Indian Patents. 278323:"REAGENT FOR DETECTING AN ANALYTE"

Title of Invention	"REAGENT FOR DETECTING AN ANALYTE"
Abstract	A reagent for use in detecting an analyle comprises a fluorescent energy donor and an energy acceptor, wherein the energy acceptor is of the general formula (I) and wherein the distance between the energy donor and the energy acceptor of the reagent is capable of modulation by a suitable analyle to be detected.

Full Text	REAGENT FOR DETECTING AN ANALYTE The present invention relates to a reagent for use in detecting an analyte, to a dye compound suitable for use in such a reagent, to a method of detecting or measuring an analyte using such a reagent and to a complex of an analyte and such a reagent. The method of detecting or measuring an analyte relies on FRET (fluorescence resonance energy transfer). FRET is a distance-dependent interaction between the electronic excited states of two dye species in which excitation energy is transferred from a donor to an acceptor without emission of a photon. The efficiency of FRET is inversely dependent on the sixth power of 'the intermolecular separation. [Refs. 1. B. Wieb Van der Meer, G. Coker III, S.-Y. Simon Chen, Resonance energy transfer; Theory and data, VCH publishers, 1994; 2. Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Ed.), 2. edition, Plenum Press, New York 1999]. Detection of FRET can therefore be used to determine the distance between a species labelled with the donor and a species labelled with the acceptor. This may be used for example to determine whether the two species are bound to one another. The requirements for FRET are: - Donor and acceptor must be in close proximity (typically 10-100 A). - The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor. - Donor and acceptor transition dipole orientations must be approximately parallel. FRET causes a decrease in intensity and lifetime of donor fluorescence. If the acceptor is fluorescent, FRET may also cause an increase in intensity of acceptor fluorescence. Where the donor and acceptor are the same, FRET causes fluorescence depolarization. FRET may be detected by illuminating a sample with light in the absorption spectrum of the donor and measuring any of these properties. A simple FRET assay may be carried out by labelling an analyte with a donor and labelling an analyte binding agent with an acceptor (or vice versa) . When the analyte is not bound to the analyte binding agent, the distance between the donor and acceptor is large and no FRET occurs. When the analyte is bound to the analyte binding agent, the distance between the donor and acceptor is small.and FRET occurs. Therefore, detection of FRET may be used to determine whether analyte is present and/or analyte concentration. A competitive FRET assay may be carried out where labelled analyte competes with non-labelled analyte. In this case, as the concentration of non-labelled analyte increases, there will be less bound labelled analyte. This means that less FRET will occur. Therefore, again detection of FRET may be used to determine analyte concentration. An assay of this type may be used for the measurement of glucose concentration. Glucose binds reversibly 'to a class of proteins called lectins. Concanavalin A is an example of a lectin. The components of the assay are for example labelled concanavalin A (the analyte binding reagent) and labeled glucose or labelled glucose analogue. Dextran is a suitable glucose analogue. When glucose (the analyte) binds to concanavalin A, it displaces labelled glucose or dextran, and less FRET will occur. Alternatively, the donor and acceptor may be attached to the same species to form a single FRET reagent. An example of such a reagent is a "molecular beacon". A molecular beacon consists of a single stranded polynucleotide or polynucleotide analogue sequence with a donor attached to one end of the sequence. A complementary acceptor is attached to the other end of the sequence. The unbound molecular beacon exists as a stem-and-loop structure. The sequences at the ends of the molecular beacon match and bind, creating the stem, while the rest of the probe is unmatched and unbound, creating the loop. While folded this way, .the donor at one end of the probe is next to the acceptor at the other end. The proximity of the donor and acceptor allows FRET to occur. When the probe recognizes and binds to a target, the molecular beacon structure unfolds. This separates the acceptor from the donor so that FRET cannot take place. Therefore, FRET may be used to determine whether a particular target is present. Molecular beacons may be used to detect complementary single stranded polynucleotide sequences. Alternatively, they may be used to detect other non-nucleotide species. Such molecular beacons are called aptamers. For example, aptamers may be used to detect proteins. The molecular beacon has the potential disadvantage that it relies on FRET occurring while the beacon is unbound. During an in vivo assay the beacon sequence may be cleaved by enzymes in the body. This will reduce the FRET. The reduction in FRET caused by beacon degradation cannot be distinguished from the reduction in FRET caused by beacon binding to the target, and the results obtained may therefore be unreliable. An alternative to a molecular beacon which deals with this problem is a dual probe. This comprises two single stranded polynucleotide or polynucleotide analogue sequences, one labelled with an energy donor and one with a complementary energy acceptor. The dual probe is used to detect a single stranded polynucleotide target sequence which is complementary to the sequences of both parts of the dual probe. When the parts of the dual probe are both bound to the target, FRET occurs. Since FRET does not occur when the dual probe is unbound, dual probe degradation will not affect the occurrence of FRET. Therefore, degradation- will not cause the results to be unreliable. Reagents similar to molecular beacons can be formed from polypeptides.FRET can also be used in immunoassays. The choice of donor and acceptor used in FRET is important, and the following factors need to be taken into account. The donor should have a high quantum yield. A suitable acceptor must have an absorption spectrum overlapping the emission spectrum of the donor. For example, QSY 21™ is a suitable donor for use with Alexa Fluor 21™. It is desirable for an acceptor to have a wide absorption spectrum so that it can be used with a variety x>f donors. If the acceptor is also a fluorescent species it is possible that background fluorescence resulting from direct acceptor excitation will occur and be detected as well as donor fluorescence. Filtering of the acceptor emission