BACKGROUND ART Optically-based assays are routinely used in diagnostic assays based on the use of enzymes, antibodies and polynucleotide probes, for the rapid and sensitive detection of many important analytes. The application of fluorescent reagents in antibody-based assays has advantages of sensitivity and safety compared with coloured and radioactive reagents respectively.

Chromophores which absorb in the far red region of the spectrum have an advantage over other chromophores in that they are much less susceptible to optical interference from components in the test sample, as most biological chromophores absorb and emit light at much lower wavelengths (Patonay and Antoine, 1991; Thompson, 1994). These far-red absorbing chromophores also have the advantage that they can be used in combination with the small and inexpensive laser diodes and detectors which have been developed for the telecommunications industry.

Significant numbers of fluorescent dyes are available commercially for use as labels for use in direct fluorescence labelling applications such as fluorescence microscopy, or as enzyme substrates in, for example, enzyme-linked immunosorbent assay (ELISA); however, almost all of these reagents have absorption maxima below 600 nm. The only far-red chromophores which have been developed previously for use in diagnostic assays are amino-reactive cyanine dyes and the protein chromophores, the phycobiliproteins.

United States Patent No. 5,268,486 to Waggoner et al. describes the characteristics of a class of arylsulphonated cyanine dyes and details their uses in various diagnostic applications.

The currently available arylsulphonated cyanine dyes (Cy 5 and Cy 5.5) have wavelength maxima of 650 nm and 670 nm. These chromophores have the disadvantage that they are not maximally excited with cheap, powerful HeNe lasers, and are not optimised for procedures based on energy transfer from phycoerythrin (Lansdorp et al., 1991). In addition, the cyanine dyes have low

Stokes shifts (about 20 nm) and suffer from photostability problems (see Anderson et al, 1996). No cyanine dyes which act as chromogenic substrates have been described.

In addition, a number of cyanine-based dyes which absorb light in the infrared region (-780-900 nm) have been developed for labelling of nucleotides (Shealy et aL, 1995) and antibodies (Boyer et aL, 1992). International application No. 95/08118 also discloses a method and apparatus for assay of biological samples using detected fluorescence in the near infrared region.

United States Patent No. 4,520,110 to Stryer et al., (1985) describes a fluorescence- based immunoassay using phycobiliproteins. These protein-based fluorophores have the disadvantage of relative poor stability during long-ter storage.

Thus there remains a need in the art for photostable chromophores which absorb in the far-red region of the spectrum and which are capable of being coupled to biologically-important molecules or can act as a substrate for an enzyme.

DISCLOSURE OF INVENTION According to a first aspect of the present invention there is provided a compound of general formula I: in which: Ru, R2, R3 and R4 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOL15, S03H, SO3Rt6, OR, nitro or NR18R9, provided that at least one of R', R2, R3 and R4 is OR, or

R1 and R2 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR15 SO3H, SO3R15, OR17, nitro or NR18R19, and R3 and R4 together form a fused aromatic ring substituted by OR17; R5, R6, R7, R8, R9 and Rl° are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR15, S03H, SO3R16, OR17, nitro or NRt8RI9; R11, R12, R13 and R14 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR15, SO3H, SO3R16, OR17, nitro or NRt8RI9, provided that at least one of R11, R12, R13 and R14 is OR17, or R"and R12 are each hydrogen, optionally substituted alkyl, halogen, COOH, COOR15, S03H, SO3R16, OR", nitro or NRI8Rl9, and R and R together form a fused aromatic ring substituted by OR"; Rls and Rl6 are each optionally substituted alkyl ; Rl'is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, PO3H2, or a sugar residue; R'8 and R'9 are each hydrogen or optionally substituted alkyl; with the proviso that when R3, and R13 are both ORl'and R', R2, R4, Rs, R6, R', R8, R9,<BR> Rlo, R", R'2 and R'4 are hydrogen, then Rl7is not alkyl; and salts thereof.

Where R'7is an acyl group, it is preferred that the acyl group be a substrate for an esterase, and more preferably an acyl group of 1 to 20, preferably 1 to 6 carbon atoms, optionally substituted by halogen, OH or NH2. Other substituents may be suitable for particular purposes, and the person skilled in the art will readily be able to determine the most suitable substituent in each case.

Preferably, R3 and R4 and/or R13 and R14, when they together form a fused aromatic ring, form a fused benzene ring.

Preferably, R's and Ru6 are each alkyl or alkyl substituted by halogen, hydroxyl, thio, carboxyl, cyano or amino. Preferably the halogen is fluorine, chlorine or bromine. Where any

one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R18 or R19 is optionally substituted alkyl these moieties are the preferred substituents. It is particularly preferred that each alkyl is Cl 6 alkyl.

Preferably, Rl7 is hydrogen; alkyl, more preferably Cl 6 alkyl; (CH2) nR2°; (CCH2O)"H; aryl or heteroaryl: aryl or heteroaryl, preferably C6 l0 heteroaryl with 1,2 or 3 carbon atoms replaced by one or more heteroatoms selected from N, O or S, substituted by OH, SH, COOH, Cour21, CN, NH2, NCS, NCO, NHCOR24, NHCOOR25, where Q is a leaving group such as a halogen group, acyl, P03H2 or a salt thereof; or a sugar residue selected from the group consisting of the furanose or pyranose forms of pentose or hexose; wherein n is an integer from 1 to 20, preferably 1 to 6, R20R20is OH, SH, COOH, NR22R23,NCS,NCO,NHCOR24,NHCOOR25,CN,

where at least one of Q or W is a leaving group such as a halo group, where Q is a leaving group such as a halo group, R21 is optionally substituted alkyl or R22 and R23 are each hydrogen or optionally substituted alkyl, and W 4 and R25 are each hydrogen or optionally substituted alkyl, typically by halogen.

It will be clearly understood that all substituents in general formula I above are independently selected. Where groups such as alkyl and acyl are able to exist in straight or branched chain forms, it will be appreciated that both forms are envisaged.

The compounds of the invention are generally photostable. For the purposes of this specification, the term"photostable"is to be understood to mean that the concentration of the compound is reduced by less than 10% on exposure to normal laboratory lighting conditions for 48 hours. However, some compounds of the invention are useful because of their high quantum yield, although they are stable to a lesser degree.

Other groups suitable for linking the chromophore to a biomolecule groups are described in US patent No. 5,268,468, the disclosure of which is incorporated herein by reference.

Preferably, R3 is OR17 and/or R13 is ORt7, Rl and R3 are OR17 and/or R11 and R13 are ORl'or Rs is ORt'and/or R8 is OR".

More preferably the compound is (Compound 6b), (Compound 9), 2,10-Dihydroxydibenzo [a, j] perylene-8,16-dione (Compound 10), 16 dioxodibenzo la, jlperylenediol) bis (dihydrogen phosphate) (Compound 11), 2,10-Diacetyloxydibenzo [a, jlperylene-8,16-dione (Compound 12), Dimethyl 16-dixoxodibenzo [aj] perylene) bis (oxy)] bisbutanoate (Compound 13), 16-dioxodibenzo [a, j] perylene) bis (oxy)] bisbutanoic acid (Compound 14), Di-N-succinimidyl 16-dioxo dibenzo [a, j] perylene) bis (oxy)] bisbutanoate (Compound 15), 2,10-Bis- (3-Aminopropyloxy) dibenzo [a, j] perylene-8,16-dione (Compound 17), or N, N'- [ [2, 10- (8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene) bis (oxy)] di-2, 1-ethanediyl]bis-2- iodoacetamide (Compound 19).

Some compounds of general formula I are suitable for use as enzyme substrates. For example, when Rl'is P03H2 or a salt thereof, the compound is a substrate for an alkaline phosphatase. When R17 is a sugar residue such as pentose or hexose, the compound is a substrate for glycosidases. When Rl7 is acyl, the compound is a substrate for an esterase.

In some compounds of general formula I, R17 comprises a reactive group which enables the compound to be attached as a fluorescent label to available amino, hydroxy and/or sulfhydryl groups on biological molecules.

Other compounds of general formula I, including those in which R3 and Rl3 are oRt7 and R17 is alkyl, are useful intermediates in the preparation of other compounds or are useful as membrane probes.

