ENERGY TRANSFER ASSAY METHOD AND REAGENT

Abstract: Disclosed is a non-fluorescent cyanine dye that may be used as an acceptor in fluorescence energy transfer assays involving the detection of binding and/or cleavage events in reactions involving biological molecules, and assay methods utilising such dyes. The non-fluorescent cyanine dye is a compound of formula (I), wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y; groups R<3>, R<4>, R<5> and R<6> are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z<1> and Z<2> ring structures; Z<1> and Z<2> each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; at least one of groups R<1>, R<2>, R<3>, R<4>, R<5>, R<6> and R<7> is a target bonding group; any remaining groups R<3>, R<4>, R<5>, R<6> and R<7> groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR<9>, COOR<9>, nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R<9> is selected from H and C1-C4 alkyl; any remaining R<1> and R<2> are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl, the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R<1>, R<2>, R<3>, R<4>, R<5>, R<6> and R<7> comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent.

Description: Energy Transfer Assay Method and Reagent The present invention relates to the field of fluorescence resonance energy transfer. In particular, the invention relates to fluorogenic assays which include a novel class of non-fluorescent quenching dyes, and to novel quenching dye compounds thereof. Fluorescence resonance energy transfer (FRET) occurs between the electronic excited states of two fluorophores when they are in sufficient proximity to each other, in which the excited-state energy of the donor fluorophore is transferred to the acceptor fluorophore. The result is a decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species. In one application of this principle, a fluorescent moiety is caused to be in close proximity to a quencher molecule. In this configuration, the energy from the excited donor fluorophore is transferred to the quencher and dissipated as heat rather than fluorescence energy. The use of fluorescence resonance energy transfer (FRET) labels in biological systems is well known. The principle has been used in the detection of binding events or cleavage reactions in assays which employ fluorescence resonance energy transfer. In the case of peptide cleavage reactions, a fluorescent donor molecule and fluorescent acceptor molecule are attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that non-radiative energy transfer between the donor and the acceptor species takes place. For example, EPA 428000 discloses a novel fluorogenic peptide substrate involving a fluorescent donor molecule and a quenching acceptor molecule attached thereto. The labelled substrate can be used in the detection and assay of a viral protease enzyme, whereby, if there is enzyme present in a test sample, the substrate is cleaved and the donor and acceptor species are thereby separated. The resultant fluorescent emission of the donor species can be measured. Suitable fluorescent donors include fluorescein derivatives, coumaπns and 5-((2-amιnoethyl)amιno)-naphthalene-1 - sulphonic acid (EDANS) . Suitable quenching acceptors include 2,4- dmitrophenyl (DNP) and 4-(4-dιmethylamιnophenyl)azobenzoιc acid (DABCYL) . Fluorescence energy transfer has also been used in the study of nucleic acid hybridisation. For example, Tyagi and Kramer (Nature Biotechnology, 14, 303-8, (1 996)) disclose homogeneous hybridisation assays which utilise fluorescent labelled probes. The hair-pin probes comprise a single-stranded nucleic acid sequence that is complementary to the target nucleic acid, together with a stem sequence formed from two complementary arms which flank the probe sequence. A fluorophore (EDANS) is attached to one arm and the non-fluorescent quencher moiety (DABCYL) is attached to the complementary arm. In the absence of target, the stem keeps the fluorescent and quenching groups in close proximity causing the fluorescence of the fluorophore to be quenched. When the probe is allowed to bind to a nucleic acid target, it undergoes a conformational change, forming a more stable hybrid with the target and forcing the arm sequences (and the fluorophore and quencher) to move apart. The fluorophore will then emit fluorescence when excited by light of a suitable wavelength. The success of the fluorescence resonance energy transfer approach is dependent upon the choice of the appropriate donor/acceptor pair. If energy transfer between the donor and acceptor can be optimised, residual fluorescence is minimised when the donor/quencher pair are in close proximity and a large change in signal can be obtained when they are separated. There is an increasing trend towards assay miniaturisation and in high throughput screening assays and, as a result, it is beneficial to use fluorophores with high extinction coefficients in order to achieve the sensitivity levels required. A further problem associated with such assays is due to colour quenching caused by the presence in the assay medium of coloured samples which tend to absorb strongly in the 350-450nm region of the spectrum. The present invention provides a non-fluorescent cyanine acceptor dye that can be used as one component of a fluorescent donor/acceptor pair for assays involving the detection of binding and/or cleavage events in reactions involving biological molecules. The fluorescent donor dye possesses a high extinction coefficient, thereby enabling the detection of low levels of the fluorophore. Moreover, the fluorescent dye pair have excitation and emission wavelengths in a range which is substantially free from auto-fluorescence associated with biological samples and from quenching due to coloured samples. Additionally, the dyes are relatively pH insensitive and they possess a high degree of spectral overlap, allowing efficient energy transfer. Accordingly, the present invention relates to a compound of formula (1 ) : ( 1 ) wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y, groups R 3 , R 4 , R 6 and R 6 are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z 1 and Z 2 ring structures; Z 1 and Z 2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Y are the same or different and are selected from bis-Cι-C alkyl- and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium, -CH = CH- and N-W wherein N is nitrogen and W is selected from hydrogen, a group -(CH_) m R 8 where m is an integer from 1 to 26 and R 8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups; at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a target bonding group; any remaining groups R 3 , R 4 , R 5 , R 6 and R 7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR 9 , COOR 9 , nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R 9 is selected from H and C1-C4 alkyl; any remaining R 1 and R 2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent; provided that the linker group Q is not a squaraine ring system. Preferably the linker group Q contains 1 , 2 or 3 double bonds in conjugation with the rings containing X and Y. Preferably Q is the group: wherein the groups R 10 are selected from hydrogen and C1-C4 alkyl which may be unsubstituted or substituted with phenyl, or two or more of R 10 together with the group: form a hydrocarbon ring system substituted with R 7 and which may optionally contain a heteroatom selected from -O-, -S-, or >NR 7 , wherein R 7 is hereinbefore defined; and n = 1 , 2 or 3. Suitably, the non-fluorescent cyanine dye for use in the present invention is a compound having the formula (2) : 99/64519 (2) wherein groups R 3 , R 4 , R 6 and R 6 are attached to the rings containing X and Y or, optionally, are attached to atoms of the Z 1 and Z 2 ring structures and n is an integer from 1 -3; Z 1 and Z 2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Y are the same or different and are selected from bis-Ct-C4 alkyl- and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium, - CH = CH- and N-W wherein N is nitrogen and W is selected from hydrogen, a group -(CH 2 ) m R 8 where m is an integer from 1 to 26 and R 8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups; at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a target bonding group; any remaining groups R 3 , R 4 , R 5 , R 6 and R 7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR 9 , COOR 9 , nitro, ammo, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R 9 is selected from H and C1-C4 alkyl; any remaining R 1 and R 2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent Suitably at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 of the dyes according to the present invention comprises a substituent which reduces the fluorescence emission of the dye such that it is essentially non-fluorescent. Suitably, at least one of groups R 3 , R 4 , R 5 , R 6 and R 7 of the non-fluorescent cyanine dyes of structures (1 ) and (2) is a nitro group which may be attached directly to the rings containing X and Y. In the alternative, a mono- or di-nitro-substituted benzyl group may be attached to the rings containing X and Y, which optionally may be further substituted with one or more nitro groups attached directly to the aromatic rings. Preferably, at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 of the non-fluorescent cyanine dyes of structures ( 1 ) and (2) comprises at least one nitro group. The target bonding group R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 can be any group suitable for attaching the non-fluorescent cyanine dye to a target material, such as a carrier material or a biological compound and as such will be well known to those skilled in the art. For example, the target bonding group may be a reactive group for reacting with a functional group on the target material. Alternatively, the target bonding group may be a functional group and the target may contain the reactive constituent. Preferably, the target bonding group is of the structure -E-F where E is a spacer group and F is the reactive or functional group. A reactive group of the dye can react under suitable conditions with a functional group of a target molecule; a functional group of the dye can react under suitable conditions with a reactive group of the target molecule, whereby the target molecule becomes labelled with the dye. Preferably, the reactive group F is selected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite. Preferably, the functional group F is selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate. By virtue of these reactive and functional groups the non-fluorescent cyanine dye may be reacted with and covalently bound to target materials. Suitable spacer groups may contain 1 -60 chain atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus. For example the spacer group may be: -{(CHR') q -O-(CHR')r-}s- -{(CHR') q -NR'-(CHR')r}s- -{(CHR' CH = CH)-(CHR') r }ε- -{(CHR')q-Ar-(CHR')r-} ε - -{(CHR') q -CO-NR'-(CHR')r-} s - -{(CHR') q -CO-Ar-NR'-(CHR')r-}s- 99/64519 where R' is hydrogen, or C1-C4 alkyl which may be optionally substituted with sulphonate, Ar is phenylene, optionally substituted with sulphonate, p is 1 -20, preferably 1 -10, q is 1 -5, r is 0-5 and s is 1 -5. Specific examples of reactive groups R 1 - R 7 and the groups with which R 1 - R 7 can react are provided in Table 1 . In the alternative, the R 1 R 7 may be the functional groups of Table 1 which would react with the reactive groups of a target molecule. Table 1 . Possible Reactive Substituents and Functional Groups Reactive Therewith Particularly suitable reactive groups R 1 - R 7 which are especially useful for labelling target components with available amino and hydroxyl functional groups include: where m is an integer from 1 -10. Aryl is an aromatic substituent containing one or two fused aromatic rings containing 6 to 10 carbon atoms, for example phenyl or naphthyl, the aryl being optionally and independently substituted by one or more substituents, for example halogen, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and alkoxy for example methoxy, ethoxy, propoxy and n-butoxy. Heteroaryl is a mono- or bicyc c 5 to10 membered aromatic ring system containing at least one and no more than 3 heteroatoms which may be selected from N, O, and S and is optionally and independently substituted by one or more substituents, for example halogen, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and alkoxy for example methoxy, ethoxy, propoxy and n-butoxy. Aralkyl is a Ct - Ce alkyl group substituted by an aryl or heteroaryl group. Halogen and halo groups are selected from fluorine, chlorine, bromine and iodine. The non-fluorescent cyanine dyes for use in the present invention may also include water solubilising constituents attached thereto for conferring a hydrophi c characteristic to the dye. They may be attached directly to the aromatic ring system of the cyanine dye or they may be attached to the spacer group E Suitable solubilising constituents may be selected from the group consisting of sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and hydroxyl Sulphonate or sulphonic acid groups attached directly to the aromatic ring of the non-fluorescent quenching dye are to be particularly preferred. Water solubility may be necessary when labelling proteins. In a second aspect, the present invention relates to a biological material labelled with a non-fluorescent cyanine dye. In a further aspect, the invention relates to a biological material which comprises two components one of which is labelled with a fluorescent dye which may act as a donor of resonance energy and the other with a non-fluorescent cyanine dye which may act as an acceptor of resonance energy transferred from the donor. In a still further aspect, the invention relates to an assay method which comprises: i) separating two components which are in an energy transfer relationship, the first component being labelled with a fluorescent donor dye and the second component being labelled with a non-fluorescent cyanine acceptor dye; and, ii) detecting the presence of the first component by measuring emitted fluorescence. In a still further aspect the invention relates to an assay method which comprises: i) binding one component of a specific binding pair with a second component of said pair, said first component being labelled with a fluorescent donor dye and said second component being labelled with a non-fluorescent cyanine acceptor dye to bring about an energy transfer relationship between said first and second components; and, n) detecting the binding of the first and second components by measuring emitted fluorescence. The non-fluorescent cyanine dyes of the present invention are employed as acceptor dyes in assay methods utilising fluorescence resonance energy transfer. When a non-fluorescent cyanine dye of the invention is in an energy transfer relationship with a fluorescent donor dye, the fluorescence emission of the donor is reduced through quenching by the acceptor. When resonance energy transfer is lost through separation of the fluorescent donor dye and the acceptor dye, the fluorescence emission due to the donor dye is restored. Effective non- fluorescent quenching dyes have a low efficiency for converting absorbed incident light into fluorescence and, as such, are unsuitable as fluorescent labels where a high degree of sensitivity is required. The relative effectiveness of the non-fluorescent cyanine dyes as quencher dyes is illustrated in Figure 1 which shows the relative fluorescence emission of representative non-fluorescent cyanine dyes of the invention compared with corresponding dyes of the same class and with similar absorption characteristics in the visible region of the spectrum. The intrinsic fluorescence of the cyanine dyes of the present invention, when they are employed as energy acceptors is preferably less than 10% of the fluorescence emission of the donor dye upon excitation at the donor excitation wavelength and detection of emission at the donor emission wavelength. Figure 3 shows the decrease in background signal obtainable by the use of Compound II and Compound XI as acceptor dyes compared with that of a standard matched fluorescent cyanine acceptor dye (Cy5), when measured at the emission maxima of the donor dye. The contribution to background fluorescence is thus minimised by the use of the non-fluorescent cyanine dyes of the invention as quencher dyes. Moreover, the present dyes are designed such that their spectral overlap with a fluorescent donor dye is maximised, thereby improving efficiency of quenching. The biological material can be a biological molecule which may be cleaved into the two component parts; or the biological material may comprise two components as hereinbefore defined which may be bound either by covalent or non-covalent