may result in loss of the donor fluorescence signal where filtering is not sharp enough. Non-fluorescent acceptors such as dabcyl and QSY can be used to avoid this problem. Alternatively, the problem can be avoided by choosing an acceptor whose emission spectrum is remote from that of the donor. The emission spectrum of some donors and absorption spectrum of some acceptors (for example QSY 21) are shifted when the dor.or or acceptor is conjugated to an analyte or analyte binding agent. This is undesirable. Where FRET assays are to be used in vivo, it is desirable for donors to fluoresce at 550 to around 600 nm and for acceptors to absorb light at around 650 nm. This avoids overlap between the donor fluorescence and in vivo autof luorescence at lower wavelengths. '"" Fluorescein fluoresces at 495 nm and is therefore not ideal for in vivo use. Alexa Fluor 594 is a dye with a suitable emission spectrum for use in vivo. This dye absorbs at 594 nm and fluoresces at 620 nm. The present inventors have now provided a new class of acceptors (HMCV dyes) for use in FRET. The acceptors are stabilized carbenium ions which are structurally related to crystal violet and malachite green. The acceptors can be derived from intermediates in the synthesis of trioxatriangulenium systems described in Bo W. Laursen et al., J. Am. Chem. Soc., 1998, 120, 12255-12263. Accordingly, the present invention provides in a first aspect a reagent for use in detecting an analyte, comprising a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula: wherein: R1, R2 and R3 are each independently H, electron donating substituents, or electron withdrawing substituents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups; R4, R5, Rs, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, 0-alkyl, S-alkyl and R , R1- ,12 R13, R14 and R15 are each independently hydrogen, 0-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and Rfi and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R and R10 and/or R5 and R11 and/or Rfi and R13 and/or R7 and R13 and/or Ra and R1 and/or R9 and Ris is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of S03", P03 OH, 0-alkyl, SH, S-alkyl, COOH, COO', ester, amide, halogen, SO-alkyl, S02-alkyl, S02NH2, S02NH-alkyl, S02Ndialkyl, SO3-alkyl, CN, secondary amine or tertiary araine, provided that not all of R10, R11, R12, R13, R14 and R1S are hydrogen; and wherein the distance between the energy donor and the energy acceptor of the reagent is capable of modulation by a suitable analyte to be detected. As used herein, the term "electron donating substituent" includes but is not limited to amino, primary amine, secondary amine, alkyl, 0-alkyl, S-alkyl, amide (NHCOR), ester (OCOR), OH, SH and electron rich aryl. As used herein, the term "electron withdrawing substituent" includes but is not limited to NO, N02, CN, COOH, ester (COOR) , COO", amide (CONR2) , CHO, keto (COR), SOalkyl, S02-alkyl, S02NH2, S02NH-alkyl, S02N-dialkyl, SO3- alkyl, and electron deficient aryl. Electron rich/deficient aryls may be defined as aryls having electron-donating or -withdrawing substituents respectively. As used herein, the term "alkyl" and "alkylene" include linear, branched and cyclic groups, saturated and unsaturated groups (including groups containing triple bonds), groups containing one or more substituents, and groups containing one or more heteroatoms. Ci to Cg alky groups are preferred. As used herein, the term "0-alkylene", "N-alkylene" and "S-alkylene" each include groups wherein the heteroatom is at any position within the group. Ci to C7 O-alkylene, Nalkylene and S-alkylene and C2 to C7 alkylene are preferred. Where an ester substituent is referred to, the substituent may be linked via the carbonyl portion (as R(C=0)OR) or via the alkoxy portion, (as RO(C=0)R). Similarly, where an amide substituent is referred to, the substituent may be linked via the carbonyl portion (as RCONR2) or the nitrogen portion (as R(NR)COR). In one preferred embodiment, the energy donor and energy acceptor are linked together by non-covalent binding. The non-covalent binding may exist between an analybe binding agent linked to one of the energy donor and the energy acceptor and an analyte analogue linked to the other of the energy donor and the energy acceptor, the non-covalent binding being disruptable by a suitable analyte so as to increase the distance between the energy donor and the energy acceptor of the reagent. Suitably, in this embodiment the analyte binding agent is a lectin and/or the analyte analogue is a glucose analogue. For example, the analyte analogue may be dextran. This system can be used for glucose detection and/or measurement as discussed above. It is preferable for the lectin to be bound to the energy donor and the analyte analogue to be bound to the energy acceptor (e.g. Dextran-HMCV and Concanavalin A-Alexa Fluor 594) . Dextran can be more heavily labelled than Concanavalin A. If dextran is labelled with a fluorophore there will be excess fluorescence which dilutes the signal in a lifetime based assay. In an alternative embodiment, the energy donor and energy acceptor are linked together by a covalent linkage. The covalent linkage between the energy donor and energy acceptor may be cleavable to increase the distance between the energy donor and the energy acceptor of the reagent. Suitably, the energy donor and energy acceptor are linked via a polynucleotide sequence or a polynucleotide analogue sequence or a polypeptide sequence, the sequence having a conformation which is capable of modulation by a suitable analyte to be detected so as to modulate the distance between the energy donor and the energy acceptor of the reagent. In this case, the reagent is a "molecular beacon" as discussed above. In a further alternative embodimejit, the energy donor and the energy acceptor are not linked in the absence of analyte. This reagent may be the "dual probe" discussed above. Preferably, a linker structure is attached to the energy acceptor at R3, or where a bridging group is present optionally the linker structure is attached to the energy donor at the bridging group. Preferably, the electron donating substituents are selected from amino, primary amine, secondary amine, O-alkyl, alkyl, S-alkyl, amide, ester, OH and SH. Preferably, one or more of R1 to R3 is dimethylamino, diethylamino or methylethylamino, optionally substituted with one or more of S03", P03 2", OH, O-alkyl, SH, S-alkyl, COOK, COO", ester, amide, halogen, SO-alkyl, SO2-alkyl, S02NH2, S02NH-alkyl, S02N-dialkyl, and S03-alkyl, CN, secondary amine or tertiary amine. In a preferred embodiment, R1 and R2 are each dimethylamino. Alternatively, R1 and R2 may each be optionally substituted