A compound of formula I may be prepared using a method comprising the steps of (a) converting a dicarboxylic acid of formula II

to its acid chloride of formula in and (b) reacting a compound of formula m with a compound of formula IV in the presence of aluminium chloride.

In this reaction when one or more of the substituents W to R4 of formula IV is MeO, the perylenedione system is formed direct, in some cases in conjunction with a dilactone. Where the latter is formed, it can be converted by hydrolysis to the corresponding base-soluble acid.

Alternatively, compounds of formula I may be prepared in using a method comprising the steps of : (a) reacting a compound of formula V

with an N-methylarylamide in the presence of butylithium, and (b) allowing the dilactone product of formula VI to rearrange in the presence of hot polyphosphoric acid.

In a particularly preferred embodiment of the invention, when R3 and Rl3 are both ORI7 and Rl7 is PO3H2 or a salt thereof, pentose or hexose, or (CH2) this group is added in a subsequent reaction.

When Rl7 is PO3H2 or a salt thereof, the compound of formula I is converted into a phenol of formula VII

preferably by demethylation of the compound of formula I wherein R'and R"are OR" and Rl7 is methyl; this may be effected by heating in the presence of aluminium chloride, and reacting with phosphorous oxychloride.

When Rl is acyl a compound of formula I wherein R3 and R'3 are OR17 and R17 is methyl is converted into a phenol in the manner described above, and treated with an acid or its anhydride.

When R17 is (CH2)nCO2R21, a compound of formula I wherein R3 and Rl3 are Ors'an R17 is methyl is converted into a phenol in the manner described above and the phenol treated with base to convert it into a salt; the salt is then reacted with a compound of formula X (CH2)"CO2Y, where X is a leaving group and Y is a protecting group, to produce a compound of formula Vin The compound of formula VIII is deprotected to produce a free acid of formula IX

and the free acid is reacted with a compound of formula R2lOH.

When R"is (CH2) NHC (O) CH2Q, a compound of formula I wherein R3 and Rl3 are OR17 and R17 is methyl is converted into a phenol in the manner described above, reacted with a protected aminoalcohol, HO (CH2) NH-BOC-t by Mitsunobu reaction, and the protecting group removed by trifluoroacetic acid to give a compound of formula X.

The free base form of compound of formula X is reacted with ClC (O) CH2Q to give the

desired compound of formula I.

Members of the classes of perylene dione compounds described above are suitable for use in a range of antibody-based, enzyme-based and DNA-based diagnostic assays. Bio-reactive (amino-reactive, hydroxyl-reactive or sulfhydryl-reactive) derivatives are suitable, for example, for coupling to antibodies or DNA for use in direct binding-based protection systems. A number of the compounds are suitable to be used as non-specific membrane probes for the determination of total cell volume in flow cytometric applications.

Accordingly, in a second aspect of the present invention there is provided an assay for a biomolecule comprising the steps of : (1) providing a compound of formula I wherein at least one group represented by Rl7 comprises a group reactive with amino, hydroxyl or sulfhydryl groups; (2) incorporating said compound of formula I into a biomolecule to form a labelled biomolecule; and (3) measuring a selected spectral property of said labelled biomolecule.

The biomolecule may be capable of binding a substrate, in which case the assay typically further comprises the step of separating labelled biomolecule bound to said substrate from unbound labelled biomolecule prior to measuring said spectral property. The presence and/or quantity present of said substrate may be determined.

Preferably, the compound of formula is a compound in which Rl7 is selected from the group consisting of substituted alkyl, substituted aryl, substituted heteroaryl and substituted acyl, and the substituent is a group reactive with amino, hydroxyl or sulfhydryl groups.

More preferably, the compound of formula I is selected from the group consisting of <BR> <BR> <BR> those compounds wherein Rl'is selected from the group consisting of (CHZ) n o, aryl, heteroaryl or acyl substituted by OH, SH, COOH, CO2R21, CN, NH2, NCS, NCO where Q is a leaving group,

wherein n is an integer from 1 to 20, R20 is selected from the group consisting of OH, SH, COOH, COOH21, CN, NR22R23, NCS, NCO, NHCOR24,NHCOOR25, where at least one of Q or W is a leaving group, where Q is a leaving group,

R21 is selected from the group consisting of optionally substituted alkyl and R22 and W3 are selected from the group consisting of hydrogen and optionally substituted alkyl, and R24 and R25 are selected from the group consisting of hydrogen and optionally substituted alkyl.

Still more preferably, the compound of formula I is selected from the group consisting of : Di-N-succinimidyl 16-dioxo dibenzo [a, j] perylene) bis (oxy)] bisbutanoate N, N'- [12, 10- (8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene) bis (oxy)] di-2,1- ethanediyl] bis-2-iodoacetamide The biomolecule may be derived from labelled precursor molecules, eg labelled nucleotides, primers, or amino acids.

The biomolecule is preferably a protein, typically an antibody and the presence of an antigen is ascertained, but the biomolecule may also be a nucleotide or polynucleotide which binds to a complementary nucleo acid sequence.

It will be clearly understood that step 3 may be either qualitative or quantitative. For a qualitative assay the presence or absence biomolecule is detected; for a quantitative assay the amount of biomolecule present is determined by reference to an appropriate standard. The assay may involve separation of labelled biomolecule bound to substrate from unbound labelled biomolecule, or may rely on the biomolecule bound to substrate having different spectral properties to those of the unbound molecule.

Preferably the assay is one of the following: Couplingof bio-reactivefluorophores to proteins (antibodies) : a) Fluorescence Immuno-Assay (FIA), wherein the fluorophore is covalently attached to a protein (e. g. antibody) and the binding of the fluorescently-labelled protein to an immobilised binding site (e. g. antigen) is detected after suitable washing steps by employing a commercially available fluorescence plate reader with appropriate filters. b) Fluorescence Microscopy, wherein fluorescently-labelled antibodies are used in conventional immunofluorescence microscopy, in confocal fluorescence microscopy applications and in flow cytometry applications. These applications require a microscope fitted with a fluorescence attachment and a suitable light source and filters. c) Immunobiosensor Applications, wherein the binding of a fluorescently labelled protein (e. g. antibody) to an immobilised binding site is detected using evanescent wave illumination employing a commercially available instrument e. g. the Naval Research Laboratories fibre waveguide-based instrument (see Abel et al*, 1996; Wadkins et aL 1995, for examples of applications).

Coupling of amino-reactive fluorophores to nucleotides or polynucleotides : a) DNA sequencing, wherein the incorporation of fluorescence-labelled nucleotides or primers into DNA fragments is detected using additional filters on standard automated sequencing systems. b) DNA-based direct-binding diagnostic applications, wherein the hybridisation of a fluorescently-labelled DNA probe to an immobilised complementary sequence is detected after suitable washing steps by employing a suitable fluorescence detector with appropriate filters.

This technique could be used for example in Southern blotting and in Fluorescence In-situ Hybridisation (FISH) applications.

According to a third aspect of the present invention there is provided an assay for a biomolecule comprising the steps of : (1) providing a compound of formula I which is a substrate for an enzyme suitable for use in an enzyme-based detection system; (2) reacting said compound of formula I with said enzyme; and (3) detecting said product by measuring a selected spectral property, wherein the product of the action of said enzyme on said compound of formula I has different spectral characteristics to said compound of formula L Typically fluorescence is measured but absorbance may also be measured, in which case the difference between the absorbance of the two compounds is measured.

Preferably, the compound of formula I is not itself fluorescent or has a fluorescence spectrum which does not substantially overlap with the fluorescence spectrum of the product of the enzyme reaction, but if the compound is fluorescent its fluorescence may also be measured and corrections made where necessary.

Preferably the assay is one of the following: (a) Enzyme-linked fluorescence immuno-assay wherein the fluorogenic substrate is used in an ELISA-type assay employing a fluorescence plate reader with appropriate filters.

These assays would have improved sensitivity over absorbance-based assays.

(b) Enzyme-linked fluorescence DNA-probe assays, wherein a nucleic acid molecule is immobilised on a surface (eg. on a 96-well plate) and the presence of a particular sequence is detected by binding of a labelled (eg. biotinylated) complementary nucleotide molecule. The bound polynucleotide is detected using an enzyme-conjugated binding material (eg. strepavidin).

The captured enzyme converts the fluorogenic enzyme substrate to a fluorescent product (see eg.