association. The present invention therefore relates to a novel fluorogenic substrate and an assay method for the detection and measurement of the cleavage of a molecule into two component parts, the first component being labelled with a fluorescent donor dye in an energy transfer relationship with a non-fluorescent cyanine acceptor dye bound to the second component. The assays may be performed according to the present invention in high throughput screening applications, including those in which compounds are to be screened for their inhibitory effects, potentiation effects, agonistic, or antagonistic effects on the reaction under investigation. Examples of such assays include, but are not restricted to, the cleavage of a peptide or protein by a protease and the cleavage of a DNA or RNA molecule by a nuclease. In this assay format, the enzyme substrate (peptide or nucleic acid) will include a sequence whose structure combines a fluorescent donor dye molecule with the non-fluorescent cyanine acceptor dye, attached to the substrate at either side of the substrate bond to be cleaved. The substrate joins the fluorescent donor and the acceptor moieties in close proximity. The intrinsic fluorescence of the donor is reduced through quenching by the acceptor due to resonance energy transfer between the pair of dyes. Resonance energy transfer becomes insignificant when the distance between the donor and acceptor moieties is greater than about 100 Angstroms. Cleavage of the substrate results in the separation between donor and acceptor dyes and concomitant loss of resonance energy transfer. The fluorescence signal of the donor fluorescent dye increases, thereby enabling accurate measurement of the cleavage reaction. Briefly, an assay for the detection of proteolytic enzyme activity may be configured as follows. A reaction mixture is prepared by combining a protease enzyme and a fluorogenic substrate which combines a fluorescent donor dye molecule with a non-fluorescent acceptor dye of formula (2) attached to the substrate at either side of the substrate bond to be cleaved. A known or a putative protease inhibitor compound may be optionally included in the reaction mixture. Typically the reaction is performed in buffered solution and the reaction is allowed to proceed to completion. The progress of the reaction may be monitored by observing the steady state fluorescence emission due to the fluorescent donor dye, which is recorded using a spectrofluorimeter. Alternatively, the invention relates to an assay method for detecting and measuring binding, by covalent or non-covalent association, of one component of a ligand/reactant pair with a second component of said pair, said first component being labelled with a fluorescent donor dye and said second component being labelled with a non-fluorescent cyanine acceptor dye. Such assays are conveniently categorised as one of two types. i) The first category comprises equilibrium binding assays, in which one component of a specific binding pair binds non-covalently to a second component of the specific binding pair. Such equilibrium binding assays may be applied to screening assays in which samples containing compounds to be screened are tested for their effect upon the binding of the first component of the specific binding pair (either antagonistic or agonistic), to the second component. Either component may be labelled with the donor dye or the acceptor dye. In the absence of binding, the labelled components are too far apart for resonance energy transfer to occur. Upon binding of one labelled component to its labelled specific binding partner, the label moieties are brought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a decrease in donor fluorescent signal. For example, the dyes used in the present invention can be used to label probes such as those described by Tyagi and Kramer (loc. cit.) for use in the detection and identification of unique DNA sequences or specific genes in a complete DNA molecule or mixtures of nucleic acid fragments. One end of the nucleic acid probe is labelled with a fluorescent dye and at the other end with a non-fluorescent cyanine acceptor dye. In the absence of specific target sequence, the fluorescent and quenching species will be held sufficiently close for energy transfer to occur. Consequently, irradiation of the fluorophore by excitation light will give reduced fluorescent signal. Interaction of the probe with a specific target nucleic acid sequence causes a conformational change to take place in the probe, such that the fluorescent donor and acceptor become separated by distance. Excitation of the fluorophore will result in a fluorescent signal which may be recorded using a spectrofluorimeter. Alternatively, the equilibrium binding assay may employ a sandwich assay format in which one component of a specific binding pair, such as a first antibody, is coated onto the wells of a microtitre well plate. Following binding of an antigen to the first antibody, a second antigen- specific antibody is then added to the assay mix, so as to bind with the antigen-first antibody complex. In this format, either the first antibody or the antigen may be labelled with the donor dye and the second antibody labelled with the acceptor dye or vice versa. In the absence of binding of the first antibody-antigen-second antibody complex, the labelled components are too far apart for resonance energy transfer to occur. Upon binding of the second antibody with the first antibody-antigen complex, the label moieties are brought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a decrease in donor fluorescent signal. Fluorescence signal is measured and the concentration of antigen may be determined by interpolation from a standard curve. Examples of specific binding pairs include, but are not restricted to, antibodies/antigens, lectins/glycoproteins, bιotιn/(strept)avιdιn, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that in the present invention, any molecules which possess a specific binding affinity may be employed, so that the energy transfer dyes of the present invention may be used for labelling one component of a specific binding pair, which in turn may be used in the detection of binding to the other component. n) In the second category, the assay may comprise detection and measurement of the addition of a fluorescent donor dye labelled moiety (the reactant) in solution in the assay medium to a non-fluorescent acceptor dye-labelled moiety (the substrate) or vice versa, by covalent attachment mediated through enzyme activity. Examples of such assays include, but are not restricted to, the joining of DNA or RNA molecules to other nucleic acid molecules by gases, the addition of a nucleotide to a DNA or RNA molecule by a polymerase and the transfer of a labelled chemical moiety from one molecule to another by a transferase such as acetyl transferase A known or a putative enzyme inhibitor may be optionally included in the reaction mixture It is to be understood that any two appropriate reactant and substrate moieties may be employed. Either of the donor or the acceptor dyes of the present invention may be used for labelling one moiety which in turn may be used in the detection and measurement of the reaction with the substrate. For example, in a DNA ligation assay, DNA molecules to be joined are mixed together in aqueous buffer containing ATP in the presence of a DNA iigase. Following incubation, the DNA strands are covalently attached in the correct configuration by the formation of standard phosphodiester linkages in both strands of the duplex. Upon joining the label moieties are bought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a signal decrease which is proportional to the amount of ligated product formed. The invention also relates to labelling methods wherein the non- fluorescent cyanine dyes of structures (1 ) and (2) including at least one reactive or functional group at the R 1 - R 7 positions covalently react with amino, hydroxyl, aldehyde, phosphoryl, carboxyl, sulphydryl or other reactive groups on target materials. Such target materials include, but are not limited to the group consisting of antigen, antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, microbiai materials, drugs and toxins. The selection of suitable fluorescent donor and acceptor pairs is generally dependent on several factors. i) Firstly, the donor and