methylethylamino. Preferably, an electron withdrawing substituent is present, and the electron withdrawing substituent is selected from NO, N02, CN, COOH, ester, COO", amide, CHO, keto, SOalkyl, SO2-alkyl, S02NH2l SOjNH-alkyl, S02N-dialkyl, and S03- alkyl. Preferably, at least one of R10, R11, R12, R13, R14 and R" is O-alkyl. Suitably, one or more pairs of groups R4 and R10 and/or R5 and R11 and/or Rs and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene , O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of S03~, PO3 2~, OH, O-alkyl, SH,' S-alkyl, COOH, COO', ester, amide, halogen, SO-alkyl, S02-alkyl, SO2NH2, SO2NH-alkyl, S02N-dialkyl, CN, secondary amine or tertiary amine. Preferably, R10 to R1S are each 0-methyl or O-ethyl. Preferably, the reagent further comprises one or more counterions selected from halide, BF4", PF6", NOa", carboxylate, ClO4", Li+, Na+, K, Mg2 and Zn2+ so that the reagent is uncharged overall. Preferably, a linker structure is present, and is formed by reaction of a linker element selected from an active ester, an isothiocyanate, an acid chloride, an aldehyde, an azide, an a-halogenated ketone and an amine with a reaction partner. Suitably, the reaction partner is selected from a polysaccharide, polynucleotide or a protein. Suitably, the linker element is an active ester, and is selected from succinimidyl and pentafluorophenyl active esters. Suitably, the energy donor is Alexa Fluor 594W. The reagent may be used for in vivo detection of an analyte. For example, the reagent may be contained within a sensor which is implanted into the body. Suitably, the sensor is implanted within the epidermis so that it will be shed from the body over time. Suitable sensor constructions are described in detail in W002/30275. In this case, it should be possible to illuminate the reagent and measure reagent fluorescence through the layers of the skin above the implanted sensor. Alternatively, the reagent may be contained within a sensor which is partially implanted within the body but extends outside the body. Such a sensor may take the form of a needle. In this case, the reagent may be illuminated down the bore of the needle and reagent fluorescence may be detected in the same way without light passing through layers of the skin. The sensor should be permeable to analyte. In this second aspect, the invention relates to a sensor comprising a semi-permeable membrane enclosing a reagent for use in detecting an analyte as described above. In a third aspect, the present invention relates to a dye compound having the general formula: wherein: R, R5, Rs, R7, Ra and R9 are each independently H, halogen, alkyl, aryl, 0-alkyl or S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, 0- alkyl, S-alkyl, or alkyl, or one or more pairs of groups R20 and R and/or R20 and R5 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, o-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3", PO3 OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide, halogen, SO-alkyl, S0z-alkyl, S02NH2, S02NH-alkyl, SO2Ndialkyl, S03-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen; R1S, R17, R18 and R19 are each independently H, alkyl (preferably Ci to Cs alkyl) or aryl, or one or more of Rls and R17 or Ria and R19 is alkylene (preferably C3 to C7 alkylene) optionally substituted with one or more of S03", P03 2', OH, 0-alkyl, SH, S-alkyl, COOH, COO', ester, amide, halogen, SO-alkyl, S02-alkyl, S02NH2, SO2NH-alkyl, S02N-dialkyl, S03-alkyl, CN, secondary amine or tertiary amine; or one or more of pairs of groups R6 and R16, R7 and R17, R8 and Ria and R9 and R19 is alkylene, 0-alkylene, Salkylene or N-alkylene optionally substituted with one or more of S03", P03 2", OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide, halogen, SO-alkyl, S02-alkyl, S02NH2, S02NH-alkyl, S02N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine and R20 is a linker element selected from an active ester, an isothiocyanate, an acid chloride, an a-halogenated ketone, an azide and an amine. Suitably, at least one of R10, R11, R12, R13, R14 and R15 is alkyl. In a preferred embodiment, R16, R17, R16 and R19 are each methyl or ethyl. Alternatively, R16 and R18 may each be methyl and R17 and R19 may each be ethyl substituted at the 2- position with S03". Suitably, one or more pairs of groups R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of S03~, P03 2', OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide, halogen, SO-, S02NH2, S02NH-alkyl, S02N-dialkyl, CN, secondary amine or tertiary amine. Preferably, R20 is a linker element having the structure: wherein R21 is H or alkyl or aryl optionally substituted with one or more of S03", P03 2~, OH, 0-alkyl, SH, Salkyl, COOH, COO", ester, amide, halogen, -SO-alkyl, SO2N-dialkyl, CN, secondary amine or tertiary amine and R22 is alkylene, O-alkylene, S-alkylene or N-alkylene (preferably C2 to Cs) or R21 and R22 are part of a ring (preferably C3 to C7) , optionally substituted with one or more of S03~, P03 2', OH, O-alkyl, SH, S-alkyl, COOH, COO", ester, amide, halogen, SO-alkyl, S02NH2, S02NHalkyl, S02N-dialkyl, S03-alkyl, CN, secondary amine or tertiary amine; and R23 is o-succinimidyl, o-pentaf luorophenyl, Cl or ahalogenated alkyl. Preferably, R1C to R15 are each 0-methyl or O-ethyl. Preferably, the dye compound further comprises one or more counterions selected from halide, BF4", PF6~, N03", carboxylate, C1O4", Li+, Na, K, Mg2t and Zn2+. In a fourth aspect, the present invention relates to a method of detecting or measuring an analyte using a reagent as described above, comprising the steps of: contacting the reagent with a sample; illuminating the reagent and sample with light of wavelength within the absorption spectrum of the energy donor; detecting non-radiative energy transfer between the energy donor and energy acceptor by measuring the fluorescence of the energy donor; and associating the fluorescence measurements with presence or concentration of analyte. Suitably, - the fluorescence of the energy donor is measured by intensity based or time resolved fluorescence measurements. Preferably, the analyte is measured by comparing sample fluorescence measurements with fluorescence measurements made using known concentrations of analyte. The light used for illumination preferably has a wavelength above 550 nm. In a fifth aspect, the present invention relates to a complex of an analyte and a reagent for detecting the analyte wherein the reagent comprises a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula: wherein: R1, R2 and R3 are each independently H, electron donating substltuents, or electron withdrawing substltuents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups; R, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, 