FIG. 1.1, at both high and low concentrations of S. epidermidis and E. coli, no increase in fluorescence was observed and results remained consistently low for both bacterial species over the 6 hour time period.

(224) FIG. 15 details the efficacy of 100 M N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin as a means of detecting staphylocoagulase and in maintaining a distinct pattern of detection between both MRSA and MSSA, and S. epidermidis and E. coli. As can be observed there is a higher absorbance pattern observed at 10.sup.6 concentrations of all bacterial types, however, there is little increase over the 6 hour time period. Further to this, at 10.sup.5, 10.sup.4, 10.sup.3 and 10.sup.2 concentrations, there is no increase or variance between bacterial strains over the 6 hour time period, and absorbance remains consistent for all samples.

(225) Table 1 displays the results of the staphylase test compared to the LGX system, which is based on the agglutination potential of bacterial species and strains, for MRSA, MSSA, E. epidermidis, Encolpia and other known coagulase negative strains of bacteria including S. hominus, S. warneri and M. luteus. The kit used has the ability to distinguish between the presence of coagulase positive and negative bacterial strains, in addition to determining whether a sample is proving to be a false positive. As demonstrated by the table, MRSA and MSSA are both coagulase positive bacteria, as furthermore confirmed by our LGX system and the staphylase test, S. epidermidis, E. coli, S. warneri, S. hominus and M. Luteus are all proven to be coagulate negative, and both LGX and staphylase test showed E. coli and S. epidermidis, S. warneri and S. hominus tested negative for coagulase activity. However, M. luteus provided a false positive result using the staphylase test, but showed to be negative using the LGX system.

(226) TABLE-US-00001 TABLE 1 LGX vs Staphylase test data for the detection of coagulase positive samples BACTERIAL COAGULASE LGX STAPHYLASE SPECIES +/ SYSTEM TEST MRSA + + + MSSA + + + S. EPIDERMIDIS E. COLI S. WARNERI S. HOMINUS M. LUTEUS False positive

(227) Based on these data, it is evident that the system according to the present invention is superior to what is already in place for rapidly detect the presence of staphylocoagulase within a bacterial sample, which is relative to the amount of bacteria within a culture.

(228) Using the LGX system an immediate and significant fluorescent response in the presence of only 10.sup.2 Staphylococcus aureus units at 525 nm (ex 488 nm) is observed. This outperforms the N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin in terms of sensitivity by several orders of magnitude (10.sup.4 bacteria).

(229) The LGX system also offers good selectivity for coagulase positive bacteria and quickly discriminates for SA against E. coli and S. epidermidis and other common bacteria, which are shown to give negligible results when tested with N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin at the low bacterial concentrations at which the LGX system shows a clear response. Culturing of SA is essential for its detection using the staphylase test and N-t-BOC-val-pro-arg-7-amido-4-methylcoumarin. The present invention can detect SA at very low bacterial concentrations and can be used at the point of care as a rapid diagnostic tool for the screening of people, materials and surfaces for SA prior to typing of the strain and/or further discriminatory microanalysis.

(230) Furthermore, the use of this method of detection has shown to overrule the provision of false positives within a sample. The rapid detection and the potential applications of this compound as a culture independent means to determine the presence of MRSA and MSSA, will provide a vital tool in diagnosing this infection and may lead to faster and more specific treatment of disease, thus demonstrating that this system has significant diagnostic potential, from both a research and clinical perspective.

Efficacy of a Compound and a Method of the Current Invention, in Combination with Prothrombin or Prethrombin, to Detect S. aureus in Patient Samples

(231) This set of experiments was undertaken to assess the efficacy of a method of the current invention in combination with either prothrombin or prethrombin using patient samples. The results were verified using gram staining, cell culturing methods and the staphylase test kit (oxoid).

(232) A population of 113 volunteers were administered a set of swabs, comprising two swabs that were pre-moistened with sterile H.sub.2O, and one swab that was dry. Each volunteer swabbed the internal periphery of each nostril with the two moistened swabs. Each volunteer then subsequently swabbed the internal periphery of each nostril with the dry swab. The swabs were then placed into amies transport medium to maintain bacterial cell viability. Each sample was anonymous and the study was randomised.