acceptor chromophores should have strong electronic transitions in the near UV to near IR spectral range. ii) Secondly, the donor and acceptor moieties should be in relatively close proximity with each other. Suitably the donor and acceptor species should be in the range 10-100 Angstroms. iii) Thirdly, there should be a suitable overlap between the donor emission spectrum and absorption spectrum of the acceptor. The greater the overlap between donor emission spectrum and the excitation spectrum of the acceptor, the greater the energy transfer. Energy transfer can occur between dyes which share minimal spectral overlap, but this is only observed when the dyes are in close proximity. Suitable fluorescent donor dyes that can be combined with the non- fluorescent cyanine acceptor dyes to form energy transfer pairs for the practice of the present invention include the well known reactive analogues of the fluorescein, rhodamine and cyanine dyes. Other low molecular weight fluorescent dyes may be selected from the derivatives of the bis-pyrromethine boron difluoride dyes, such as 3,3',5,5'-tetramethyl- 2,2'-pyrromethine-1 , 1 '-boron difluoride, sold under the trademark BODIPY by Molecular Probes Inc. Particularly preferred are the cyanine dyes. Suitable fluorescein donor dyes include: 5- and 6-carboxyfluorescein and 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein. Suitable rhodamine dyes include: 6-carboxyrhodamine (Rhodamine 1 10), 5- carboxyrhodamine-6G (R6G-5 or REG-5), 6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or TMR), 6-carboxy-X-rhodamine (ROX) . Suitable cyanine donor dyes include the CyDyes™: Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. (CyDye and Cy are trademarks of Amersham Pharmacia Biotech UK Limited.) Cyanine dyes suitable for use as the donor component in the assay of the present invention are disclosed in US Patent No.5268486 (Waggoner et al), or the rigidised cyanine dyes such as those disclosed in GB Patent No.2301 832 (Waggoner et al). Alternatively the fluorescent donor species used as the donor component may be a fluorescence energy transfer dye cassette. Examples of such fluorescence energy transfer dye cassettes are to be found in GB Patent No.2301833 (Waggoner et al) . Table 2 below shows examples of fluorescent donor dyes and corresponding non-fluorescent cyanine acceptor dyes which are suitable for use in the methods according to the present invention. Table 2 * salt form, undefined. The non-fluorescent cyanine dyes of formulae ( 1 ) and (2) may be prepared by a process comprising- a) reacting a first compound having the formula (A) : (A) where X, Z , R , R and R are hereinbefore defined, b) a second compound which is the same or different from the first compound and having the formula (B) : (B) where Y, Z 2 , R 2 , R 4 , R 6 are hereinbefore defined, and c) a third compound suitable for forming a linkage between the first and second compounds, wherein a), c) and b) are reacted either in a two- or single step process to form the compounds of formulae (1 ) and (2) . In the case of a two step synthesis, an intermediate dye compound is first formed by reacting an indolenine compound of structure (A) with a compound suitable for forming the linkage wherein the reaction is performed in a suitable polar solvent such as ethanol or acetic acid. In the second stage, the intermediate dye is reacted with the second indolenine compound of structure (B) in a medium such as pyπdine, acetic acid and acetic anhydride at room temperature Such reaction conditions are also suitable for the preparation of non-fluorescent cyanine dyes of the present invention by a one step process. Reagents c) which may be used for forming the linkage between the indolenine moieties are those suitable for forming a polymethine chain. Reagents and methods suitable for forming cyanine dyes containing polymethine linkages will be well known to those skilled in the art and include tπethyl orthoformate, malondialdehyde bis- (phenylimme) hydrochioπde and N-[(5-phenylamιno)-2,4- pentadienylidenejani ne hydrochloπde. (See for example, Fry D.J, Cyanine Dyes and Related Compounds, in Rodd's Chemistry of Carbon Compounds, Elsevier 1 977, page 369-422) . The non-fluorescent cyanine dyes of the present invention may be used to covalently label a target material such as a component of the assay system as hereinbefore described. Covalent labelling using compounds of the present invention may be accomplished with a target having at least one functional or reactive group as hereinbefore defined. The target may be incubated with an amount of a compound of the present invention having at least one of R 1 - R 7 that includes a reactive or functional group as hereinbefore defined that can covalently bind with the functional or reactive group of the target material. The target material and the compound of the present invention are incubated under conditions and for a period of time sufficient to permit the target material to covalently bond to the compound of the present invention. The invention is further illustrated by reference to the following examples and figures. Figures Figure 1 illustrates the relative fluorescence emission of Cy3 and Compounds V and VII (Fig 1 a) and of Cy5 and Compounds IX and XI (Fig 1 b) according to Example 1 2. Figure 2 illustrates nucleic acid hybridisation assays in which a reduction in fluorescence signal is observed, resulting from the binding of a Cy3-labelled oligonucleotide and its complementary oligonucleotide labelled with a non-fluorescent Cy5 analogue (Compound II) and with Dabcyl, according to Example 1 3. Figure 3 illustrates the effect of using non-fluorescent cyanine dyes as acceptor dyes on signal to noise increase (and background reduction) compared with a standard matched fluorescent cyanine acceptor dye (Cy5) in an oligonucleotide hybridisation study according to Example 14. Figure 4 illustrates the course of protease cleavage assays utilising the substrate, Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys-Compound III, in the presence and in the absence of pepsin, measured with respect to time and fluorescence intensity, according to Example 1 5. 99/64519 Examples Example 1 Preparation of 2-{5-[1 -(5-carboxypentyl)-3,3-dιmethyl-5- sulpho-1 ,3-dιhydro-2 -ιndol-2-ylιdene]-1 ,3-pentadιenyl}-1 ethyl-3,3-dιmethyl-5-nιtro-3/V-ιndolιnιum, salt (Compound i) 5-Nιtro-2,3,3-trιmethylιndole Sodium nitrate (3.84g, 45.2mmol) was dissolved in concentrated sulphuric acid (100ml) . After cooling in ice, this solution was added to a solution of 2,3,3-trιmethylιndole (6.65g, 41 .8mmol) in concentrated sulphuric acid ( 100ml) such that the temperature was maintained in the range 0-5°C. The reaction was stirred at 0-5°C for 90 minutes after completing the addition, then allowed to warm to room temperature and stirred for a further 1 6 hours. The mixture was poured onto ice (200g) then made basic by the addition of 50% aqueous sodium hydroxide solution to pH 1 2 (pH paper) . The crude product was collected by filtration and washed with water until the washings were neutral ( ~ 1000ml) . The off-yellow solid was dried at the pump, dissolved in ethyl acetate (250ml) and dried further (MgSO4> The solution was filtered and the red filtrate was rotary evaporated to dryness. The solid was dissolved in chloroform:ethyl acetate (95:5, 30ml) and purified by silica flash chromatography This gave a dark yellow solid, 5.1 2g, 25mmol, 60% yield UV analysis (methanol) showed a single peak with λmax = 300nm. Mass spectrometry (MALDI-TOF with a gentisic acid matrix) gave m/z = 203.8 (for C11H1-N-O- = 204.23) . 