0-alkyl, S-alkyl and R10, R11, R12, R13, R14 and R1S are each independently hydrogen, O-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R and R10 and/or R5 and R11 and/or Rs and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, 0-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of S03", P03 OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide, 17 halogen, SO-alkyl, S02-alkyl, S02NH2, SOjNH-alkyl, S02Ndialkyl, and S03-alkyl, CN, secondary amine or tertiary amine, provided that not all of Rxo, R11, R12, R13, R14 and R15 are hydrogen; and wherein the presence of the analyte modulates the distance between the energy donor and the energy acceptor. The invention will be further described with reference to preferred embodiments illustrated by the Examples, and as illustrated in Figure 1, which shows a glucose dose-response curve, and Figure 2, which shows normalised absorption and emission spectra of Alexa Fluor 594 and HMCV-1-dextran in aqueous PBS buffer 50 tnM, pH=7.4 (Exitation of AF594 at 570 nm in a 0.7 uM solution). Example Preparation of Dye Compound 4 Synthetic Methods and Materials All reagents used were standard grade unless otherwise mentioned. Ether and THF were dried by distillation from sodium/benzophenone under nitrogen. Benzene was dried by standing over sodium. NMR spectra were on a 250 or 400 MHz spectrometer. FABMS spectra were obtained using 1,4-dicyanobutane as matrix. Elemental analysis was done at the University of Copenhagen, Department of Chemistry, Elemental Analysis Laboratory, Universitetsparken 5, 2100 Copenhagen, Denmark. Tris (2,4, 6-trimethoxyphenyl) carbenitun Tetrafluoroborate (1- BP4) . A solution of phenyllithium was prepared by addition of bromobenzene (24.5 g, 156 mmol) in dry ether (50 raL) to lithium wire (2.30 g, 328 mmol) in dry ether (100 ml). 1,3,5-Trimethoxy-benzene (25.1 g, 149 mmol) in dry benzene (100 mL) was added, and the reaction mixture was stirred at room temperature under argon for 70 h. Diethyl carbonate (5.30 g, 45 mmol) in dry benzene (150 mL) was added , and the reaction mixture was refluxed for 3 days. The cooled reaction mixture was poured into NaOH solution (300 mL, 1 M) . The phases were separated, and the water phase was extracted with ether. The combined organic phases were dried over MgS04 and filtered yielding a clear yellow solution. Addition of aqueous HBF4 solution (12 mL, 50% approximately 100 mmol) resulted in immediate precipitation of the deep blue carbenium salt. The dark purple precipitate was filtered off, washed thoroughly with dry ether, and dried over solid KOH to yield 26.0 'g (96%) of the crude product. Reprecipitation by addition of water to an acetonitrile solution followed by recrystallisation from methanol gave the pure compound in 70% overall yield. 1H NMR (250 MHz, CDC13) ; 8 6.05 (6H,s), 3.99 (9H,s), 3.59 (18H,s). 13C NMR (400 MHz, CDC13) : 5 169.70, 166.47, 163.93, 118.62, 91.53, 56.52, 56.36. MS (FAB) : m/z 513 (M+) . UV-vis Xnax (nm (log e)) (CH2C12) : 584 (4.55), 467 (3.98), 322 (3.68), 287 (4.02). Anal. Cacd for CaaHasOgBF^: C, 56.01; H, 5.50. Found: C, 55.69; H, 5.64. Tris(2,4,6-trimethoxyphenyl)carbenium Chloride (1-C1). This compound was prepared analogously to 1-BF4 (starting with 15.0 g of 1,3,5-trimethoxybenzene). Instead of HBF4, gaseous HC1 was bubbled through the hydrolysed and dried reaction, mixture. The dark purple crystalline precipitate was filtered off, washed thoroughly with dry ether, and dried over aolid KOH to yield 10.1 g (66%) of the crude product. The MS (FAB) , H NMR, and 13C NMR spectra were identical with those of the BF4 salt. Synthesis of HMCV-1: 'Schema 1. I) 4-(M-methylamino)-bvitanic acid hydrochloride (1 eq.), Diisopropylethylaroine, in acetonitri'leT 20 "C, 20 hours. II) Dimethylamine '(excess). Ill) TSTU, Diisopropylethylamine, in acetonitrile, 20 °C, 2 hours. 4a (BF«~) : 4-(methylamino)butyric acid hydrochloride (1.36 g; 8.8 mmol), 1 (5.0 g; 8.3 mraol) , and diisopropylethylamine (5 tnL) was dissolved in acetonitrile (120 mL) . The reaction mixture was stirred at 30-35 °C in a dry nitrogen atmosphere for 22 h. Aqueous dimethylamine (40 mL of a 40% solution) was added and the reaction mixture was stirred for four more days. Solvent and excess dimethylamine were removed in vacuo and the remaining material dissolved in chloroform. The chloroform solution was washed twice with brine and dried over MgSOj before evaporation of the solvent and reprecipitation of the product from CH2Cl2/ether. Yield: 4.4 g (70%) of a dark blue powder. MS (FAB+) : m/z 624 (M+) (400 MHz, DMSO-ds) : 8 8.34. (1H, bs) , 6.03 (2H, s) , 5.83 (4H, s) , 3.49 (2H, m) , 3.46 (6H, s) , 3.44 (12H, s) , 3.12 (3H, s (masked)), 3.08 (12H, a), 1.94 (2H, t) , 1.70 (2H, m) . HMCV-1 (Cl~) : TSTU (2-succinimido-l , 1 , 3 , 3 -tetraraethyluronium tetraf luoroborate; 0.8 g, 2.6 mmol) was added to a solution of 4a (0.9 g, 1.26 mmol) and diisopropylethylamine (0.55 g, 4.5 mmol) in acetonitrile (15 mL) . The reaction mixture was stirred in a closed flask for 2 h, before it was poured into an ice-cold nearly sat. NaCl, solution (approx. 150 mL) acidified with HCl-aq (4 mL, 2 M) . The water phase was extracted with chloroform (2 x 150 mL) . The combined chloroform phases was washed with brine (2 x 50 mL) -and dried over MgSO4 . Evaporation of the solvent and reprecipitation from CH2Cl2/ether gave a dark blue powder (0.80 g, 84%) . MS (FAB+) : m/z 721 (M+) '•H-NMR '•H-NMR br.(400 MHz, DMSO-d6) : 8 5.88 (2H, s) , 5.85 (4H,s), 3.60 (2H, s) , 3.46 (12H, s) , 3.45 (6H, s) , 3.15 (12H, s) , 3.12 (3H, s) , 2.85 (4H, s) , 2.80 (2H, t) , 1.95 (2H, m) . Synthesis of HMCV-2: Scheana 2. I) Piperidine-4-carboxylic acid (1 eq.), Diisopropylethylamine, in NMP, 80 °C. II) Dimethylamine (excess), 20 °C. Ill) TSTU, Diiaopropylethylamine, in acetonitrile, 20 °C, 2 hours. 