Huang et al, 1992; 1993; Diwu et al, 1997).

(c) As in (a) and (b) but using changes in the absorbance between reactant and product to monitor the reaction as is commonly used in ELISA-type assays.

In one embodiment of the invention the compound of formula I which is a substrate for the enzyme is one in which R'is POsH, or a salt thereof.

In another embodiment of the invention the compound of formula I which is a substrate for the enzyme is one in which R17 is a sugar residue.

In still another embodiment of the invention the compound which is a substrate for the enzyme is one in which wherein Rl7 is optionally substituted acyl, where the acyl group is a substrate for an esterase.

Preferably, the compound of formula I which is a substrate for the enzyme is 2,10- (8,16- Dihydro-8,16 dioxodibenzo [a, j] perylenediol) bis (dihydrogen phosphate) or 2,10- Diacetyloxydibenzo [a, j] perylene-8,16-dione.

According to a fourth aspect of the present invention there is provided the use of a compound of general formula I: in which: R', R, R3 and W are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COLORIS, S03H, SO3R16, OR, nitro or NRt8RI9, provided that at least one of Rl, R2, R3 and R4 is oRt7, or R1 and R2 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR'S, SO3H, SO3R16, OR17, nitro or NR18R19, and R3 and R4 together form a fused aromatic ring substituted by OR" ;

Rs, R6, R7, R8, R9 and Rl° are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COLORIS, SO3H, SO3R16, OR17, nitro orNR18R19 ; R11, R12, R13 and R14 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COLORIS, S03H, SO3R16, OR, nitro or NR18R 9, provided that at least one of R", R12, R and R is OR, or R"and R'2 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOL5, S03H, SO3R16, OR17, nitro or NRt8RI9, and Rl3 and R'4 together form a fused aromatic ring substituted by oRI7; R'S and R16 are each optionally substituted alkyl; R17 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, P03H2, or a sugar residue; Rl8 and R'9 are each hydrogen or optionally substituted alkyl as a label in a biological system.

The use may be in fluorescence immuno-assays, fluorescence microscopy, immunobiosensor applications, DNA sequencing, DNA-based direct-binding diagnostic applications, enzyme-linked fluorescence immuno-assays, ELISA-type assays, enzyme-linked DNA-probe assays or as a membrane probe. Preferred compounds for use as a membrane probe are 2,10-Dihydroxydibenzo [a, j] perylene-8,16-dione 2,10-Bis-(3-Aminopropyloxy) dibenzo [a, j] perylene-8,16-dione.

According to a fifth aspect of the present invention there is provided a method of labelling a component of a biological system comprising the steps of : (1) providing a compound of general formula I:

in which: R1, R2, R3 and R4 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR'S, S03H, SO3R16, OR, nitro or NR18R19, provided that at least one of R', and R4 is OR, or R'and R2 are selected from the group consisting of hydrogen, optionally substituted COOH,COOR15,SO3H,SO3R16,OR17,nitroalkyl,halogen, or NR18R19, and R4togetherand form a fused aromatic ring substituted by OR17; R5, R6, R7, R8, R9 and Rl° are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR'S, S03H, SO3R16, OR17, nitro orNR18R19 ; R11, R12, R13 and Rl4 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR'S, S03H, SO3R16, OR17, nitro or NR18R19, provided that at least one of R", R12, R'3 and Rl4 is oRt7, or Rll and R12 are selected from the group consisting of hydrogen, optionally substituted alkyl, halogen, COOH, COOR'S, S03H, SO3R16, OR, nitro or NR18R'9, and R13 and R together form a fused aromatic ring substituted by OR17 ; R'S and R16 are each optionally substituted alkyl; R17 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, PO3H2,

or a sugar residue; R'8 and R'9 are each hydrogen or optionally substituted alkyl; and (2) bringing the component of the biological system and the compound of formula I into physical and/or chemical association.

Typically the chromophores in the molecules described above can be excited using a range of light sources, including white light sources (eg. quartz halogen lamp with suitable filters) or a suitable laser (eg. He/Ne 633 nm laser line or Kr/Ar 647 nm laser line or a 635 nm diode laser). The chromophores of the present invention absorb in the far red region of the spectrum, and offer the advantage of a low level of interference from biological components in the assay system as well as the possibility of using inexpensive light sources and detectors which have been developed for the telecommunications industry.

Typically the perylene diones described herein have absorption maxima in the far red region, including but not limited to maxima in the range 590 nm-640 nm. For example, under alkaline conditions in solution (pH 12), Compound 10 exhibits a spectral shift with an absorption maximum at 740 nm which reflects the ionisation of the hydroxy group (s).

The chromophores which absorb at 630 nm are particularly useful, as they can be maximally excited with cheap and powerful HeNe lasers as well as with 635 nm diode lasers.

These perylene dione dyes could be used in combination with additional fluorophores such as fluorescein rhodamine and phycoerythrin in three-colour fluorescence applications (Lansdorp et at 1991). The perylene dione dyes have high Stokes shifts (approx 70 nm between the excitation and emission wavelengths). This facilitates the separation of excitation and emission wavelengths.

The perylene diones also possess relatively large spectral bandwidths (-110 nm full- width at half maximum) which are three-fold greater than those of the cyanine dyes. This permits their use over a much larger spectral range compared to the cyanine dyes. For example, compounds exhibiting absorption maxima well removed from the 630 nm line of the HeNe laser may still exhibit substantial absorbance at this wavelength. In addition, the combination of a larger Stokes shift and increased spectral bandwidths means that a greater proportion of the emission of the perylene diones can be measured using appropriate filters, compared to the cyanine dyes.

For the purposes of this specification it will be clearly understood that the word "comprising"means"including but not limited to", and that the word"comprises"has a corresponding meaning.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows Reaction Scheme 1 for synthesis of perylenedione intermediates as disclosed in Example 1.

Figure 2 shows Reaction Scheme 2 for synthesis of perylenedione system intermediates as described in Example 2.

Figure 3 shows Reaction Scheme 3 for synthesis of phosphate derivatives.

Figure 4 shows Reaction Scheme 4 for synthesis of acetate esters.

Figure 5a shows Reaction Scheme 5a for synthesis of succinimidyl esters. Figure 5b shows Reaction Schemes 5b and 5c for synthesis of iodoacetamide derivatives.

Figure 6a is a plot of excitation and emission spectra of a preferred compound in accordance with the present invention. Figure 6b is a plot of the absorption spectrum and the corrected emission spectrum of Compound 10 in DMSO.

Figure 7 is an illustration of an alkaline phosphatase assay in accordance with the present invention.

Figure 8a shows the fluorescence emission generated in several alkaline phosphatase assays. Figure 8b shows the time-dependent change in the absorption spectrum of Compound 11 following incubation with alkaline phosphatase for the indicated amount of time.

Figure 9 is a schematic illustration of the manner in which compounds in accordance with the present invention can be coupled to biomolecules.

Figure 10 shows the effect of pH on the absorption spectrum of Compound 10 in aqueous solutions.

Figure 11 shows the effect of temperature on the polarisation of fluorescence emission of Compound 6a following its incorporation into lipid bilayers composed of dimyristoyl phosphatidylcholine.

Figure 12 shows an example of flow cytometric analysis of red blood cell ghosts and red blood cells which have been labelled with Compound 17.

Figure 13 shows confocal fluorescence microscopy of red blood cell ghosts which have

been labelled with Compound 17.

BEST MODE FOR CARRYING OUT THE INVENTION Two strategies were employed in the synthesis of the perylenedione system: acid (3) was prepared according to a literature procedure (Karpen et aL, 1986) except that dimethylformamide was used instead of benzyl cyanide in the cyanation step. This is summarized in Figure 1, showing Reaction Scheme 1. The perylenedione system was then prepared from the acid chloride of this diacid by aluminium chloride catalysed reaction with a suitable aromatic compound, by a variation of a literature method (Scholl et aL, 1932). In this method, with benzene as the aromatic compound, a mixture of a dilactone and a phenyl ketone is formed. The former can be isolated by hydrolysis to the base- soluble derived acid, which was subsequently cyclized to the perylenedione in two steps by reaction with (i) red phosphorus/hydroiodic acid and (ii) sulfuric acid. In the present work with methoxy substituted benzenes, the perylenedione system was formed directly in the aluminium chloride-catalysed step. With 1,3-dimethoxybenzene, no dilactone was detected while, with anisole, a 1: 1 mixture of 5 and 6a was formed.