1 H NMR : δ = 1 .97 (s, 6H), 2.95 (s, 3H), 8.58 (d, J = 10Hz, 1 H), 8.70 (s, 1 H), 8.78 (d, J = 10Hz, 1 H) . ii) 1 -Ethyl-5-nitro-2,3,3-trimethylindolium iodide 5-Nitro-2,3,3-trimethylindole (51 8mg, 2.54mmoi) and ethyl iodide (2.5ml, 4.85g, 31 mmol, excess) were mixed and heated to reflux for 8 hours. After cooling to room temperature the excess ethyl iodide was removed by a gentle nitrogen stream and the residue was dissolved in chloroform and filtered through a plug of silica. The silica was washed with chloroform (1 volume) and the combined filtrates were rotary evaporated to give the product as a yellow oil, (486mg, 1 .35mmol, 53% yield) . iii) 1 -(5-Carboxypentyl)-5-sulpho-2,3,3-trimethylindolium bromide To 5-sulpho-2,3,3-trimethylindole (potassium salt) (5.0g, 1 8mmol) was added 6-bromohexanoic acid (10.56g, 53.8mmol) in 1 ,2-propanediol ( 1 5ml) and heated at 80°C for 72 hours. The mixture was cooled and diluted with water (60ml) and a solution of NaOH (10% in water, 60ml) with stirring . The product was purified using preparative HPLC (Dynamax, Ci8 column, gradient of TFA/H 2 0 to TFA/MeCN) to yield a grey/pink solid (2.4g, 28%) . iv) 2-(4-Anilinobuta-1 ,3-dienyl)-1 -(5-carboxypentyl)-3,3-dimethyl-5- sulpho-indolium, salt To the product from stage iii) (400mg, 0.85mmol) was added malondialdehyde bis-(phenylimine) HCI (322mg, 1 .24mmol) in acetic acid (5ml) and the mixture heated at 140°C for 1 6 hours with stirring. The product was purified by HPLC (C4 column, TFA/H_O to TFA/MeCN gradient) to yield a red/grey solid (1 94.4mg, 38%) . v) Synthesis of Compound (I) 1 -Ethyl-5-nitro-2,3,3-trimethylindolium iodide ( 100mg, 0.277mmol) and the product from stage iv) (50mg, O.1 04mmol) were dissolved in pyridine:acetic acid:acetic anhydride (9:9:2, 5ml) and the reaction was stood at room temperature in the dark for 24 hours. After removing the solvents by a gentle nitrogen stream the residue was dissolved in water:acetonitrile (7:3, 5ml) and the product was isolated by reverse phase HPLC. UV (methanol) : λmax abs = 652nm, λmax em = 670nm. MS: m/z = 621 .2 (for C33H40N3O7S = 622.8) . Example 2. Preparation of 2-{5-[1 -(5-carboxypentyl)-3,3-dimethyl-5- sulpho-1 ,3-dihydro-2//-indol-2-ylidene]-1 ,3-pentadienyl}-1 ■ (3,5-dinitrobenzyl)-3,3-dimethyl-3/V-indolinium, salt (Compound II) i) 1 -(3,5-Dinitrobenzyl)-2,3,3-trimethylindolium iodide 3,5-Dinitrobenzyl chloride (100mg, 0.46mmol), 2,3,3-trimethylindole (74μl, 73.2mg, 0.46mmol) and sodium iodide (69mg, 0.46mmol) were heated at 100°C in sulpholane (5ml) for 1 6 hours. After cooling to room temperature the product was isolated by reverse phase HPLC (89.7mg, 0.26mmol, 57% yield) . n) 2-(5-π -(5-Carboxypentyl)-3,3-dιmethyl-5-sulpho-1 ,3-dιhydro-2ry- ιndol-2-ylιdene]-1 ,3-pentadιenyl}-1 -(3,5-dιnιtrobenzyl)-3,3-dιmethyl- 3H-indolinium, salt (II) N-(3,5-Dιnιtrobenzyl)-2,3,3-trιmethylιndolιum iodide (20mg, 0.059mmol) and the product from Example 1 iv) (20mg, 0.041 mmol) were dissolved in pyrιdιne:acetιc acιd:acetιc anhydride (2:2: 1 , 2.5ml) and the reaction was allowed to stand in the dark at room temperature for 1 6 hours. The product was isolated by reverse phase HPLC to give 1 7.6mg, 0.024mmol, 59% yield of compound (II) MS- m/z = 730 (for 111) 2-{5-[1 -(5-Carboxypentyl)-3,3-dιmethyl-5-sulpho-1 ,3-dιhvdro-2H- ιndol-2-ylιdene]-1 ,3-pentadιenyi}-1 -(3,5-dιnιtrobenzyl)-3,3-dιmethyl- 3H-ιndolιnιum, salt, N-hydroxysuccinimidyl ester O-(N-Succιnιmιdyl)-N,N,N' ,N'-bιs-(tetramethylene)-uronιum hexafluorophosphate (10mg, 0.04mmol) was dissolved in N,N- dimethylformamide ( 1 mi) and N,N-dιιsopropylethylamιne (1 1 μl, 0.063mmol) added. From this solution 100μl was taken and added to the carboxylic acid (1 mg) from n) above After 3hrs, mass spectrum analysis indicated that the NHS ester had been formed The product was used without further purification for labelling . MS: m/z = 797 (for C43H49N4O-S = 797.9) . Example 3. Preparation of 1 -Butyl-2-{5-[1 -(5-carboxypentyl)-3,3-dimethyl- 6-sulpho-1 ,3-dihydro-2r/-benzo[e]indol-2-ylidene]-1 ,3- pentadienyl}-3,3-dimethyl-5-nitro-3H-indolinium, salt (Compound III) 1 -Butyl-5-nitro-2,3,3-trimethylindolium iodide (2.45mg, 9.37μmol) was added to 2-(2-anilinobutenyl)-1 -(5-carboxypentyl)-3,3-dimethyl-6- sulpho-benz(e)indolium, salt (5mg, 9.37μmol) in pyridine:acetic acid:acetic anhydride (9:9:2; 200μl) . The reaction mixture was stored at room temperature in the dark for 4 days. The solvents were removed under reduced pressure and the residue was purified by reverse phase HPLC to give the desired product (2.0mg, 2.85μmol, 30% yield) . Example 4. Preparation of 1 -butyl-2-{7-[1 -(5-carboxypentyl)3,3-dimethyl- 5-sulpho-1 ,3-dihydro-2r -indol-2-ylidene]-1 ,3,5-heptatrienyl)- 3,3-dimethyl-5-nitro-3r7-indolinium, salt (Compound IV) i) 1 -Butyl-5-nitro-2,3,3-trimethylindolium iodide 5-Nitro-2,3,3-trimethylindole (250mg, 1 .22mmol) and iodobutane (10ml, 1 6.1 7g, 87.87mmol) were mixed and heated to reflux for 1 6hrs. The solvent was removed and the impure material stored at -20°C for use in later reactions. ii) 1 -Butyl-2-(7-[1 -(5-carboxypentyl)3,3-dimethyl-5-sulpho-1 ,3-dihvdro- 2r -indol-2-ylidene]-1 ,3,5-heptatrienyl}-3,3-dimethyl-5-nitro-3 - indolium, salt (IV) 1 -(5-Carboxypentyl)-5-sulpho-2,3,3-trimethylindolium, salt (5mg, 0.0141 mmol), 1 -butyl-5-nitro-2,3,3-trimethylindolium iodide (4.02mg, 0.0141 mmol) and N-[(5-phenylamino-)2,4-pentadienylidene]ani!ine monohydrochloride (3.69mg, 0.0141 mmol) were dissolved in pyridine:acetic acid:acetic anhydride (9:9:2, 1 ml) and the reaction was allowed to stand at room temperature in the dark for 1 week. UV analysis at 752nm over the week indicated the reaction was complete at the end of the week. The product was then isolated by reverse phase HPLC to give a blue solid (yield = 4.3mg, 45%) . UV (methanol) : λmax. abs = 752nm. MS: m/z = 676 (for C37H46N3O7S = 676.9) . Example 5. Preparation of 2-{3-[1 -(3-aminopropyl)-3,3-dimethyl-5-sulpho- 1 ,3-dihydro-2r -indol-2-ylidene]-1 -propenyl}-3-(3,5- dinitrobenzyQ-1 ,3-benzothiazol-3-ium, salt (Compound V) 3-(3,5-Dinitrobenzyl)-2-methyl-1 ,3-benzothiazol-3-ium iodide 2-methylbenzothiazole (3ml, 20.1 mmol) was dissolved in acetone (25ml), to which was added 3,5-dinitrobenzyl iodide (7.4g, 24.1 mmol) and heated with stirring to reflux for 1 5 hours. The reaction was allowed to cool and added drop-wise to ether (500ml) to produce a fine yellow solid which was filtered and dried to constant weight in vacuo to give 3-(3,5- dinitrobenzyl)-2-methyl- 1 ,3-benzothiazol-3-ium iodide (5g, 10.9mmol, 45% yield). MS: m/z = 328 (Ci5Hι_N 3 O 4 S = 330.3) . 1 H NMR (200MHz - DMSO-de) : δ 2.85 (s, 1 H); 3.7 (s, 3H); 6.35 (s, 2H); 7.37 (m, 2H); 8.23 (d, 1 H); 8.53 (d, 1 H); 8.62 (s, 2H) : 8.8 (s, 1 H) . ii) 1 -(3-Aminopropyl)-2,3,3-trimethyl-5-sulpho-3/7-indolium bromide a) 2,3,3-Trimethylindolenium-5-sulphate (20g, 84mmol) and N-(3- bromopropyOphthalimide (50g, 1 86mmols) were stirred in sulpholane (75ml) at 1 10°C for 28 hours. Further amounts of N-(3- bromopropyDphthalimide (2.5g, 84mmol) were then added after 28 hours and after 45 hours. The reaction mixture was heated for a total of 65 hours then cooled and added to ethyl acetate (800ml) forming a precipitate which was filtered and washed with ethyl acetate (3x300ml) and acetonitrile (5x400ml) to give a pink solid which was dried in vacua to give 2, 3, 3-trimethyl-1 -(3-phthalimidopropyl)-5-sulfo-indolium bromide (25g, 49mmols, 59%yield) . b) 2,3,3-Trimethyl-1 -(3-phthalimidopropyl)-5-sulpho-indolinium bromide (14g, 27.6mmols) was dissolved in concentrated HCI (100ml) with methanol (50ml) . The reaction mixture was stirred at 100°C for 32hrs. The reaction mixture was then cooled and the solvent removed under reduced pressure and dissolved in acetonitrile forming a precipitate. The solution was neutralised with concentrated ammonia ( ~ 10ml) and the precipitate was filtered, dissolved in a minimum amount of water and purified by running the solution through a 2g plug of C1 8 silica. The pink solid was freeze dried to give 1 -(3-aminopropyl)-2 3,3-trimethyl-5-sulpho- 3H-indolium bromide (8g, 21 .2mmols, 77%yield) . UV (methanol): λmax = 254nm. MS: m/z = 294 {C14H21N2O3S = 297) . 'H NMR (200MHz - DMSO-de) : δ 1 .5 (2s, 6H); 2.1 (m, 2H); 2.98 (s, 3H); 3.3 (m, 2H,); 3.6 (t, 2H); 6.6 (d, 1 H, J = 7.8Hz); 7.5 (m, 2H); 8.2 (dd, 1 H, J = 4 and 6Hz). iii) Synthesis of Compound V N,N'-Diphenylformamidine (236mg, 1 .2mmol) was added to a solution of 3-(3,5-dinitrobenzyl)-2-methyl-1 ,3-benzothiazol-3-ium iodide (500mg, 1 .09mmol) and 1 -(3-aminopropyl)-2,3,3-trimethyl-5-sulpho-3H- indolium bromide (41 2.5mg, 1 .09mmol) dissolved in pyridine (4.5ml), acetic acid (4.5ml) and acetic anhydride ( 1 .0ml) . The reaction was heated at 100°C for 3.5 hours then cooled to room temperature and purified by reverse phase HPLC. The pink solid isolated was then dissolved in aqueous hydrochloric acid (2.0M) and refluxed for 95 hours. The solution was then cooled to room temperature, evaporated and then re-purified by reverse phase HPLC. The pink solid collected was dried in vacuo. UV (ethanol): λmax = 559nm. MS: m/z = 638 (CsoHsoNsS.O? = 636.6) . Example 6 Preparation of 2-[3-(3-(5-carboxypentyl)-1 ,3-benzothiazol- 2(3A )-ylidene)-1 -propenyl]-3-(3,5-dinitrobenzyl)-1 ,3- benzothiazol-3-ium, salt (Compound VI) i) 3-(5-Carboxypentyl)-2-methyl-1 ,3-benxothiazol-3-ium bromide To 2-methylbenzothiazoie (80ml, 93.84g, 0.629mol) was added 6- bromohexanoic acid (250g, 1 .28mol) . The reaction mixture was stirred at 140°C for 24 hours. A yellow precipitate was formed. The reaction was allowed to cool, dissolved in methanol (350ml) and added dropwise to ethyl acetate (2 I) . A beige precipitate was collected and washed with ethyl acetate (3 x 50ml) . The solid was then dried to constant weight in vacuo to give 3-(5-carboxypentyl)-2-methyl-1 ,3-benxothiazol-3-ium bromide (182.35g 0.53mol, 84% yield) . UV (methanol): λmax = 237, 277nm. MS: gave m/z = 262 (C HisNO.S = 264). 