4b (PF6")s A suspension of piperidine-4-carboxylic acid (Isonipecotic acid) (0.215 g,- 1.7 mmol) and diisopropylethylamine (1 mL) in NMP {N-methyl-2-pyrrolidone, 20 mL) was added to a solution of 1 (1.0 g; 1.7 nvmol) in 30 mL of NMP. The reaction mixture was heated in an 80-100 °C warm oil bath for 2 h (until the reaction mixture was bright and left at room temperature overnight. Dimethylarrdne (10 mL of a 33% solution in absolute ethanol) was added and the reaction mixture was stirred for another 24 h at room temperature. The reaction mixture was poured into an aqueous KPF6 solution (400 mL, 0.2 M) and KCl-aq (2M) was added in small portions until precipitation. The blue precipitate was filtered off and washed with pure water, dried, dissolved in DCM, filtered and reprecipitated by addition of ether. Yield: 0.85 g of a dark blue powder (64%) . MS (FAB+): m/z 636 (M+) XH-NMR (CD3CN, int. solvent ref. 1.94 ppm) : 6 6.03 (2H, s) , 5.81 (4H, s), 3.90 (2H, m), 3.48 (12H, s) , 3.47 (6H, s), 3.14 (12H, s) , 3.04 (2H, m) , 2.61 (1H, m) , 2.15 (1H, br) , 1.97 (2H, m), 1.71 (2H, m). HMCV-2 (PFS~) : TSTU (2-succinimido-l, 1, 3,3-tetramethyluronium tetrafluoroborate; 0.72 g, 2.4 mtnol) was added to a solution of 4b (0.8 g, 1 mmol) and diisopropylethylaraine (0.47 g) in acetonitrile (15 mL) . The reaction mixture was stirred in a closed flask for 2 h, before it was poured into an cold NaCl solution (ca 100 mL) acidified with HCl-aq (4 mL, 2 M solution, ca 4 mmol}. The water phase was extracted with chloroform (2 x 100 mL) . The combined chloroform phases were washed with brine (2 x 50 mL) and dried over MgS04. Filter aid (10 g) was added to the filtered chloroform phase .and the solvent removed. Workup by column chromatography (silica, CHCl3/MeCN 5:1) gave, after evaporation of the solvent (the first blue fraction was collected) and re-precipitation from CH2Cl2/ether a dark blue powder (0.55 g, 62%) . MS (FAB+): m/Z 732 (M+) XH-NMR (DMSO, int. solvent ref 2.50 ppm): S 6.08 (2H, s) , 5.84 (4H, s), 4.93 (2H, m), 3.45 (12H, s ) , 3.44 (6H, s), 3.14 (12H, s) , 3.14 (2H, m (masked)), 2.82 (4H) , 2.03 (2H, m) , 1.74 (2H, m) . (CDC13/ int. solvent ref 7.76 ppm ) 8 5.94 (2H, s) , 5.70 (4H, S) , 3.80 (2H, m) , 3.53 (12H, s) , 3.18 (6H) , 3.16 (2H, m (masked)), 2.97 (1H, m) , 2.85 (4H, s), 2.19 (2H, m) , 2.04 (2H, m) . Synthesis of HMCV-3: Synthesis of the HMCV-3 precursor 4c has been accomplished by two synthetic routes. A: by a "one-pot" procedure analogous to the one used in the synthesis of HMCV-1 and HMCV-2, where the linker carrying atnino group (4-(N-methylamino)-butanic acid) is introduced first, followed by the two sulfonic acid substituted amino functions (N-methyl-taurine) (Scheme 3). In method B the sequence is reversed and performed in two steps with isolation of the double substituted product 3c (Scheme 4). Scheme 3 (Method A).- I) 4-(N-methylamino)-butanic acid hydrochloride (1 eg.), Diisopropylethylamine, in DMSO, 20 °C, 24 hours. II) sodium N-methyltaurine (excess), 70 Schema 4 (Mathod B) : I) sodium N-methyltaurine (excess), 20 "C, 3 days. II) 4-(N-methylamino)-bXicanic acid hydrochloride (excess), NajC03, in DMSO, 70 "C, • 3 days . 4c (Na) (method A): A solution of 4-(methylamino)butyric acid hydrochloride (0.275 g; 1.8 mmol) , 1 (1.0 g; 1.7 mmol) , and diisopropylethylamine (I mL) in DMSO (25 mL) was stirred for 20 h. at room temperature before sodium N-methyltaurine (2.0 g, 12 mmol) , diisopropylethylamine- (1 mL) and DMSO (20 mL) was added. The reaction mixture was then stirred for two days at about 70 °C and two days at room temperature. The reaction mixture was filtered through silica followed by thoroughly washing with methanol. The blue filtrate was concentrated and the crude product precipitated, as a sticky blue wax, by addition of ethyl acetate. Column chromatography (silica, MeCN/MeOH 5:2)' gave a blue solid (first blue fraction/band), which was further purified by dissolution in hot ethanol followed by filtration (hot). After cooling the ethanol solution was filtered again and the precipitate was washed with ethanol (leaving a white material) . The blue ethanol solution was evaporated yielding 0.3 g of 4c (18%) . MS (FAB+, Glycerol): m/z 812 (MH2 +) , 834 (MNaH) , 856 (MNa/) XH-NMR (CD3OD, int. solvent ref 3.35 ppm) : 8 5.98 (6H, s) , 3.95 (4H, t ) , 3.62 (2H, t), 3.57 (6H, s) , 3.56 (12H, s), 3.21 (3H, s), 3.20 (6H, s) , 3.15 (4H, t) , 2.41 (2H, t ) , 1.99 (2H, m ) . 4c by Method B: 3o (Na) : A solution of sodium N-methyltaurine (1.3 g, 8 mmol) , and 1 (1.0 g; 1.7 tnraol) in DMSO (25 mL) was stirred for three days at room temperature. Addition of ethyl acetate (approx. 500 mL) gave a blue precipitate (and a red mother liquor). The solid material was heated to reflux in absolute ethanol (approx. 150 mL) and left overnight at room temperature. The precipitate was filtered off, dissolved in methanol and filtered through silica (5 cm layer), which was washed with methanol. Evaporation of the solvent and reprecipitation from methanol/ethyl acetate yielded a blue powder (1.1 g, 88%). MS (FAB+, Glycerol) : m/z 727 (MH2 +) , 749 (MNaH+) , 765 (MNaK+) , 771 (MNa2 '•H-NMR (CD3OD, int. solvent ref 3.35 ppm): 8 6.20 (2H, s) , 5.98 (4H, s), 4.00 (4H, t), 3.87 (3H, s) , 3.58 (12H, s), 3.56 (6H, S), 3.28 (6H, s), 3.17 (2H, t). 4c (Na+) : A solution of 3c (0.5 g; 0.67 mmol), 4-(Nmethylamino)- butanic acid hydrochloride (0.5 g, 3.26 mmol), and dry Na2CO3 (0.5 g) in DMSO (7 mL) was stirred for 3 days at 70 °C. The crude product was precipitated from the cooled reaction mixture by addition of ethyl acetate, giving a sticky blue material (ca 1.1 g.). Column, chromatography 26 (silica, MeCN/MeOH 5:2) gave the product 4c as a blue solid after evaporation of the solvents (0.25 g, 44%). MS (FAE+, Glycerol) : m/z 812 (MH2+) (CD3OD, int. solvent ref 3.35 pptn) : 5 5.98 (6H, s) , 3.95 (4H, t) , 3.62 (2H, t) , 3.57 (6H, s) , 3.56 (12H, s) , 3.21 (3H, a), 3.20 (6H, s) , 3.15 (4H, t) , 2.41 (2H, t) , 1.99 (2H, m) . Scheme 5. Ill) TSTU, Diisopropylethylamine, DMSO. HMCV-3 (Na+) : TSTU (2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate; 0.20 g, 0.6 mmol) was added to a solution of 4c (0.25 g, 0.3 mmol) and diisopropylethylamine (0.06 g) in dry DMSO (10 mL) . The reaction mixture was stirred in a closed flask for 2 h, before the crude product was precipitated by addition of ethyl acetate. The sticky blue material was washed from the filter with methanol and dried at high vacuum. Workup by column chromatography (silica, H2O/MeCN 1:10) gave, after evaporation of the solvent (high vacuum at room temperature) and re-precipitation from methanol/ethyl acetate a dark blue powder (0.25 g, 90%). MS (FAB+, Glycerol) : m/z 909 (MH2 +) , 931 (MNaH+) , 947 (MKH4) , D, int. solvent ref 3,35 ppm) : 5 5.99 (4H, s) , 5.94 (2H, s) , 3.95 (4H, t) , 3.68 (2H, t) , 3.57 (12H, s) , 3.58 (2H, masked), 3.55 (6H, s) , 3.21 (9H, s) , 3.16 (4H, t) , 2.88 (4H, s) , 2.80 (2H,s), 2.13 (2H, m) . Attachment of KMCV-1 to Aminodextran Aminodextran (130 mg, Mw 110.000, 0.5 mmol NH2/g dextran) was dissolved in 6.5 mL aqueous sodium carbonate (15 mM, pH = 8.5 to give a solution with dextran concentration 182 uM, 20 mg/mL) . To 3.0 mL of this solution under stirring was added HMCV-1 (5.65 mg in 200 uL DMSO) in 10 uL portions over 10 minutes. The solution was stirred at room temperature for 1 hour and dialysed.(3 mL dialysis slide, 12-14.000 MwCO membrane) extensively against an aqueous phosphate buffer (10 mM H2P04"/HPO4 2', 4 mM K+, 145 mM Na+, 0 . 1 mM Ca2, 0 . 