2. A substituted anthraquinone was reacted with an N-methylarylamide in the presence of butyl lithium, by a literature method (Carissimo-Rietsch et al*, 1991). This is shown in Reaction Scheme 2, Figure 2. The intermediate dilactone 8 was rearranged to the perylenedione with hot polyphosphoric acid.

Synthesis of alkaline phosphatase substrate Demethylation of compound 6a was achieved by reaction with aluminium chloride in refluxing benzene, as seen in Reaction Scheme 3, Figure 3. Treatment of 10 with freshly distille phosphorous oxychloride in dry pyridine under nitrogen gave 11. The 31P NMR spectrum showed peaks at-2.76 and 2.38 ppm. The latter is no doubt due to pyridine-solvated phosphate species, as reported in a similar preparation (Organic Synthesis, Coll. Vol. VISU, p 50), and treatment with boiling 6M hydrochloric acid converted all to the phosphoric acid 11.

Synthesis of esterase substrate Acetylation of the dihydroxy compound 10 with acetic anhydride and pyridine gave 12 in high yield, as shown in Reaction Scheme 4, Figure 4.

Synthesis of amino-reactive fluorescent label The sodium salt of 10 was prepared and reacted with methyl 4-chlorobutyrate to give the ester 13 which, on mild basic hydrolysis, gave diacid 14. This was converted to the succinimidyl ester 15 by reaction with N-hydroxysuccinimide,. This is summarized in Reaction Scheme 5a, Figure 5a. The structure was confirmed from the characteristic peak at 2.85 ppm for the succinimide methylene protons in the 1H NMR spectrum.

Synthesis of thiol-reactive fluorescent label Compound 10 was coupled with t-BOC protected 3-aminopropanol by Mitsunobu reaction. Cleavage of the BOC group with trifluoroacetic acid gave the amine 17 as the trifluoroacetate salt. 2,6-Lutidine was a suitable basic solvent for coupling of 17 with chloroacetyl chloride, and the thiol-reactive iodoacetamide derivative 19 was then obtained from 18 by conventional halogen exchange. This is summarized in Reaction Schemes 5b and 5c, Figure 5b.

1H NMR spectra were recorded at 300 MHz, in deuterated dimethyl sulfoxide unless 31 otherwise stated. P NMR spectra were recorded at 121.5 MHz, in deuterated dimethyl sulfoxide, with chemical shifts in ppm relative to phosphoric acid. IR spectra were recorded on a Perkin-Elmer 1720X FTIR spectrometer, using a diffuse reflectance accessory with KBr background.

Example 1 Preparation of perylenedione intermediates according to Reaction Scheme 1 Reaction Scheme 1 is summarized in Figure 1.

9, 10-Dihydro-9, 10-dioxo-1, 5-anthracenedicarbonitrile (2) A mixture of 1,5-dichloro-9,10-anthraquinone (10 g) and cuprous cyanide (8.5 g) in DMF (50 ml) was heated under reflux for 3 h. The reaction mixture was then poured onto ice and stirred. The solid which separated was filtered off, and the filter cake was washed with water and air dried to give a dark brown solid. This was stirred in dilute nitric acid (200 ml of a solution consisting of 70 mi conc. nitric acid in 130 ml of water) at 120 °C for 0.5 h. The mixture was then filtered, washed with water and the filter cake was dried to give the product 2 (9.2 g, 98%), mp >300 °C; IR vo, ax 2210 cm~l.

9, 10-Dihydro-9, 10-dioxo-1, 5-anthracenedicarboxylic acid (3)

Compound 2 (9.0 g) in aqueous sulfuric acid (100 ml conc. sulfuric acid in 20 ml water) was stirred at 165 °C for 1.5 h and then poured onto finely crushed ice (500 ml) with stirring. The light brown solid which separated was filtered off at the pump, washed with water (3 x 100 ml), and air dried to give the acid 3 (9.0 g, 87%), mp >300 °C. 1H NMR 8 7.8, d, H-2; 7.9, t, H-3; 8.2, d, H-4; 13.3, br s, COOH.

2,4, 10, 12-Tetramethoxydbenzola, jlperylene-8, 16-done (6b) Compound 3 (1.0 g) in thionyl chloride (100 ml) was heated under reflux for 4 h. The excess thionyl chloride was removed by evaporation under reduced pressure to afford the crude acid chloride 4. To this was added aluminium trichloride (2.0 g) and nitrobenzene (30 ml) and the mixture was stirred under a nitrogen atmosphere at room temperature for 0.5 h and then cooled to 0 °C. A solution of 1,3-dimethoxybenzene (1.0 g) in nitrobenzene (5 ml) was added dropwise to the reaction mixture and stirring was continued for 24 h at 0 °C, then 24 h at room temperature.

The reaction mixture was quenched with water (50 ml) and the nitrobenzene was removed by steam distillation. The remaining solid was collected, washed with ethanol and diethyl ether and then extracted with chloroform using a Soxhlet apparatus. The chloroform extract was evaporated to dryness and the residue was flash column chromatographed (silica/chloroform).

The appropriate fractions were collected and evaporated to dryness under reduced pressure. The residue (0.5 g) was washed with toluene and then methanol to give the dione 6b (0.2 g), mp >300 °C. 1H NMR (CDC13) 8 3.7, s, CH3-4; 4.0, s, CH3-2; 6.6, d, J=2.5 Hz, H-3; 7.5, t, J=7.2 Hz, H-6; 7.6, d, J=2.5 Hz, H-1; 8.3, d, J=8.7 Hz, H-5; 8.6, d, J=7.1 Hz, H-7.

The following diones were prepared in a similar manner: 2,10-Dmethoxydbenzola, jlperylene-8, 16-done (6a) This was prepared from acid chloride 4 (1.0 g) and anisole (1.0 g). The residue from the Soxhlet extraction was heated under reflux in 10% methanolic potassium hydroxide (70 ml) for 0.5 h with stirring. Water (100 ml) was added and the mixture was filtered at the pump. The cake was washed with water to give crude 6a (0.5 g, 37%), mp >300 °C. 1H NMR (CDC13) 8 4.0, s, OCH3; 7.3, dd, J=8.8,2.6 Hz, H-3; 7.8, t, J=8.5 Hz, H-6; 8.0, d, J=2.6 Hz, H-1; 8.1, d, J=8.9 Hz, H-4; 8.8, d, J=6.8 Hz, H-5; 8.9, d, J=8. 8 Hz, H-7. The filtrate was acidified to pH 1 to give the intermediate dilactone 5 (0.5 g, 38%), mp >300 °C. 1H NMR (CDC13) 8 3.6, s, OCH3; 6.3, d, J=8. 8 Hz, H-2', 6' ; 6.6, d, J=8. 8 Hz, H-3', 5' ; 7.7, t, J=7. 5 Hz, H-3; 7.9, d, J=7. 9 Hz, H-4; 8.0, d, J=7. 0 Hz, H-2.

Example 2 Preparation of perylenedione intermediates according to Reaction Scheme 2 Reaction Scheme 2 is summarized in Figure 2.

7,15-Dimethoxydibenzo [a, jlperylene-8, 1 6-dione (9) To a solution of N-methylbenzamide (1 g) in freshly distille dry tetrahydrofuran (50 ml) at 0 °C in a nitrogen atmosphere, butyl lithium (8.5 ml, 1.6 M solution in hexane) was added dropwise over 15 min. After a further 0.5 h at this temperature, a solution of 2,6- dimethoxyanthraquinone (0.78 g) in dry tetrahydrofuran (80 ml) was added over 15 min and the solution was stirred at 0 °C for 2 h, then refluxed for 2 h. The solvent was then evaporated at reduced pressure, 10% hydrochloric acid (20 ml) was added and the solid which separated was filtered off, dried, and washed with diethyl ether to give the crude product (0.8 g, 56%) which contained ca. 10% of unreacted anthraquinone. The latter was separated by extraction with hot <BR> <BR> <BR> <BR> toluene to leave the insoluble dilactone 8 as a white powder (0.7 g). mp >300 °C. 1H NMR 8 2.9, s, OCH3; 6.2, d, J=2.4 Hz, H-1; 6.8, d, J=8.8 Hz, H-4; 7.0, dd, J= 8.8,2.4 Hz, H-3; 7.4, d, J=7.5 Hz, H-4' ; 7.8, m, J=7.4 Hz, H-2', 3' ; 8.1, d, J=7.4 Hz, H-1'.