1 H NMR (200MHz - DMSO-de) : δ 1 .49 (m, 4H); 1 .9 (m, 2H) ; 2.24 (t, 2H J = 6.35Hz); 3.32 (s, 3H); 4.72 (t, , 2H J = 7.81 Hz); 7.85 (m, 2H); 8.4 (dd, 2H, J = 8.31 Hz) . ii) 2-(2-Anilinoethenyl)-3-(3,5-dinitrobenzyl)-1 ,3-benzothiazol-3-ium, salt N,N'-Diphenylformamidine (42.9mg, 0.29mmol) was added to 3- (3,5-dinitrobenzyl)-2-methyl-1 ,3-benzothiazol-3-ium iodide d OOmg, 0.29mmol), (prepared as in Example 5 i)) and sodium acetate (excess) in ethanol (1 ml) . The reaction was stirred at 40°C for 4 hours to give a red solution. The solvent was removed under reduced pressure and the residue was purified by reverse phase HPLC to yield an orange gum. iii) Synthesis of Compound VI 3-(5-Carboxypentyl)-2-methyl-1 ,3-benzothiazol-3-ium bromide (7.9mg, 0.02mmol) was added to 2-(2-anilinoethenyl)-3-(3,5- dinitrobenzyl)-1 ,3-benzothiazol-3-ium salt (10mg, 0.02mmol) and sodium acetate (excess) in ethanol ( 1 ml) . The reaction was stirred at 45°C and a pink solution was observed. The solvent was removed in vacuo and the residue was purified by reverse phase HPLC to yield a dark pink solid. UV (water/acetonitrile) : λmax = 558nm. MS: m/z = 604 (C3oH 2 N4θeS2 = 603.7) . Example 7 Preparation of 3-[4-(carboxymethyl)benzyl]-2-{3-[1 -(3,5- dinitrobenzyl)-3,3-dimethyl-5-sulpho-1 ,3-dihvdro-2//-indol-2- ylidene]-1 -propenyl)-1 ,3-benzoxazol-3-ium, salt (Compound VII) i) 3-[4-(Carboxymethyl)benzyl]-2-methyl- 1 ,3-benzoxazol-3-ium bromide 2-Methylbenzoxazole (yellow) was distilled under vacuum to give a colourless liquid (33ml) . 4-(Bromomethyl)phenylacetic acid (20g, 87.3mmol) and 2-methylbenzoxazole (distilled, 33ml, 278mmol) were dissolved in 1 ,2-dichlorobenzene (70ml) . The reaction mixture was heated at 80°C for 2.5 days to produce a thick yellow precipitate. The reaction mixture was then cooled to room temperature, the precipitate collected via filtration and washed with 1 ,2-dichlorobenzene (3x100ml) and diethyl ether (4x200ml) . The cream solid collected was dried in vacuo to give 3- [4-(carboxymethyl)benzyl]-2-methyl-1 ,3-benzoxazol-3-ium bromide (20g, 55.3mmol, 63% yield) . UV (methanol) : λmax = 277nm. MS: m/z = 282 (C17H16NO3 = 282.32) . Normal phase TLC (ether) indicated that the starting 2-methylbenzoxazole had an Rt = 0.9 ± 0.05 and the product an Rt = 0. 1 H NMR (200MHz - DMSO-de) : δ 1 .75 (s, 2H); 3.2 (s, 3H); 5.85 (s, 2H); 7.31 (d, 2H, J = 6.8Hz); 7.53 (d, 2H, J = 6.8Hz); 7.72 (m, 2H, J = 6.3Hz); 7.89 (d, 1 H, J = 8.3Hz); 8.1 7 (d, 1 H, J = 7.3Hz); 9.8 (s, 1 H). 1 -(3,5-Dinitrobenzyl)-5-sulpho-2,3,3-trimethyl-3r/-indolium bromide 3,5-Dinitrobenzyl chloride (22.1 g, 102mmoi) and sodium bromide (10.5g, 102mmol) were added to 2,3,3-trimethyl-5-sulpho-indole, potassium salt (5. Og, 20.4mmol) in sulpholane (25ml) . The reaction mixture was stirred at 100°C for 5 hours. The solution was then cooled to room temperature and acetone (300ml) was added to the reaction flask. A brown precipitate was formed which was collected by filtration and then purified by reverse phase HPLC. This gave a beige solid which was dried in vacuo. UV (water/acetonitrile) : λmax = 283nm. MS: m/z = 420 (C1-H18N3O7S = 420.4) . iii) 2-{2-Anilinoethenyl)-3-[4-(carboxymethyl) benzyl]- 1 ,3-benzoxazol-3- ium, salt Ethyl N-phenylformimidate (0.94g, 6.27mmol) was added to 3-[4- (carboxymethyl)benzyl]-2-methyl-1 ,3-benzoxazole-3-ium bromide (1 .Og, 2.76mmol) dissolved in 2-methoxyethanol (10ml) and heated at 100°C for 1 .5 hours. The solution was then cooled to room temperature and purified by reverse phase HPLC. The bright yellow solid obtained was dried in vacuo. iv) Synthesis of Compound VII 1 -(3,5-Dinitrobenzyl)-2,3,3-trimethyl-5-sulpho-_?/V-indolium bromide (1 30mg, 0.26mmol) was added to 2-(2-anilinoethenyl)-3-[4- (carboxymethyl)benzyl]-1 ,3-benzoxazol-3-ium bromide (100mg, 0.26mmol) dissolved in pyridine and acetic anhydride (20:0.6, 1 0ml) . The reaction was kept in the dark for 24 hours at room temperature. The solvent was then removed and the product was isolated after purification by reverse phase HPLC. UV (ethanol) : λmax = 51 2nm. MS: m/z = 714 (C36H31 N4O10S = 71 1 .7) . Example 8 Preparation of 1 -(5-carboxypentyl)-2-{3-[1 -(5-carboxypentyD- 3-methyl-5-nitro-1 ,3-dihydro-2 7-benzimidazole-2-ylidene]-1 - propenyl}-3-methyl-5-nitro-3r/-benzimidazol-1 -ium, salt (Compound VIII) i) 1 -(5-Carboxypentyl)-2,3-dimethyl-5-nitro-3r/-benzimidazol-1 -ium iodide 6-lodohexanoic acid (6.33g, 26.1 5mmol) was added to 2,3- dimethyl-5-nitrobenzimidazole (1 .0g, 5.23mmol) dissolved in sulpholane (10ml) and stirred for 24 hours at 100°C. The reaction mixture was then cooled to room temperature and added dropwise to ethyl acetate (200ml) . A yellow precipitate was formed which was collected by filtration and dried in vacuo. This gave 1 -(5-carboxypentyl)-2,3-dimethyl-5-nitro-3r/- benzimidazol-1 -ium iodide as a yellow solid (1 .77g, 4.09mmol, 78% yield). UV (ethanol) : λmax = 285nm. MS: m/z = 306 (Ci 5 H 2 oN 3 O4 = 306). Normal phase TLC (ethanol) indicated the starting material with Rt = 0.6 ± 0.05 and the product with Rt = 0. 1 H NMR (200MHz - DMSO-de) : δ 2.3 (m, 8H); 2.95 (s, 3H); 4.0 (s, 3H); 4.6 (t, 2H); 8.24 (d, 1 H, J = 9.28Hz); 8.5 (dd, 1 H, J = 9.28 and 1 .95Hz); 9.09 (d, 1 H, J = 1 .95Hz) . ii) 2-(2-Anilinoethenyl)-1 -(5-carboxypentyl)-3-methyl-5-nitro-3r/- benzimidazol-1 -ium, salt Ethyl N-phenylformimidate (384.9mg, 2.58mmol) was added to 1 - (5-carboxypentyl)-2,3-dimethyl-5-nitro-3A -benzimidazol-1 -ium iodide (500mg, 1 .29mmol) dissolved in 2-methoxyethanol (5ml) . The reaction was heated at 100°C for 1 hour, cooled to room temperature and purified by reverse phase HPLC. The yellow solid was dried in vacuo for reacting further. iii) Synthesis of Compound VIII 1 -(5-Carboxypentyl)-2,3-dimethyl-5-nitro-3H-benzimidazol-1 -ium iodide (9.4mg, 0.024mmol) was added to 2-(2-anilinoethenyl)-1 -(5- carboxypentyl)-3-methyl-5-nitro-3 -benzimidazol-1 -ium salt (1 Omg, 0.024mmol) dissolved in pyridine, acetic anhydride and triethylamine (20:0.6:0.7, 2ml) . The reaction was kept in the dark for 24 hours. The solvent was then evaporated and the product was isolated after purification by reverse phase HPLC. UV: λmax = 535nm. MS: m/z = 622 (C31 H37N6O8 = 621 .6) . Example 9 Preparation of 2-{5-[1 -(5-carboxypentyl)-3,3-dimethyl-5,7- disulpho-1 ,3-dihydro-2//-benzo[e]indol-2-ylidene]-1 ,3- pentadienyl)-3-(3,5-dinitrobenzyl)-1 ,3-benzothiazol-3-ium, salt (Compound IX) i) 1 (5-Carboxypentyl)-2,3,3-trimethyl-5,7-disulpho-3r/- benzo[e]indolium bromide 6-Bromohexanoic acid (30g, 0.1 53mol) was added to 2,3,3- trimethyl-3 y-benzo[e]indole-5,7-disulphonic acid (50g, 0.1 1 2mol) in nitrobenzene (200ml) and stirred for 24 hours at 1 20°C. The reaction mixture was cooled to room temperature. A brown precipitate was formed which was collected by filtration and washed with 2-propanol (250ml) . The solid was then recrystallised from 2-propanol. The taupe solid was filtered and purified by reverse phase HPLC. This gave a grey solid which was dried in vacuo. UV (water/acetonitrile) : λmax = 273nm, 283nm. MS: m/z = 484 (C-iH.eNOsS- = 484.5) . 