1 mM Mr.2*, pH =7.4) . The concentration of labeled dextran was determined from the dilution during dialysis. The degree of labelling (the DOL- value) was determined from a series of UV-Vis spectra of the resulting solution (3.2 mL, concentration of dextran 168 uM, DOL = 10) . Attachment of Alexa Fluor 594™ to Concanavalin A (ConA) A solution of Con A (Type III, Sigma) was made as follows. Methyl -a-D-mannopyranoside (0.97 g) , 30 uL aqueous CaCl2 (0.1 M) and 30 p,L aqueous MnCl2 (0.1 M) were mixed in 20 mL 0.5 M Na2CO3 buffer (pH = 8.5). Then ConA (1.00 g ~ 150 mg protein) was added and the suspension stirred vigorously for 1 h. The solution was centrifuged and the supernatant collected. To 13.0 mL of this solution was added Alexa Fluor 594™ (10.0 mg in 800 mL dry DMSO) in 20 \iL portions over 10 min. The resulting blue solution was stirred for 1 h and then succinic anhydride (18.2 mg in 867 uL dry DMSO) was added in 20 [iL portions over 10 min. The solution was stirred for another 1.5 h after which it was transferred to a 15 mL dialysis slide and dialysed against the same buffer as described for the HMCV-1-Dextran synthesis. To the buffer was added 2 mM NaN3 to prevent growth. (15.4 mL, concentration of protein dimer 59 ^M, DOL =4.0) Use of analyte binding reagent labelled with Dye Compound 4 in FRET assay The glucose measurement assay chemistry of the preferred embodiment is based on the competition between binding of dextran and glucose to Concanavalin A (Con A) as discussed above. Con A is labelled with Alexa Fluor 594 (AF594) (donor) and dextran is labelled with a non-fluorescent dye, HMCV-1 (acceptor), absorbing within the emission band of AF594. The fluorescence lifetime is measured by frequencydomain fluoritnetry. The intensity of the excitation light is modulated causing the excited donor to emit light modulated with the same frequency and delayed by the lifetime of the excited state. This results in a phase shift between the excitation light and the emitted fluorescence. We chose AF594-labelled (ConA) as the sugar binding lectin and the HMCV-1 labelled dextran as a glucose analogue. The bulk concentrations of the donor and acceptor dyes used, [AF594] and [HMCV-1] were constant during the whole experiment and their ratio was 1: 6. This is consistent with the Forster theory condition that the donor concentration is much smaller than the acceptor concentration. [Ref. : C.J, Rolinski, D.J.S. Birch, L.J. McCartney, J.C. Pickup, J. Photochem. Photobiol. B: Biol. 54, 26-34, 2000] It is our finding that the Forster theory is best fulfilled when ConA is labelled with the donor and the poly-sugar dextran is labelled with the acceptor. Measurements The donor gpnjugate, the acceptor conjugate and the PBSbuffer (50 mK PBS-buffer, pH = 7.4, ionic strength adjusted to 150 mM with NaCl) were mixed so that the final concentration in the assay chemistry of ConA was 20 /xM and the concentration of dextran was 50 fiM. The molar ratio ([ConA]/[Dex] ) was 0.40 and the concentration ratio ([AF594]/[HMCV-1]) was 0.16. Two hollow cellulose fibres (Spectrum Laboratories, Inc., regenerated cellulose, fibre outer diameter 216 /J.mf fibre inner diameter 200 ^m, MWCO 13 kDa Reorder No. 132294) were filled with assay chemistry, and were then mounted in a custom-made holder placed in a fluorescence cell containing PBS-buffer. The phase excitation shift was measured using a phase and modulation fluorimeter (Koala from ISS, Inc., Champaign, Illinois). The light source was a yellow LED. A XF1067 band pass filter, which transmits light from 540 to 590 rim (Glenn Spectra Ltd) was placed in the excitation channel as well as in the reference channel. The emission channel was fitted with a XF3061 band pass filter, which transmits light from 610 to 690 nm (Glenn Spectra Ltd) . The glucose dose-response was measured by successive replacement of the PBS buffer with PBS buffer containing increasing glucose concentration from 0 mM to 50 mM glucose. The results are shown in Table 1 and in Fig. 1. (Table Removed)The above assay system was tested in vivo in a clamped pig experiment. The assay was loaded in a hollow fiber which by a needle was placed in the subcutus of the skin. The results from the phase measurements showed a slight phase shift compared with the in vitro experiment but could be correlated very well with reference values for whole blood glucose. The acceptors of the preferred embodiment of the present invention have a number of advantages over known acceptors. First, the HMCV acceptors have a broad and intense absorption spectrum (550 to 700 nm) . This means that they can be used with a variety of donors (See Fig. 2) . Second, the acceptors absorb at high wavelengths. This means that they are suitable for in vivo use at wavelengths where autofluorescence does not interfere with the measurements. Third, the center carbon atom of the dyes is shielded by ortho substituents on the aromatic moities prohibiting degradation processes that initiates by either reductive, oxidative or nucleophilic attack at this position. Fourth, the absorption spectrum of the acceptors is not significantly affected by conjugation to analyte or analyte binding reagent. This means that the performance of the acceptors is predictable. Fifth, the acceptors do not fluoresce and will not contribute to background fluorescence signal. We Claim; 1. A reagent for detecting an analyte, comprising a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula: (Formula Removed) wherein: R1, R2 and R3 are each independently H, electron donating substituents, or electron withdrawing substituents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups; R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl, S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more -of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, S03-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen; and wherein the distance between the energy donor and the energy acceptor of the reagent is capable of modulation by a suitable analyte to be detected. 2. A reagent as claimed in Claim 1, wherein the energy-donor and energy acceptor are linked together by a covalent linkage. 3. A reagent as claimed in Claim 2, wherein the covalent linkage between the energy donor and energy acceptor is cleavable to increase the distance between the energy donor and the energy acceptor of the reagent. 4. A reagent as claimed in Claim 2, wherein the energy donor and energy acceptor are linked via a polynucleotide sequence or a polynucleotide analogue sequence or a polypeptide sequence, the sequence having a conformation which is capable of modulation by a suitable analyte to be detected so as to modulate the distance between the energy donor and the energy acceptor of the reagent. 