The dilactone 8 (0.1 g) was mixed with polyphosphoric acid (10 g) and heated at 100 C for 3 h, with stirring, then poured on to ice. The solid which separated was filtered, washed with water and dried to give the product (0.05 g, 60%), mp >300 °C. 1H NMR (CDC13) 8 4.2, s, OCH3; 7.4, d, 1H; 7.6, m, 2H; 7.8, d, 1H; 8.5, m, 2H.

Example 3 Preparation of alkaline phosphatase substrate according to Reaction Scheme 3 Reaction Scheme 3 is summarized in Figure 3.

2,10-Dihydroxydibenzo [a, j] perylene-8, 16-dione (10) To a boiling solution of crude 6a (1.0 g) in dry benzene (80 ml) was added aluminium trichloride (4.0 g) and the mixture was heated under reflux for 1.5 h. The solvent was evaporated under reduced pressure. 10% Hydrochloric acid (100 ml) was added to the residue and the mixture was digested at 80 °C for 1.5 h. The solid was filtered off at the pump and washed with water, methanol and finally with chloroform to give the product 10 (9.0 g, 96%), mp >300 °C. 1H <BR> <BR> <BR> <BR> NMR S 7.2, dd, J=8.7,2.7 Hz, H-3; 7.7, d, J=2.6 Hz, H-1; 7.8, t, H-6; 7.9, d, J=8.9 Hz, H-4; 8.5, d, J=7.0 Hz, H-5; 8.8, d, J=8. 6 Hz, H-7; 10.5, s, OH.

2,10- (8, 16 Dihydro-8, 16-dioxodibenzo [a, j] perylenediol) bis (dihydrogen phosphate) (11)

To a solution of 10 (0.1 g) in dry pyridine (3.0 ml) at 90 °C under a nitrogen atmosphere was added, with stirring, freshly distilled phosphorous oxychloride (1.0 ml). The resulting mixture was heated at 90 °C for 0.5 h. Excess pyridine and phosphorous oxychloride were evaporated under reduced pressure. The residual solid was boiled with 6 M hydrochloric acid (20 ml) for 10 min and then cooled rapidly. The black solid was filtered, washed with water, and dried to give the product 11 (0.13 g, 95%), mp >300 °C. 1H NMR 8 7.5, d, J= 8.3 Hz, H-3; 7.7, t, H-6; 7.9, d, J= 8.5 Hz, H-4; 8.1, s, H-1; 8.5, d, J= 6.8 Hz, H-5; 8.7, d, J= 8.1 Hz, H-7.31P NMR 8- 2.76, s,-OPO (OH) 2.

Example 4 Preparation of esterase substrate according to Reaction Scheme 4 Reaction Scheme 4 is summarized in Figure 4.

2,10-Diacetyloxydibenzo [a, j] perylene-8, 16-dione (12) To a solution of compound 10 (0.2 g) in pyridine (3.0 ml) at 80 °C was added acetic anhydride (2.0 ml) with stirring. Heating at 80 °C was continued for 1.5 h. Excess pyridine and acetic anhydride were removed by evaporation at reduced pressure. The residual solid was washed with water and then methanol to give the diacetate 12 (0.2 g, 84%), mp 305 °C (dec.). 1H <BR> <BR> <BR> NMR (CDC13) 8 2.4, s, COCH3; 7.5, dd, J=8.7,2.5 Hz, H-3; 7.7, t, H-6; 8.1, d, J=8.7 Hz, H-4; 8.2, d, J=2.6 Hz, H-1; 8.8, d, J=6.9 Hz, H-5; 8.9, d, J=8. 6 Hz, H-7.

Example 5a: Preparation of amino-reactive fluorescent label according to Reaction Scheme 5a Reaction Scheme 5a is summarized in Figure 5a.

Dimethyl 4, 4'-2, 10- (8, 16-Dihydro-8, 16-dioxodibenzo [a, j] perylene) bis (oxy) Jbis butanoale (13) Compound 10 (0.3 g) was dissolved in sodium hydroxide solution [sodium hydroxide (0.08 g) in 20 ml water] and the mixture was then freeze dried overnight. To a solution of the resulting dry sodium salt in dry DMSO (10 ml) was added methyl 4-chlorobutyrate (1.0 ml) with stirring at room temperature. Stirring was continued for 20 h. The reaction mixture was poured onto ice, the solid which separated was filtered off, washed with water, and dried to give a black solid (0.4 g). This was extracted with cold chloroform (3 x 20 ml). The chloroform extracts were evaporated under reduced pressure to give the diester 13 (0.18 g), mp 170-175 °C. 1H NMR (CDC13) 8 2.2, m, CH2; 2.6, t, COCH2; 3.7, s, COOCH3; 4.2, t, OCH2; 7.3, dd, J=8.7,2.6 Hz, H-3; 7.7, t, H-6; 8.0, d, J=2.6 Hz, H-1; 8.1, d, J=8. 8 Hz, H-4; 8.8, d, J=6.9 Hz, H-5; 8.9, d, J=8. 5 Hz, H-

7. The black chloroform insoluble material (0.16 g) contained unreacted 10.

4,4'-[2,10-(8,16-Dihydro-8,16-dioxodibenzo [a, jlperylene) bis (oxy) Ibishutanoic acid (14) Compound 13 (0.07 g) was heated at 90 °C for 3 h in sodium hydroxide solution (20 ml of a 0.1 M solution), cooled to room temperature and left to stand overnight. The mixture was then extracted with chloroform (3 x 20 ml). The aqueous fraction was acidified with dilute hydrochloric acid. The black solid which separated was centrifuged and filtered off to give the <BR> <BR> <BR> <BR> diacid 14 (0.05 g, 90%), mp 200-205 °C. 1H NMR 8 2.0, m, CH2; 2.4, t, COCH2; 4.1, t, OCH2; 7.2, d, J= 8.4 Hz, H-3; 7.6, s, H-1; 7.7, t, H-6; 7.7, d, J= 8.3 Hz, H-4; 8.4, d, J= 6.8 Hz, H-5; 8.5, d, J= 8.4 Hz, H-7. <BR> <BR> <BR> <BR> <BR> <BR> <P>Di-N-succinimidyl 4, 4'-2, 10- (8, 16-Dihydro-8, 16-dioxodibenzo [a, j] perylene) bis (oxy) Jbis butanoate (15) A mixture of the diacid 14 (0.04 g), N-hydroxysuccinimide (0.025 g) and dicyclohexylcarbodiimide (0.04 g) in dimethyl formamide (1 ml) was stirred for 20 h at room temperature. A mixture of acetic acid and methanol (1: 5,10 ml) was added. The solid which separated was filtered, washed with methanol and dried to give the ester 15 (0.04 g) as a black solid, mp 150-153 °C; 1H NMR (CDC13) 8 2.3, m, CH2; 2.8, s, COCH2CH2CO; 2.9, t,-COCH2; 4.3, t, OCH2; 7.3, dd, J=8. 7,2.6 Hz, H-3; 7.7, t, H-6; 8.0, d, J= 2.6 Hz, H-1; 8.1, d, J= 8.8 Hz, H- 4; 8.8, d, J= 6.9 Hz, H-5; 8.9, d, J= 8.5 Hz, H-7.

Example 5b: Preparation of thiol-reactive fluorescent label according to Reaction Schemes 5b and 5c Reaction Schemes 5b and 5c are summarized in Figure 5b 2,10-Bis- (3-Aminopropyloxy) dibenzo [a, j] perylene-8, 16-dione (17) To a stirred mixture of 10 (0.8 g), triphenyl phosphine (1.07 g) and 3-t-BOC-amino-1- propanol (0.7 g) in dry DMF (10 ml) at room temperature under nitrogen, a solution of diethyl azodicarboxylate (0.7 g) in dry DMF (3 ml) was added dropwise over a period of 20 min. Stirring was continued at room temperature for 16 h. and the mixture was then poured onto ice. The solid which separated was filtered off, washed with water and dried to give 16, 1H NMR (CDC13) 8 1.46 (s, t-butyl), 2.08 (m, CH2), 3.41 (br s, CH2N), 4.23 (t, OCH2), 4.9 (br s, NH), 7.21-7.25 (m, H- 3), 7.69 (t, J = 7.8 Hz, H-6), 7.90 (d, J= 2.6 Hz, H-1), 8.00 (d, J= 8.8 Hz, H-4), 8.69 (d, J = 6.9 Hz, H-5), 8.78 (d, J= 8.9 Hz, H-7).