'H NMR (200MHz- DMSO-de) : δ 1 .7 (m 14H) ; 2.2 (t, 2H); 2.9 (s, 2H); 8.2 (d, 1 H, J = 9.28Hz); 8.4 (d, 2H, J = 4.39Hz), 9.1 5 (d, 1 H, J = 9.28Hz) . ii) Synthesis of Compound IX Malonaldehydebisphenylimine monohydrochloride (141 mg, 0.55mmol) was added to 1 -(5-carboxypentyl)-2,3,3-trimethyl-5,7-disulpho- 3/Y-benzo[e]indolium bromide (286mg, 0.55mmol) and 3-(3,5- dinitrobenzyl)-2-methyl-1 ,3-benzthiazole-3-ium iodide (200mg, 0.55mmol), (prepared as in Example 5 i)) dissolved in pyridine (1 8ml), acetic acid (1 8ml) and acetic anhydride (4ml) . The reaction was stirred at 80°C for 4 hours and cooled to room temperature. The solvent was evaporated and the mixture was purified by reverse phase HPLC. The product, obtained as a blue solid, was dried in vacuo. UV (ethanol) : λmax = 676nm. MS: m/z = 853 (C 39 H37N4θι 2 S3 = 849.9) . Example 10 Preparation of 1 -butyl-2-[7-(3-(5-carboxypentyl)-1 ,3- benzothiazol-2-ylidene)-1 ,3,5-heptatrienyl]-3,3-dimethyl-5- nitro-3A/-indolenium, salt (Compound X) i) Synthesis of Compound X N-[5-(Phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (1 8mg, 0.064mmol) was added to 1 -butyl-2,3,3-trimethyl-5-nitro-3/¥- indolium iodide (25mg, 0.064mmol) and 3-(5-carboxypentyl)-2-methyl-1 ,3- benzothiazol-3-ium bromide (22mg, 0.064mmol) in acetic acid (0.5ml) and pyridine (0.5ml) . The reaction was heated at 40°C for 4 hours. The solution was then cooled to room temperature and the solvent evaporated under reduced pressure. The dark green residue was dissolved in dimethyl sulphoxide and purified by reverse phase HPLC to yield a green solid. UV (methanol) : λmax = 768nm. MS: m/z = 587 (C34H40N3O4S = 586.7) . Example 1 1 Preparation of 2-{5-[1 -(5-carboxypentyl)-3,3-dimethyl-5- sulpho-1 ,3-dihydro-2r/-indol-2-ylidene]-1 ,3-pentadienyl}-1 (3,5-dinitrobenzyl)-3,3-dimethyl-5-sulfo-3A -indolium, salt (Compound XI) i) Synthesis of Compound XI The reaction was carried out by an analogous method to that reported for Compound IX using 1 -(3,5-dinitrobenzyl)-2,3,3-trimethyl-5- sulfo-3r/-indolium bromide (50mg, O.1 2mmol) and 1 -(5-carboxypentyl)- 2,3,3-trimethyl-5-sulfo-3/¥-indolium bromide (43mg, 0.1 2mmol) in the presence of malonaldehydebisphenylimine monohydrochloride (32mg, 0.1 2mmol) in acetic acid (0.9ml) , pyridine (0.9ml) and acetic anhydride (0.2ml) . The product was isolated as a blue solid. UV (ethanol) : λmax 651 nm. MS: m/z = 81 1 (C 38 H4iN4θi2S 2 = 809.9) . Example 1 2 Relative Fluorescence Emission of Non-fluorescent Cyanine Dyes Compounds V, VII, IX and XI were prepared as solutions in ethanol (5x10 "δ M) . For comparison, solutions of the fluorescent dyes Cy3 and Cy5 were prepared in ethanol at the same concentration. The fluorescence intensity of each of the dye solutions was measured using a fluorescence plate reader. Serial dilutions ( 1 : 1 ) with ethanol of all dye solutions were then made and the fluorescence intensity measured for each dilution. The fluorescence emission of the non-fluorescent cyanine dyes relative to the normal fluorescent cyanine dyes is shown in Figure 1 . Example 1 3 Nucleic Acid Hybridisation Assay 1 3.1 Probe Preparation Oligonucleotide A (5'-C6-amιno modmer-TAC CCA GAC GAG CAA- bιotιn-3') and the complementary oligonucleotide B (5'-TTG CTC GTC TGG GTA-C7-amιno modιfιer-3') were synthesised on an Applied Biosystems 391 DNA synthesiser using standard methods and materials. The oligonucleotides were deprotected for 1 7 hours at 40°C and purified by reverse phase HPLC using C1 8 column and a 40% TEAA/acetonitπle gradient. The desired peaks were collected, freeze dried and the samples were resuspended in sterile H O. Oligonucleotides A and B were incubated with a 10-fold molar excess of Cy3 NHS-ester dye and 2-{5-t1 -(5-carboxypentyl)-3,3-dιmethyl- 5-sulpho-1 ,3-dιhydro-2/Y-ιndol-2-ylιdene]-1 ,3-pentadιenyl}-1 -(3,5- dιnιtrobenzyl)-3,3-dιmethyl-5-nιtro-3/V-ιndolιnιum salt (Compound ID-NHS- ester respectively, in 0.1 M sodium bicarbonate buffer (pH9) , overnight at 22°C. The following morning, the oligonucleotides were precipitated using ethanol and the resulting pellets were resuspended in water. Labelled oligonucleotides were purified by reverse phase HPLC using a C1 8 column and a 60% TEAA/acetonitrile gradient and the desired peaks collected and freeze dried. Residues were resuspended into H_O and concentration of recovered material was determined. A control oligonucleotide C (5'-TTG CTC GTC TGG GTA-Dabcyl-3') was synthesised as above. The DABCYL moiety was added using a 3'- Dabcyl cpg column. Following synthesis, oligonucleotide C was deprotected for 1 7 hours at 40°C and purified by HPLC as described. 13.2 Binding Assay Wells of a black, streptavidin-coated 96-well plate were coated with Cy3-labelled oligonucleotide A d OOpmol/well diluted in 100μl PBS/1 MgCb) for 120 minutes at ambient temperature. Any unbound material was removed by washing wells vigorously with buffer (PBS/1 mM MgCI 2 /0.1 % BSA). The Compound ll-labelled oligonucleotide B and Dabcyl-labelled oligonucleotide C were diluted to 0.2pmol/μl in buffer, and 100μl was incubated with the coated wells at ambient temperatures for 1 20 minutes. Wells were washed vigorously with PBS and fluorescence intensity was measured on a fluorescence plate reader using filter sets appropriate for Cy3. Signals from wells coated with Cy3-labelled oligonucleotide A alone were compared with those coated with Cy3-oligonucleotide A and incubated with either Compound ll-labelled oligonucleotide B or control oligonucleotide C. The results are shown in Figure 2. Wells coated with Cy3-oiigonucleotide A, gave a strong fluorescence signal. This signal was reduced by 95% following hybridisation with Compound ll-labelled oligonucleotide B and by 75% with oligonucleotide C. Example 14 Use of Non-fluorescent Cyanine Dyes Compared with a Standard Matched Fluorescent Cyanine Acceptor Dye (Cy5) in an Oligonucleotide Hybridisation Study An oligonucleotide (5'-TAC-CCA-GAC-GAG-CAA-3') , labelled at the 3'- terminus with biotin and at the 5'-terminus with Cy3, was bound to the wells of a microtitre plate coated with streptavidin. The coated wells were then incubated with complementary oligonucleotide probes labelled individually with non-fluorescent cyanine acceptor dyes (Compound II and Compound XI) . Following hybridisation, the wells were washed and the relative fluorescence measured upon excitation of the Cy3 donor dye. Control measurements were made on complementary unlabelled-, Cy5- labelled and 2,4-dinitrobenzoyl-labelled oligonucleotides. The results are shown in Figure 3. The data indicates that the use of the non-fluorescent cyanine dyes (Compound II and Compound XI) is able to increase the signal to noise ratio by approximately a factor of 2 over that obtained with Cy5. Example 1 5. Protease Cleavage Assay 1 5.1 Preparation of Protease Substrate The peptide Ala-Ala-Phe-Phe-Ala-Ala-Lys was synthesised on a Perkin Elmer 433A peptide synthesizer using standard Fmoc chemistry. Cy5-NHS-ester dye (in slight molar excess) was coupled to the N-terminus of the peptide on the resin during overnight incubation in DMSO containing 2% v/v diisopropyl-ethylamine. The labelled peptide resin was washed sequentially with DMSO ( ~ 10 ml), methanol ( ~ 1 0 ml) and dichloromethane ( ~ 5ml) and dried . A 90 minute incubation in 95%TFA/2.5% triisopropylsilane/2.5% H 2 O facilitated side-chain deprotection and cleavage from the resin. After filtering through glass wool the peptide was isolated as a blue precipitate in ice-cold diethylether. The product was re-suspended in DMSO and purified by reverse-phase HPLC (gradient: water + 0.1 %TFA to 70% MeCN + 0.1 % TFA), the desired fractions being collected and freeze-dried. Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys was incubated with 1 -butyl-2-{5- [1 -(5-carboxypentyl)-3,3-dimethyl-6-sulpho-1 ,3-dihydro-2H-benzo[e]indol- 2-ylidene]-1 ,3-pentadienyl}-3,3-dimethyl-5-nitro-3 y-indolinium salt (Compound III), NHS-ester, in slight molar excess in DMSO containing 2% v/v diisopropylethylamine overnight. The dual-labelled peptide was again isolated by reverse-phase HPLC and freeze-dried. The residue was resuspended into H 2 O and the concentration determined . 1 5.2 Assay for Protease Enzyme The protease substrate (Cy5-Ala-Ala-Phe-Phe-Ala-Ala-Lys-Compound III) was diluted to 0.02 absorbance units/cm (at 650nm) in citrate buffer d OOmM pH 3.0) . To the solution ( 1 ml) of the labelled substrate in a cuvette, 25ng pepsin ( 10μl volume) was added . The fluorescence baseline prior to protease addition and the subsequent increase in intensity were recorded at appropriate wavelengths. A control experiment (minus pepsin) was performed in a similar manner. The results are shown in Figure 4. Efficient quenching of Cy5 fluorescence in the intact substrate results in a low signal. Protease-catalysed hydrolysis of the substrate removes this quenching, restoring the Cy5 fluorescence. The increase in fluorescence intensity can be continuously monitored and is proportional to protease activity.