5. A reagent as claimed in Claim 1, wherein the energy donor and energy acceptor are linked together by non-covalent binding. 6. A reagent as claimed in Claim 5 wherein the non-covalent binding exists between an analyte binding agent linked to one of the energy donor and the energy acceptor and an analyte analogue linked to the other of the energy donor and the energy acceptor, the non-covalent binding being disruptable by a suitable analyte so as to increase the distance between the energy donor and the energy acceptor of the reagent. 7. A reagent as claimed in Claim 6, wherein the analyte binding agent is a lectin. 8. A reagent as claimed in Claim 6 or Claim 7, wherein the analyte analogue is a glucose analogue. 9. A reagent as claimed in Claim 8, wherein the analyte analogue is dextran. 10. A reagent, as claimed in Claim 1, wherein the energy donor and the energy acceptor are not linked in the absence of analyte. 11. A reagent as claimed in any one of the preceding claims, wherein a linker structure is attached to the energy acceptor at R3, or where a bridging group is present optionally the linker structure is attached to the energy acceptor at the bridging group. 12. A reagent as claimed in any one of the preceding claims, ' wherein the electron donating substituents are selected from amino, primary amine, secondary amine, O-alkyl, alkyl, S-alkyl, amide, ester, OH and SH. 13. A reagent as claimed in Claim 12, wherein one or more of R1 to R3 is dimethylamino, diethylamino or methylethylamino, optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine. 14. A reagent as claimed in any one of the preceding claims, wherein an electron withdrawing substituent is present, and the electron withdrawing substituent is selected from NO, NO2, CN, COOH, ester, COO", amide, CHO, keto, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, and SO3-alkyl. 15. A reagent as claimed in any one of the preceding claims, wherein at least one of R10, R11, R12, R13, R14 ANd R15 is O-alkyl. 16. A reagent as claimed in any one of the preceding claims, wherein one or more pairs of groups R4 and R10 and/or R5 and R11 and/or R6 and Rx2 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, .SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine. 17. A reagent as claimed in any one of Claims 1 to 14, wherein R10 to R15 are each O-methyl or O-ethyl. 18. A reagent as claimed in any one of the preceding claims, further comprising one or more counterions selected from halide, BF4-, PF6-, NO3-, carboxylate, C104-, Li+, Na+, K+, Mg2+ and Zn2+. 19. A reagent as claimed in any one of the preceding claims, wherein a linker structure is present, and is formed by reaction of a linker element selected from an active ester, an isothiocyanate, an acid chloride, an aldehyde, an azide, an α-halogenated ketone and an amine with a reaction partner. 20. A reagent as claimed in Claim 19, wherein the reaction partner is selected from a polysaccharide, a polynucleotide and a protein. 21. A reagent as claimed in Claim 19 or Claim 20, wherein the linker element is an active ester, and is selected from succinimidyl and pentafluorophenyl active esters. 22. A reagent as claimed in any one of the preceding claims, wherein the energy donor is Alexa Fluor-594™. 23. A dye compound having the general formula: (Formula Removed) wherein: R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl or S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O- alkyl, S-alkyl, or alkyl, or one or more pairs of groups R20 and R4 and/or R20 and R5 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkyiene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen; R15, R17, R18 and R19 are each independently H, alkyl or aryl, or one or more of R16 and R17 or R18 and R19 is alkylene, optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine; or one or more of pairs of groups R6 and R16, R7 and R17, R8 and R18 and R9 and R19 is alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine and R20 is a linker element selected from an active ester, an isothiocyanate, an acid chloride, an α-halogenated ketone, an azide and an amine. 24. A dye compound as claimed in Claim 23, wherein at least one of R10, R11, R12, R13, R14 and R15 is alkyl. 25. A dye compound as claimed in Claim 24, wherein one or more pairs of groups R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3, PO32-, OH, o-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine. 26. A dye compound as claimed in any one of Claims 23 to 25, wherein R20 is a linker element having the structure: (Structure Removed) R21 is H or alkyl or aryl optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2N-dialkyl, CN, secondary amine or tertiary amine and R22 is alkylene, O-alkylene, S-alkylene or N-alkylene or R21 and R22 are part of a ring, optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine; and R23 is o-succinimidyl, o-pentafluorophenyl, C1 or α-halogenated alkyl. 27. A.dye compound as claimed in any one of Claims 23 to 26, wherein R10 to R15 are each O-methyl or O-ethyl. 28. A dye compound as claimed in any one of Claims 23 to 27, further comprising one or more counterions selected from halide, BF4-, PF6-, NO3-, carboxylate, C1O4-, Li+, Na+, K+, Mg2+ and Zn2+. 29. A method of detecting or measuring an analyte using a reagent as claimed in any one of Claims 1 to 22, comprising the steps of: contacting the reagent with a sample; illuminating the reagent and sample with light of wavelength within the absorption spectrum of the energy donor; detecting non-radiative energy transfer between the energy donor and energy acceptor by measuring the fluorescence of the energy donor; and associating the fluorescence measurements with presence or concentration of analyte. 30. A method as claimed in Claim 29, wherein the fluorescence of the energy donor is measured by measuring making intensity based or time resolved fluorescence measurements. 31. A method as claimed in Claim 29 or 30, wherein the analyte is measured by comparing sample fluorescence measurements with fluorescence measurements made using known concentrations of analyte. 32 . A complex of an analyte and a reagent for detecting the analyte wherein the reagent comprises a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula: (Formula Removed) wherein: R1, R2 and R3 are each independently H, electron donating substituent.s, or electron withdrawing substituents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups; R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl, S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N- dialkyl, and SO3-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen; and wherein the presence of the analyte modulates the distance between the energy donor and the energy acceptor.