(233) A 100 L solution of 100 M LGX with either 1prothrombin or 1prethrombin (buffered to pH 8.5 with Tris base and NaCl), was pipetted into sterile 1.5 mL eppendorf tubes. Each moistened swab was placed into the eppendorf tube, and cut to a length to facilitate closure of the tube, with a pair of sterilised scissors. Each tube was subjected to vigorous agitation and the swab tips were removed with sterile forceps. The remaining solution was subsequently pipetted into a microtitre plate (Nunclon 96 well plates). The relative fluorescence of the sample was then recorded every fifteen minutes over a six hour time period (ex.=488 nm and em=525 nm).

(234) In tandem with this, the remaining swabs (in amies transport medium) were transferred onto pre warmed mannitol salt agar (chromogenic agar for the determination of S. aureus) plates and incubated for up to 48 hours at 37 C. The colour change and colony appearance of the bacteria was noted. A colour change from red to yellow demonstrates the presence of S. aureus, due to fermentation of the mannitol, causing a change in pH. Following this, 20 separate, discrete colonies were sub-cultured onto nutrient agar and also onto Baird Parker Agar (agar for the determination of S. aureus and S. epidermidis). After an overnight culture at 37 C., colony morphology and appearance was observed (S. aureus colonies are large with a pale halo surrounding the colonies, S. epidermidis colonies are small and black).

(235) Additionally, isolates from the nasal swabs cultured on nutrient agar were tested for coagulase activity using a staphylase test kit (oxoid). Loopfuls of bacteria were assayed based on coagulase mediated clumping of fibrinogen-sensitised ovine blood cells. A coagulase positive bacteria is indicative of S. aureus, and as little as 1 positive isolate is subsequently deemed a S. aureus carrier.

(236) A final test to determine the identification of the bacteria within each sample, a gram stain was performed. This is based on the presence of peptidoglycan in gram positive cells and their ability to retain crystal violet. S. aureus is a gram positive bacterium. Briefly, a drop of sterile dH.sub.2O was placed on a microscope slide and a small amount of culture was applied to this and allowed to air dry. The slide was passed though a flame to fix the bacteria. Crystal Violet was applied to each slide, followed by washing with dH.sub.2O. Iodine was then added to the slides, followed by washing with dH.sub.2O and subsequent decolourisation with ethanol and another wash with dH2O. Each slide was counterstained with carbol fushchin (for gram negative), rinsed again and allowed to dry. Each slide was observed for gram positive bacteria using 40magnification using a light microscope.

Results

(237) Each swab sample was examined for the presence of S. aureus based on the above identification techniques, and termed positive or negative. The results were compared to the finding of the assay of the current invention (LGX test), with either prethrombin or prothrombin, and plotted on a bar chart as a function of relative fluorescence units.

(238) FIGS. 16A to 16C illustrate the results using 100 M LGX with 1prothrombin at 15, 30, and 60 minutes. The lower threshold limit for relative fluorescence units of 20,000 RFU was used to determine if a sample was positive or negative for S. aureus using the method of the invention. This threshold was based on previous data using clinical strains at a concentration of 10.sup.2 colony forming units per ml (CFU/mL.sup.1). As can be seen from FIGS. 16A and 16C approximately 29 samples tested positive for S. aureus using a method of the current invention after 15 minutes and approximately 34 samples tested positive for S. aureus after 60 minutes using a method of the current invention. As illustrated in FIG. 16C a number of samples tested negative for S. aureus using an assay of the present invention, which tested positive for S. aureus (by demonstrating a prevalence of 5% coagulase+bacteria) using other identification techniques. It is considered that this false negative result was due to the colony numbers being insufficient to yield a positive results with the method of the current invention. Overall, the use of prothrombin in the assay of the current invention was efficient in detecting S. aureus in as little as 15 minutes.