To a solution of crude 16 in chloroform (15 ml) at room temperature was added TFA

(10 ml), dropwise with stirring, and stirring was continued for a further 24 h. The solvents were evaporated and the residual solid was washed with chloroform and dried to give 17 as the <BR> <BR> <BR> <BR> <BR> bistrifluoroacetate salt (1.3 g, 86%). 1H NMR [ (CD3) 2SO] 8 2.12 (br s, CH2), 3.06 (br s, CH2N), 4.24 (br s, OCH2), 7.32 (d, J= 7.0 Hz, H-3), 7.60-7.78 (m, 2 H, H-1,6), 7.92 (br s, 4 H, H-4, NH3), 8.50 (d, J = 6.8 Hz, H-5), 8.71 (d, J = 8.4 Hz, H-7).

N,N'- ( (2, 10- (8, 16-Dihydro-8, 16-dioxodibenzo [a, j] perylene) bis (oxy) ldi-2, 1-ethanediylJbis-2- iodoacetamide (19) To a stirring suspension of 17 (0.1 g) in freshly distilled 2,6-lutidine (10 ml), under nitrogen at room temperature was added dropwise a solution of chloroacetyl chloride (0.04 g) in dry dioxane (5 ml), and the stirring was continued for a further 16 h. The reaction mixture was poured onto ice and stirred, and the solid which separated was filtered off, washed with water and <BR> <BR> <BR> <BR> dried to give the bis-2-chloroacetamide 18 (0.08 g, 89%). 1H NMR (CDC13) 5 2.17 (m, CH2), 3.62 (m, CH2N), 4.10 (s, CH2Cl), 4.3 (t, J= 5.4 Hz, OCH2), 7.15 (br s, NH), 7.3 (dd, J= 8.7,2.6 Hz, H- 3), 7.78 (t, J = 7.4 Hz, H-6) 8.0 (d, J =2.6 Hz, H-1), 8.09 (d, J = 8.6 Hz, H-4), 8.78 (d, J = 6.1 Hz, H- 5), 8.90 (d, J = 8.5 Hz, H-7).

A mixture of crude 18 and anhydrous sodium iodide (0. lg) in dry butanone under nitrogen was heated under reflux for 1 h. The solvent was removed at reduced pressure and the residual solid was washed with water, dried and washed with cold chloroform to give 19, (0.1 g, 88%). 1H NMR [ (CD3) 2SO] 6 1.97 (br s, CH2), 3.3 (br s, CH2N), 3.67 (s, CH2I), 4.19 (br s, OCH2), 7.35 (br d, H-3), 7.7-7.8 (m, 2 H, H-1,6), 7.96 (d, J= 8.6 Hz, H-4), 8.43 (br s, H-5 (7)), 8.56 (br s, H-7 (5)), 8.78, (br s, NH).

Example 6: SPECTRAL CHARACTERISTICS OF THE COMPOUND The spectral characteristics of each of the compounds have been studied, and conditions have been devised for solubilising the compounds in solutions which are compatible with aqueous media. The phosphoric derivative 11, has been shown to be a substrate for alkaline phosphatase and the formation of the product of this reaction has been detected by the generation of a fluorescence signal or by alterations in its absorption spectrum. This indicates that this class of chromogenic derivatives could be developed for use in ELISA-type assays. The succinimide derivative 15 and the iodoacetamide derivative 19, have been successfully conjugated to antibodies, and have been shown to retain fluorescence upon conjugation. This indicates that this class of compound is suitable for applications which require direct labelling with a fluorescent tag.

Example 7: SOLUBILITY STUDIES: All the perylene dione compounds described herein, apart from the phosphoric acid

and hydroxy derivatives 11 and 10, are soluble in dimethylsulphoxide (DMSO), dimethyl formamide (DMF) and chloroform (Table 1). However, Compound 10 does have limited solubility in chloroform if stock solutions in DMSO are diluted into chloroform (final DMSO concentrations less than 2% (vol/vol)).

The above solvents are generally unsuitable solvents for use in biological assays.

Therefore an attempt was made to identify a solvent system which is compatible with aqueous media. Compounds 9,6a, 6b and 15 were found to retain their fluorescent properties in neutral or alkaline pH buffers (phosphate-buffered saline, pH 7.4 and 0.1M Tris-HCl, pH 8) when in the presence of 1% Triton X-100 (Table 1). Fluorescence was also retained in a range of other mild detergents, including 1% CHAPS, Brij 35 and n-octylglucoside. Low levels of detergent are not expected to interfere with biological assays.

The absorbance and fluorescence characteristics of compound 10 are sensitive to the concentration of Triton X-100 in the range 0.2-1%.

Compound 10 also exhibits some solubility in aqueous solutions in the absence of detergent under basic conditions (10 mM NaOH). Stock solutions of Compound 10 in DMSO (1 mg/ml) diluted 50-fold into 10 mM NaOH exhibit a stable absorbance (with maximum absorbance at 730 nm) for at least 30 minutes. This reflects the ionisation of the hydroxyl group (s). The compound is not fluorescent under these conditions.

Table 1. Solubility and fluorescence characteristics of some perylene dione chromophores. Compound DMSO DMF Chloroform PBS \ 1% Triton X-100 soluble, fluorescent soluble, fluorescent soluble, fluorescent soluble, fluorescent 6b soluble, fluorescent soluble, fluorescent soluble, fluorescent soluble, fluorescent 6a soluble, fluorescent soluble, fluorescent soluble, fluorescent soluble, fluorescent 11 soluble, fluorescent soluble, fluorescent insoluble soluble, poorly fluorescent 10 soluble, fluorescent soluble, fluorescent insolublea soluble, poorly fluorescent' 15 soluble, fluorescent soluble, fluorescent soluble, fluorescent soluble, fluorescent Does have limited solubility in chloroform if it is added to chloroform from a stock solution in DMSO. Under these conditions, the compound is also fluorescent. b Compound 10 is soluble to some extent in basic aqueous solutions (10 mM NaOH) in the absence of detergent, but is not fluorescent under these conditions.

Example 8: Spectral Parameters Fluorescence excitation and emission spectra were obtained using an Hitachi 650-1OS spectrofluorimeter equipped with a red-sensitive photomultiplier. The spectra for 6a in 1% Triton X-100 are shown in Figure 6a. The excitation and emission wavelength maxima for the

perylene dione compounds in a range of solvent systems are listed in Table 2a.

Absorption spectra were recorded on a Cary-lE spectrophotometer. The absorption maxima are reported in Table 2b. Additional measurements of the emission maxima were also made for calculation of the Stokes shift. These data are also included in Table 2b.

The absorption spectrum of compound 10 in DMSO is shown in Figure 6b. Also shown is the corrected emission spectrum of the compound in the same solvent recorded on a Perkin- Elmer LS50b fluorometer equipped with a red-sensitive photomuRiplier. The emission correction factors were supplied by Perkin-Elmer.