Claims: Claims A compound having the formula: wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y; groups R 3 , R 4 , R 5 and R 6 are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z 1 and Z 2 ring structures; Z 1 and Z 2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Y are the same or different and are selected from bis-Cι-C 4 alkyl- and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium, -CH = CH- and N-W wherein N is nitrogen and W is selected from hydrogen, a group -(CH 2 ) R 8 where m is an integer from 1 to 26 and R 8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups; at least one of groups R\ R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a target bonding group; any remaining groups R 3 , R 4 , R 5 , R 6 and R 7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR 9 , COOR 9 , nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R 9 is selected from H and C1-C4 alkyl; any remaining R 1 and R 2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent; provided that the linker group Q is not a squaraine ring system. 2. A compound according to claim 1 wherein Q contains 1 , 2 or 3 double bonds in conjugation with the rings containing X and Y. 3. A compound according to claims 1 or 2 wherein Q is the group: wherein the groups R 10 are selected from hydrogen and C1-C4 alkyl which may be unsubstituted or substituted with phenyl, or two or more of R 10 together with the group form a hydrocarbon ring system substituted with R 7 and which may optionally contain a heteroatom selected from -O-, -S-, or >NR 7 , wherein R 7 is hereinbefore defined; and n = 1 , 2 or 3. 4. A compound having the formula: wherein groups R 3 , R 4 , R 5 and R 6 are attached to the rings containing X and Y or, optionally, are attached to atoms of the Z 1 and Z 2 ring structures and n is an integer from 1 -3; Z 1 and Z 2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Y are the same or different and are selected from bis-d-C4 alkyl- and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium, -CH = CH- and N-W wherein N is nitrogen and W is selected from hydrogen, a group -(CH 2 ) m R 8 where m is an integer from 1 to 26 and R 8 is selected from hydrogen, ammo, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted ammo, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with ammo, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups; at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is a target bonding group; any remaining groups R 3 , R 4 , R 5 , R 6 and R 7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR 9 , COOR 9 , nitro, ammo, acylammo, quaternary ammonium, phosphate, sulphonate and sulphate, where R 9 is selected from H and C1-C4 alkyl; any remaining R 1 and R 2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups; characterised in that at least one of the groups R 1 , R 2 , R 3 , R 4 , R 6 , R 6 and R 7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent. 5. The compound according to any of claims 1 - 4 wherein said target bonding group comprises a reactive group for reacting with a functional group on a target material, or a functional group for reacting with a reactive group on a target material. 6. The compound of claim 5 wherein said reactive group is selected from carboxyl, succinimidyl ester, sulpho-succmimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite. 7. The compound of claim 5 wherein said functional group is selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate. 8. A compound according to any one of claims 1 -7 wherein at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 has the structure -E-F where E is a spacer group and F is the reactive or functional group. 9. A compound according to any one of claims 1 -8 wherein the spacer group is a chain of 1 -60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus. 10. The compound according to claim 1 -9 wherein at least one of groups R 1 , R 2 , R 3 , R 4 , R 6 , R 6 and R 7 comprises at least one nitro group. 1 1 . A biological material labelled with a non-fluorescent cyanine dye according to any one of claims 1 -10. 1 2. A biological material which comprises two components one of which is labelled with a fluorescent dye which may act as a donor of resonance energy and the other with a non-fluorescent cyanine dye according to any one of claims 1 -10 and which may act as an acceptor of resonance energy transferred from the donor. 1 3. A biological material according to claims 1 1 or 1 2 selected from the group consisting of antigen, antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, microbial materials, drugs and toxins. 14. A method for detecting the presence of a biological material which method comprises use of a compound according to any one of claims 1 - 10. 15. A method for labelling a biological material comprising: i) adding to a liquid which contains a biological material selected from the group consisting of an antigen, antibody, lipid, protein, peptide, carbohydrate, nucleotides, microbial materials, drugs, toxins and combinations thereof a compound according to any one of claims 1 -10; ii) reacting said compound with said biological material wherein said compound covalently binds to and labels said biological material. 16. An assay method which comprises: i) separating two components which are in an energy transfer relationship, the first component being labelled with a fluorescent donor dye and the second component being labelled with a non-fluorescent cyanine acceptor dye according to any one of claims 1 -10 and, ii) detecting the presence of the first component by measuring emitted fluorescence. 1 7. A method according to claim 1 6 wherein the assay is selected from proteolytic enzyme cleavage assays and nuclease enzyme cleavage assays. 18. An assay method which comprises: i) binding one component of a specific binding pair with a second component of said pair, said first component being labelled with a fluorescent donor dye and said second component being labelled with a non-fluorescent cyanine acceptor dye according to any one of claims 1 -10 so as to bring about an energy transfer relationship between said first and second components; and, ii) detecting the binding of the first and second components by measuring emitted fluorescence. 1 9. A method according to claim 1 8 wherein said specific binding pair is selected from the group consisting of antibodies/antigens, lectins/glycoproteins, biotin/(strept)avidin, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein. 20. A method according to claim 1 8 wherein said binding assay is selected from the group consisting of immunoassays, nucleic acid hybridisation assays, protein binding assays, hormone receptor binding assays and enzyme assays.

Inventors: BIRCH MARTYN N, BOSWORTH NIGEL, HAMILTON ALAN L, HATCHER MALCOLM J, SCOTT BRIAN, BIRCH, MARTYN, N., BOSWORTH, NIGEL, HAMILTON, ALAN, L., HATCHER, MALCOLM, J., SCOTT, BRIAN
Applicants: AMERSHAM PHARM BIOTECH UK LTD, BIRCH MARTYN N, BOSWORTH NIGEL, HAMILTON ALAN L, HATCHER MALCOLM J, SCOTT BRIAN, AMERSHAM PHARMACIA BIOTECH UK LIMITED, BIRCH, MARTYN, N., BOSWORTH, NIGEL, HAMILTON, ALAN, L., HATCHER, MALCOLM, J., SCOTT, BRIAN
Application year: 1999
Main classification: C09B23
IPC codes: C07D209, C07D233, C07D277, C07D413, C07D417, C07H21, C09B23, G01N33