(239) FIGS. 17A to 17C illustrate the results using 100 M LGX with 1prethrombin at 15, 30, 60 and 120 minutes. The lower threshold limit for relative fluorescence units of 20,000 RFU was used to determine if a sample was positive or negative for S. aureus. This threshold was based on previous data using clinical strains at a concentration of 10.sup.2 colony forming units per ml (CFU/mL.sup.1). As can be seen from FIGS. 17A and 17C approximately 49 samples tested positive for S. aureus after 15 minutes using a method of the current invention and approximately 49 samples tested positive for S. aureus after 60 minutes using a method of the current invention. As illustrated in FIG. 17C a number of samples tested negative for S. aureus using an assay of the present invention, which tested positive for S. aureus (by demonstrating a prevalence of 5% coagulase bacteria) using other identification techniques. It is considered that this false negative result was due to the colony numbers being insufficient to yield a positive results with the method of the current invention. Overall, the use of prethrombin in the assay of the current invention was efficient in detecting S. aureus in as little as 15 minutes.

Conclusion

(240) As can be observed from FIGS. 16 and 17, using prethrombin in the assay of the invention provided higher sensitivity and specificity than the use of prothrombin, i.e. there were more true positive results and less false positive and false negative results. It is considered that this result is due to the fact that prothrombin is not metabolised as quickly as prethrombin and therefore does not demonstrate as potent specificity in terms of detection of coagulase positive bacteria. This is further illustrated by FIG. 18 which shows the mean values for S. aureus positive and S. aureus negative samples at each time interval using the method of the invention with prothrombin and prethrombin.

Synthetic Experimental

Boc-NH-Val-Pro-OMe

(241) ##STR00026##

(242) To a solution of NH-Pro-OMe (3.00 mmol, 0.50 g, 1 eq) and Boc-NH-Val-OH (3.00 mmol, 0.65 g, 1 eq) in anhydrous DMF (6 mL) was added NEt.sub.3 (9.00 mmol, 0.91 g, 1.25 ml, 3 eq) and the solution was cooled to 0 C. COMU (3.00 mmol, 1.28 g, 1 eq) was then added to the reaction mixture and stirred for 1 hr at 0 C. before warming to ambient temperature over 4 hrs. The reaction was then diluted with EtOAc (12 mL), washed with saturated aqueous NaHCO.sub.3 (24 mL), 1M aqueous LiCl (24 mL) then dried over NaSO.sub.4 and concentrated to give product as a light brown oil which was used without further purification (2.07 mmol, 0.68 g, 69%).

Rho-(Arg-(Z)2-NHBoc)2

(243) ##STR00027##

(244) To a solution of HO-arg(Z).sub.2NHBoc (4.61 mmol, 2.5 g, 6 eq), EDCI (4.61 mmol, 883 mg, 6 eq), oxyma (4.60 mmol, 654 mg, 6 eq) in anhydrous DMF (5 mL) was added freshly distilled anhydrous pyridine (5 mL) followed by rhodamine 110 (0.763 mmol, 280 mg, 1 eq) and the solution was stirred at room temperature under a nitrogen atmosphere for 10 days. The reaction was then diluted with EtOAc (20 mL), washed with saturated aqueous NaHCO.sub.3 (210 mL), 1M aqueous LICl (210 mL) and 10% aqueous CuSO.sub.4 (310 mL), dried over MgSO.sub.4 and concentrated to give a green oily solid. This crude product was then purified by column chromatography over silica gel, eluting with 25% EtOAc in CHCl.sub.3 to yield the product as a white amorphous solid (0.14 mmol, 200 mg, 18%): .sub.H(400 MHz, CDCl.sub.3), 9.45 (2H, s, broad, NH-23), 9.29 (2H, s, Broad, NH-21), 9.01 (2H, s, broad, NH-15), 8.02 (1H, dd, .sup.3J.sub.HH=6.02 Hz, .sup.4J.sub.HH=2.01 Hz, CH-12), 7.56-7.69 (2H, m, CH-10 and CH-11), 7.43, (2H, s, CH-2), 7.38-7.20 (20H, m, CH-27, CH-28, CH-29, CH-33, CH-34 and CH-35), 7.05 (1H, dd, .sup.3J.sub.HH=6.02 Hz, .sup.4J.sub.HH=2.01 Hz, CH-9), 6.77 (2H, d, .sup.3J.sub.HH=6.02 Hz, CH-4), 6.69 (2H, dd, .sup.3J.sub.HH=6.02 Hz, CH-5), 5.70, (2H, s, broad, NH-36), 5.30-5.02 (8H, m, CH.sub.2-25 and CH.sub.2-31), 4.39, (2H, m, CH-17) 4.07-3.87 (4H, m, CH.sub.2-20), 1.86-1.62 (8H, m, CH.sub.2-18 and CH.sub.2-19), 1.41 (18H, s, CH.sub.3-39); .sub.c (100 MHz) 171.1, 171.0, 169.5, 163.6, 160.8, 156.2, 155.7, 153.1, 139.6, 136.4, 135.1, 134.4, 134.4, 129.7, 128.9, 128.8, 128.5, 128.4, 128.3, 128.0, 128.0, 126.2, 124.9, 124.1, 115.4, 114.2, 107.9, 82.4, 80.4, 69.1, 67.2, 54.8, 44.0, 28.5, 28.3, 25.01.