Table 2a. Fluorescence excitation and emission wavelength maxima for the perylene dione compounds in various solvents Compound DMSO Chloroform 1% Triton X-100 #ex#em#ex#em#ex#em 9 590 nm 620 nm 590 nm 620 nm 590 nm 620 nm 6b 630 nm 700 nm 630 nm 700 nm 630 nm 700 nm 6a 620 nm 670 nm 620 nm 670 nm 620 nm 670 nm 11 580 nm 700 nm non-fluorescenta non-fluorescent 10 620 nm 700 nm non-fluorescenta non-fluorescentb a Compounds insoluble in chloroform (see Table 1). In the case of Compound 10, both solubility and fluorescence increases substantially if it is diluted into chloroform from a stock solution in DMSO. b Very low fluorescence Table 2b. Absorption maxima, emission maxima and Stokes shifts of the perylene dione compounds in various solvents. Compound Solvent Absorption Emission Stokes maximum (nm) maximum (nm) shift (nm) 9 DMSO 590 620 30 Chloroform 590 620 30 DMF 585 613 28 PBS\Tx-100 590 620 30 ............................................................ ............................................................ .......................................................... 6b DMSO 615 710 95 Chloroform 620 705 85 DMF 620 700 80 PBS\Tx-100 620 680 60 ............................................................ ............................................................ ........................................................... 6a DMSO 620 670 50 Chloroform 620 670 50 Table 2b continued

Compound Solvent Absorption Emission Stokes maximum (nm) maximum (nm) shift (nm) DMF 610 660 50 PBS\Tx-100 620 665 45 ............................................................ ............................................................ .......................................................... 15 DMSO 620 670 50 Chloroform 620 660 40 DMF 620 660 40 PBS\Tx-100 620 665 45 19 DMSO 623 ND ND 17 water\Tx-100 626 680 60 Example 9: Photostability: Fluorophores are normally stored in the darkThe photostability of the perylene dione compounds was measured following exposure to daylight. Samples (10 Lg/ml, 3 ml) were stored in clear polypropylene tubes (15 ml) and left exposed to laboratory lighting conditions. The level of photostability was estimated by measuring the fluorescence intensity before and after exposure.

All of the fluorophores except compound 9 were photostable for more than 48 hours. Compound 9 underwent noticeable photobleaching after only a few hours.

Example 10: Extinction coefficients: The molar absorption coefficients or extinction coefficients for all compounds were measured by preparing a series of dilutions of known molar concentrations and measuring in a 1 cm path length cuvette the absorbance of each dilution at the wavelength of maximum absorbance for the compound. A plot of absorbance against concentration was prepared for each compound, and the value of the extinction coefficient was calculated by measuring the slope of the linear portion of the graph. The data obtained originally is shown in Table 3a but it was suspected that

the compounds measured may have been impure and/or not completely dissolved, so further measurements were made. They are summarized in Table 3b.

Molar absorption coefficient (l. mol-1.cm-1) = Absorbance Molar concentration x path length Table 3a. Extinction coefficients for perylene dione compounds (i. mol-l. cm~l) in various solvents Compound DMSO Chloroform DMF 9 1, 600 5, 300 n. d. 6b 3, 700 8, 900 n. d. 6a 7, 700 28, 000 n. d. 11 7, 800 n. d. n. d. 10 4, 490 n. d. 7,500 15 8, 100 n. d. 10,000

Extinction coefficient calculations were repeated more accurately with results shown in Table 3b.

Table 3b. Extinction coefficients for perylene dione compounds (l. mot cmf') in various solvents Compound DMSO Chloroform DMF PBS\Tx-100 13,50012,5003,100910,000 6b 17, 000 25, 000 22, 000 2,800 6a 21, 500 21, 000 19, 000 2,700 10 20, 000 17, 500 20, 000 7,000 15 40, 000 17, 000 25, 000 15,000 17 22, 000 ND ND 19, 000a 19 24, 000 ND ND ND

aMeasurement made after Compounds were diluted from a stock solution in DMSO to the indicated solvent (final DMSO concetration < 2% (vol/vol)).

Example 11: Quantum yields: The uncorrected quantum yields of the perylene dione compounds were measured relative to the cyanine dye (Cy5), which absorbs and fluoresces in the far-red region of the

spectrum (Table 4a). A dilution of each fluorophore was prepared in DMSO such that an equal absorbance reading was achieved for each compound at its absorption wavelength maximum.

Further dilutions of these stocks were prepared and the fluorescence emission spectra were collected for excitation at the excitation wavelength maximum using a Hitachi 650-10S fluorimeter (2 nm excitation/emission slit widths). The uncorrected relative quantum yields (RQu) were calculated using the following equation and correcting for the appropriate dilution factors: Fref As Fref As Where Are, = Absorbance of Cy5 (at its wavelength maximum) As = Absorbance of perylene dione compound (at its wavelength maximum) Fref f = Fluorescence intensity of Cy5 (area under the emission spectrum) F, = Fluorescence intensity of perylene dione compounds (area under the emission spectrum) The above calculations of the quantum yields do not reflect the true relative quantum yields of the compounds because they do not take into account the wavelength-dependent variation in the intensity of the fluorometer light source, nor the wavelength-dependent factors in the emission monochromator and photomultiplier tubes which affect the accuracy of the measured emission spectrum. The true corrected relative quantum yield (RQ¢) of one of the compounds (Compound 10) was therefore measured using the method described below.

Stock solutions of compound 10 (-1 mg/ml in DMSO) were diluted into various solvents so that the maximum absorbance was less than 0.1. Absorption spectra of the samples were recorded on a Caryl3E spectrophotometer (the absorption spectrum in DMSO is shown in Figure 6b). Emission spectra were recorded on a Perkin-Elmer LS50b fluorometer up to wavelengths of 850 nm using 625 nm excitation. The use of a single excitation wavelength avoids the problem of wavelength-dependent variation in light source-intensity. The emission spectra were then corrected using correction factors supplied by Perkin-Elmer. The corrected emission spectrum of Compound 10 in DMSO is shown in Figure 6b. Similar measurements and corrections were made with a solution of Cy5.5 in PBS.

The corrected quantum yield of Compound 10 was calculated relative to Cy5.5 using the following equation:

(1-10(-Aref)n2sFsRQc= Fr(1-10(As)n2ref where Aref and As represent the absorbance of the Cy5.5 and perylene dione samples at 625 nm, respectively; Fs and Fre, represent the integrated area of the corrected emission spectra of the compound and reference, respectively, and n. and nref is the refractive index of the solvent in which the sample and reference are present, respectively. The latter parameters correct for differences in fluorescence intensity arising from the physical properties of the solvent.

The corrected relative quantum yields of Compound 10 in various solvents is reported in Table 4b. The absorption maximum, corrected emission maxima and Stokes shifts obtained from the spectra obtained during this analysis are also reported in this table.

Table 4a Relative quantum yields (uncorrected) for perylene dione compounds in DMSO Compound Roqua 9 4. 8 6b 0.92 6a 1. 6 11 0. 25 10 0.33 15 0.83 a Uncorrected quantum yields relative to Cy5 Table 4b. Relative quantum yields (corrected) for Compound 10 in various solvents" AbsorptionEmissionStokesShiftSolventRQcb maximum (nm) maximum (nm) (nm) DMSO 0. 71 637 707. 5 70. 5 Chloroform I. 2 616 683, 5 67.5 Methanol 0.10 623. 5 711 87 PBS (pH 7.1) 0c 618 - - PBS (pH 7.1), 1% Triton X-100 0. 13 635 704 68 10 mM NaOH oc 729-- 10 mM NaOH, 1% Triton X-100 oc 744-- ° Solvents other than DMSO contained > 1% DMSO (vol/vol) b Corrected quantum yield relative to Cy5.5 c No fluorescence detected in these samples

Example 12: Alkaline phosphatase assay: To determine whether compound 11 would be suitable for use in enzyme-linked assays, it was necessary to demonstrate that it can act as a substrate for alkaline phosphatase and that the product of the reaction, 10, could be distinguished from the substrate by spectrophotometric measurement. As compound 10 exhibits some solubility and fluorescence in organic solvents, while the phosphoric acid precursor 11 is largely insoluble in organic solvents (see Table 1), a two- phase system was used to separate the substrate from the product of the alkaline phosphatase reaction. Figure 7 shows the design of the reaction format. The reaction was performed by incubating the phosphate derivative (at a concentration of-20 ~ 20 FM) with varying amounts of alkaline phosphatase (Sigma, Bovine Type VH) enzyme (0-18 units) in an appropriate buffer (100 mM NaCI, 5 mM MgCl2,100 mM diethanolamine, pH 9.5). A control reaction was performed in the absence of alkaline phosphatase. To measure the end-product of the reaction (ie. the OH compound 10), chloroform was added, mixed with the sample and the fluorescence of the two-phase system was measured. Production of the fluorescent compound 10 was found to increase with time until a plateau was achieved as shown in Figure 8. The rate of formation of the fluorescent product was dependent on the amount of enzyme added. A low level of background fluorescence was observed in the control sample (no enzyme), due to the limited solubility/low fluorescence of compound 11 in the organic solvent. This should be subtracted from the test sample in any assay system based on this fluorogenic compound.

The requirement for an organic solvent in the above assay procedure presents a major disadvantage for this assay. Hence alternative procedures for the assay were developed. Figure 10 shows the absorption spectrum of Compound 10-the product of the alkaline phosphatase assay- in solution under neutral (PBS) and basic (10 mM NaOH) conditions. The change in the absorption spectrum under basic conditions is accompanied by an increase in the stability of the absorption of the compound, and reflects the formation of a more soluble, ionised form of the compound. An examination of the pH dependence of the absorption of Compound 10 indicates the pKa for this transition is approximately 10. Hence, under the conditions of the alkaline phosphatase assay (pH 9.5), a signficant proportion of the population of compound 10 exists in the ionised form. The different absorption characteristics of this compound compared to that of the other perylene diones offers the possibility of measuring the phosphatase activity using a standard ELISA set-up with Compound 11 as the substrate. This is demonstrated in Figure 8b, which shows the absorption spectrum of Compound 11 in 0.1 M Na2C03, pH 11 at various times following the addition of 20 U alkaline phosphatase (bovine intestinal mucosa, Sigma). The change in the absorbance with time demonstrates that Compound 11 is a substrate for the enzyme, and the similarity of the absorption spectrum at the longer incubation times with that of the ionised form of Compound 10 (Figure 10), demonstrates that this is indeed the product. Hence this can be used as a quantitative assay using Compound 10 as a standard to quantitate the amount of product

formed.

An alternative strategy for the assay utilising fluorescence is as follows. The assay may be performed at basic pH as above. However, since neither substrate nor compound are fluorescent under these conditions, the reaction cannot be monitored in real time using fluorescence. However, the reaction may be quenched by neutralising the buffer or acidifying the reaction mixture and then adding detergent. Since compound 10 is fluorescent under these conditions (Table 4b), the fluorescence of the product may be measured. Again, this can be related to the activity of the enzyme with reference to a standard curve employing Compound 10.

Coupling of antibodies to the succinimide and iodoacetamide derivatives : To determine whether the succinimide derivative of compound 6a was suitable for labelling of antibodies for use in immunodiagnostic applications, a protocol was developed for protein conjugation. This is illustrated in Figure 9. Polyclonal anti-mouse IgG (Sigma, 1 mg) in 1 ml of 0.1 M sodium carbonate buffer, pH 9.3, containing 1% Triton X-100, was mixed with 30 RI the succinimide derivative of 6a (from a lmg/ml stock in DMSO), and incubated at room temperature with gentle agitation for >1 hour. The mixture was applied to a Sephadex PD-10 gel filtration column, and the void volume fraction was collected and analysed fluorometrically. The absence of non-covalently bound chromophore was established using a Folch extraction procedure, ie., a sample of the conjugated antibody was mixed with an equal volume of chloroform and vortexed. The mixture was then centrifuged at 20,000 g for 2 min to separate the two phases.

Conjugated antibody is found at the interface between the chloroform (lower) and aqueous (upper) fractions, whilst any free compound would remain in either the upper or lower phases.

The fluorescence signal was largely retained (decreased by approximately 10%) upon conjugation of the chromophore to antibody and there appeared to be no non-covalently attached fluorophore remaining after the labelling procedure.

An alternative procedure was sought to couple the iodoacetamide derivative to antibodies which avoids the use of detergent. The iodoacetamide derivative (Compound 19) was solubilised in DMSO (concentration 4.3 mM) and 20 RI added to 0.5 ml of 2.5 mg/ml sheep anti- rabbit IgG (Sigma) and incubated for 6 hrs in 0.1 M Na2CO3. Any unreacted iodoacetamide derivative was removed by passing the sample through a Sephadex G-100 column (Pharmacia) and collecting the void volume peak. The peak fractions were pooled and the absorption spectrum measured on a Caryl3E spectrophotometer. The spectrum showed the characteristic absorbance of the perylene diones at-600 nm, and the characteristic protein absorption at-280 nm. The concentration of the chromophore was calculated using the absorbance at 600 nm, and the concentration of antibodies using the 280 nm absorbance after applying corrections for the

absorbance of Compound 19 at this wavelength. The calculations indicated the presence of-7 molecules Compound 19 per IgG molecule. This was similar to the initial ratio of the two molecules, an indication that Compound 19 quantitatively labels the antibody. This is confirme by the absence of any detectable unreacted Compound 19 during the Sephadex chromatography.

The fluorescence characteristics of the Compound 19-antibody compounds were also examine. Although non-fluorescent in PBS, the sample did become fluorescent in the presence of 1% Tx-100 indicating the usefulness of these compounds.

Coupling of nucleotidesloligonucleotides to the succinimide derivative: Coupling of the succinimide derivative to oligonucleotides or nucleotides may be effected by conventional methods, for example, using an aminolink phosphoramidite or small oligonucleotide containing the aminolink. Covalent attachment of the succinimide derivative to the aminolink may be conformed by standard methods such as thin layer chromatography or gel filtration.

Perylene diones as membrane probes : The perylene diones generally do not exhibit fluorescence in water in the absence of detergent. This property makes the neutral compounds useful as membrane probes. This was tested by incorporating Compound 17 into sonicated lipid vesicles composed of dimiristoyl phosphatidylcholine (DMPC). The presence of a fluorescence signal demonstrated that the compound was associated with the bilayers.

The polarisation of a fluorophore is measured by exciting the fluorophore with vertically polarised light and measuring the proportion of the emitted light which remains vertically polarised. The polarisation (P) is defined according to the following equation: P V-GH V+GH where V and H are the fluorescence intensities measured through vertically and horizontally oriented polarisers in front of the emission detector, and G is an instrumental factor calculated by measuring the ratio of the V and H intensities following excitation with horizontally polarised light.

Figure 11 shows the effect of temperature on the polarisation of Compound 6a following its incorporation into DMPC bilayers. Compound 6a (30 pl of a 0.5 mg/ml stock solution in DMSO) was added to 1 mg of DMPC in 1 ml chloroform. The chloroform was evaporated using a stream of nitorogen, and the sample resuspended in 5 ml PBS by sonication at 37°C (10 minutes using intervals of one minute followed by one minute of"recovery"). The emission polarisation of the sample exhibits a sharp change around the phase-transition temperature of DMPC (~ 23 °C).

Since the polarisation is sensitive to the dynamics of the fluorophore, this demonstrates that Compound 6a incorporates into the bilayer and reports on the physical properties of the bilayer.

The suitability of the perylene diones as general membrane probes in cellular systems was examine by incorporating them into red blood cell ghosts and performing confocal microscopy and flow cytometric measurements. Red blood cell ghosts (100 ml) or intact red blood cells (100 ml) were mixed with 10 ml of a lmg/ml stock of Compound 17 in DMSO in a total volume of 1.5 ml of 5mM sodium phosphate, pH7. The sample was incubated for 45 minutes at 37°C and then washed by pelleting and suspending in fresh buffer. Flow cytometric measurements were performed using a HeNe laser to excite the samples at 630 nm. Figure 11 (Panel B) shows ghost membranes labelled with Compound 17 as above. A high level of fluorescence was observed in comparison with the background signal obtained for a sample of unlabelled membranes (Panel A). Intact red blood cells labelled with compound 17 (Panel F) also showed a very high level of fluorescence signal compared with unlabelled red blood cells (Panel G).

Red-blood cell ghosts labelled with Compound 17 were also analysed by confocal fluorescence microscopy utilising the red line of a KrAr laser on a Leica TCS-NT laser scanning confocal microscope. Figure 13 shows that the compound is associated with the cell membrane, demonstrating that these compounds may be useful in multi-color fluorescence microscopy applications.

INDUSTRIAL APPLICABILITY The compounds of the present invention are useful in optically-based diagnotic assays and techniques including but limited to antibody-based assays such as fluorescence immuno-assays (FIA), fluorescence microscopy and immunobiosensor applications, DNA-based assays such as DNA sequencing and DNA-based direct-binding diagnostic applications, enzyme-based assays such as enzyme-linked fluorescence immuno-assays (EL-FIA), ELISA-type assays and enzyme- linked fluorescence DNA-probe assays (EL-FDA), and as a membrane probe, for example, in flow cytometry and confocal microscopy.

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