Rho-(Arg-(Z)2NH2)2

(245) ##STR00028##

(246) To a solution of Rho-(Arg-(Z).sub.2-NHBoc).sub.2 (0.14 mmol, 200 mg, 1 eq) in DCM (2 mL) was added TFA (0.3 mL) and the reaction was stirred under nitrogen atmosphere for 6 hours. The reaction was then quenched with 1M aqueous NaOH (1 mL) and extracted. The organic layer was then washed with saturated aqueous NaCl (31 mL), dried over MgSO.sub.4 and concentrated to yield product as an amorphous solid which used immediately (0.13 mmol, 156 mg, 94%).

Rho-(Arg-(Z)2-Pro-Val-NHBoc)2

(247) ##STR00029##

(248) A solution of Rho-(Arg-(Z).sub.2NH.sub.2).sub.2 (0.132 mmol, 156 mg, 1 eq), HO-Pro-Val-NHBoc (0.264 mmol, 92 mg, 2 eq) and NEt.sub.3 (0.396 mmol, 40 mg, 55 L, 3 eq) in anhydrous DMF (1 mL) was cooled to 0 C. before the addition of COMU (0.264 mmol, 114 mg, 2 eq). The reaction was stirred under a nitrogen atmosphere for 1 hour at 0 C. before warming to room temperature over a further 3 hours. The reaction was then diluted with EtOAc (2 mL), washed with saturated aqueous NaHCO.sub.3 (21 mL), 1M aqueous LiCl (31 mL) then dried over NaSO.sub.4 and concentrated to give crude product. Product was purified by preparative HPLC to yield the pure product as a colourless glassy solid (0.0182 mmol, 32 mg, 14%): .sub.H(600 MHz; CDCl.sub.3) 9.42 (2H, m, NH-23), 8.83, (2H, s, NH-21), 8.05 (1H, d, .sup.3J.sub.HH=7.6, CH-12), 7.77 (1H, s, CH-2b), 7.70-7.61 (3H, m, CH-2, CH-10 and CH-11), 7.57 (1H, s, NH-36), 7.51 (1H, s, NH-36b), 7.41-7.36 (10H, m, CH-27, CH-28 and CH-29), 7.34-7.30 (4H, m, CH-33), 7.26-7.20 (6H, m, CH-34 and CH-35), 7.10 (1H, d, .sup.3J.sub.HH=6.4, CH-9), 7.09 (1H, d, .sup.3J.sub.HH=8.4, CH-4), 7.03 (1H, d, .sup.3J.sub.HH=8.4, CH-4b), 6.69 (1H, d, .sup.3J.sub.HH=8.4, CH-5), 6.68 (1H, d, .sup.3J.sub.HH=8.4, CH-5b), 5.27 (4H, s, CH.sub.2-31), 5.20 (2H, d, .sup.3J.sub.HH=12.0, CH.sub.2-25), 5.16 (2H, d, .sup.3J.sub.HH=8.6, NH-47), 5.06 (2H, d, .sup.3J.sub.HH=12.0, CH.sub.2-25b), 4.53 (2H, m, CH-17), 4.34 (2H, m, CH-38), 4.25 (2H, m, CH-44), 4.07 (4H, m, CH.sub.2-20), 3.73 (2H, m, CHH-41), 3.58 (2H, m, CHH-41), 2.07-1.66 (18H, m, CH.sub.2-18, CH.sub.2-19, CH.sub.2-39, CH.sub.2-40, CH-45), 1.47 (18H, s, CH.sub.3-50), 0.97-0.84 (12H, m, CH.sub.3-46); .sub.c (125 MHz; CDCl.sub.3) 172.0, 170.0, 163.5, 161.1, 155.9, 151.6, 140.0, 136.2, 135.0, 134.5, 129.7, 128.9, 128.9, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 126.3, 125.1, 123.9, 114.2, 108.2, 82.6, 79.7, 69.2, 67.2, 60.5, 60.3, 57.3, 57.2, 54.0, 47.7, 44.0, 31.3, 29.7, 28.4, 25.2, 25.0, 19.4, 17.5, 17.5, 14.2.

Rho-(Arg-Pro-Val-NHBoc)2

(249) ##STR00030##

(250) To a solution of Rho-(Arg-(Z).sub.2-Pro-Val-NHBoc).sub.2 (0.0182 mmol, 32 mg, 1 eq) in anhydrous DMF (0.5 mL) and anhydrous MeOH (0.5 mL) was added 5% Pd/C (1 mg) and the mixture was stirred under a hydrogen atmosphere for 48 hours. The reaction mixture was then concentrated, dissolved in methanol and filtered through celite. Concentration yielded the product as an amorphous white solid (0.0140 mmol, 22 mg, 77%): .sub.H(600 MHz; CD.sub.3OD) 10.11 (2H, d, .sup.3J.sub.HH=7.7, NH-24), 8.48 (2H, d, .sup.3J.sub.HH=7.0, NH-25), 8.17 (2H, s, NH-15), 8.05 (1H, d, .sup.3J.sub.HH=7.6, CH-12), 7.89 (1H, s, CH-2b), 7.82 (1H, s, CH-2), 7.79 (1H, dd, .sup.3J.sub.HH=8.5, .sup.4J.sub.HH=6.9, CH-11) 7.73 (1H, dd, .sup.3J.sub.HH=8.5, .sup.4J.sub.HH=6.9, CH-10), 7.41 (2H, s, NH-21), 7.22 (1H, d, .sup.3J.sub.HH=7.8, CH-4), 7.21 (1H, d, .sup.3J.sub.HH=7.8, CH-9), 7.19 (1H, d, .sup.3J.sub.HH=7.8, CH-4b), 6.75 (2H, d, .sup.3J.sub.HH=7.8, CH-5), 6.75 (2H, d, .sup.3J.sub.HH=7.8, CH-5b), 6.65 (2H, .sup.3J.sub.HH=7.8, NH-35), 4.52-4.96 (4H, m, CH-17 and CH-27), 4.20 (2H, m, CH-32), 3.93 (2H, m, CHH-30), 3.70 (2H, m, CHH-30) 3.24 (4H, m, CH.sub.2-20), 2.28 (4H, m, CH.sub.2-18), 2.12 (2H, m, CHH-28), 2.04-1.96 (6H, m, CHH-28, CHH-29 and CH-33), 1.87 (2H, m, CHH-29) 1.45 (18H, s, CH.sub.3-38) 1.01 (6H, m, CH.sub.a-34) 0.96 (6H, m, CH.sub.3-34); .sub.c(125 MHz; CD.sub.3OD) 173.2, 172.2, 171.0, 169.8, 163.8, 157.4, 156.7, 151.5, 140.7, 135.4, 130.0, 128.0, 126.2, 124.6, 123.7, 115.6, 114.1, 107.4, 82.7, 79.2, 73.3, 60.4, 58.0, 53.7, 48.6, 48.3, 44.1, 40.7, 35.6, 30.2, 29.4, 29.2, 28.9, 27.3, 24.9, 24.7, 22.3, 18.4, 17.2. (3 additional peaks for C-2b, C-4b and C-5b); m/z (Cl) 618.3317 [M+2H]; HRMS: Found 618.3322 (z=2) [M+2H], C.sub.62H.sub.86N.sub.14O.sub.13 [M] requires 1235.4322 (z=1), 617.7161 (z=2).

An Optional Synthetic Route for the Compounds of Embodiment 2

(251) ##STR00031##

Relevant References for Start Material Synthesis

(252) ##STR00032## ##STR00033##