Full Text

REAGENT FOR DETECTING AN ANALYTE
The present invention relates to a reagent for use in
detecting an analyte, to a dye compound suitable for use in
such a reagent, to a method of detecting or measuring an
analyte using such a reagent and to a complex of an analyte
and such a reagent.
The method of detecting or measuring an analyte relies on
FRET (fluorescence resonance energy transfer).
FRET is a distance-dependent interaction between the
electronic excited states of two dye species in which
excitation energy is transferred from a donor to an acceptor
without emission of a photon.
The efficiency of FRET is inversely dependent on the
sixth power of 'the intermolecular separation. [Refs. 1. B.
Wieb Van der Meer, G. Coker III, S.-Y. Simon Chen, Resonance
energy transfer; Theory and data, VCH publishers, 1994; 2.
Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz
(Ed.), 2. edition, Plenum Press, New York 1999]. Detection of
FRET can therefore be used to determine the distance between
a species labelled with the donor and a species labelled with
the acceptor. This may be used for example to determine
whether the two species are bound to one another.
The requirements for FRET are:
- Donor and acceptor must be in close proximity
(typically 10-100 A).
- The absorption spectrum of the acceptor must overlap
the fluorescence emission spectrum of the donor.
- Donor and acceptor transition dipole orientations must
be approximately parallel.
FRET causes a decrease in intensity and lifetime of donor
fluorescence. If the acceptor is fluorescent, FRET may also
cause an increase in intensity of acceptor fluorescence.
Where the donor and acceptor are the same, FRET causes
fluorescence depolarization.
FRET may be detected by illuminating a sample with light
in the absorption spectrum of the donor and measuring any of
these properties.
A simple FRET assay may be carried out by labelling an
analyte with a donor and labelling an analyte binding agent
with an acceptor (or vice versa) . When the analyte is not
bound to the analyte binding agent, the distance between the
donor and acceptor is large and no FRET occurs.
When the analyte is bound to the analyte binding agent,
the distance between the donor and acceptor is small.and FRET
occurs.
Therefore, detection of FRET may be used to determine
whether analyte is present and/or analyte concentration.
A competitive FRET assay may be carried out where
labelled analyte competes with non-labelled analyte. In this
case, as the concentration of non-labelled analyte increases,
there will be less bound labelled analyte. This means that
less FRET will occur.
Therefore, again detection of FRET may be used to
determine analyte concentration.
An assay of this type may be used for the measurement of
glucose concentration. Glucose binds reversibly 'to a class
of proteins called lectins. Concanavalin A is an example of
a lectin. The components of the assay are for example
labelled concanavalin A (the analyte binding reagent) and
labeled glucose or labelled glucose analogue. Dextran is a
suitable glucose analogue. When glucose (the analyte) binds
to concanavalin A, it displaces labelled glucose or dextran,
and less FRET will occur.
Alternatively, the donor and acceptor may be attached to
the same species to form a single FRET reagent. An example
of such a reagent is a "molecular beacon".
A molecular beacon consists of a single stranded
polynucleotide or polynucleotide analogue sequence with a
donor attached to one end of the sequence. A complementary
acceptor is attached to the other end of the sequence.
The unbound molecular beacon exists as a stem-and-loop
structure. The sequences at the ends of the molecular beacon
match and bind, creating the stem, while the rest of the
probe is unmatched and unbound, creating the loop. While
folded this way, .the donor at one end of the probe is next to
the acceptor at the other end. The proximity of the donor and
acceptor allows FRET to occur.
When the probe recognizes and binds to a target, the
molecular beacon structure unfolds. This separates the
acceptor from the donor so that FRET cannot take place.
Therefore, FRET may be used to determine whether a
particular target is present.
Molecular beacons may be used to detect complementary
single stranded polynucleotide sequences. Alternatively,
they may be used to detect other non-nucleotide species.
Such molecular beacons are called aptamers. For example,
aptamers may be used to detect proteins.
The molecular beacon has the potential disadvantage that
it relies on FRET occurring while the beacon is unbound.
During an in vivo assay the beacon sequence may be cleaved by
enzymes in the body. This will reduce the FRET. The
reduction in FRET caused by beacon degradation cannot be
distinguished from the reduction in FRET caused by beacon
binding to the target, and the results obtained may therefore
be unreliable.
An alternative to a molecular beacon which deals with
this problem is a dual probe. This comprises two single
stranded polynucleotide or polynucleotide analogue sequences,
one labelled with an energy donor and one with a
complementary energy acceptor. The dual probe is used to
detect a single stranded polynucleotide target sequence which
is complementary to the sequences of both parts of the dual
probe. When the parts of the dual probe are both bound to
the target, FRET occurs. Since FRET does not occur when the
dual probe is unbound, dual probe degradation will not affect
the occurrence of FRET. Therefore, degradation- will not
cause the results to be unreliable.
Reagents similar to molecular beacons can be formed from
polypeptides.FRET can also be used in immunoassays.
The choice of donor and acceptor used in FRET is
important, and the following factors need to be taken into
account.
The donor should have a high quantum yield.
A suitable acceptor must have an absorption spectrum
overlapping the emission spectrum of the donor. For example,
QSY 21™ is a suitable donor for use with Alexa Fluor 21™. It
is desirable for an acceptor to have a wide absorption
spectrum so that it can be used with a variety x>f donors.
If the acceptor is also a fluorescent species it is
possible that background fluorescence resulting from direct
acceptor excitation will occur and be detected as well as
donor fluorescence. Filtering of the acceptor emission may
result in loss of the donor fluorescence signal where
filtering is not sharp enough. Non-fluorescent acceptors
such as dabcyl and QSY can be used to avoid this problem.
Alternatively, the problem can be avoided by choosing an
acceptor whose emission spectrum is remote from that of the
donor.
The emission spectrum of some donors and absorption
spectrum of some acceptors (for example QSY 21) are shifted
when the dor.or or acceptor is conjugated to an analyte or
analyte binding agent. This is undesirable.
Where FRET assays are to be used in vivo, it is desirable
for donors to fluoresce at 550 to around 600 nm and for
acceptors to absorb light at around 650 nm. This avoids
overlap between the donor fluorescence and in vivo
autof luorescence at lower wavelengths. '"" Fluorescein
fluoresces at 495 nm and is therefore not ideal for in vivo
use.
Alexa Fluor 594 is a dye with a suitable emission
spectrum for use in vivo. This dye absorbs at 594 nm and
fluoresces at 620 nm.
The present inventors have now provided a new class of
acceptors (HMCV dyes) for use in FRET. The acceptors are
stabilized carbenium ions which are structurally related to
crystal violet and malachite green. The acceptors can be
derived from intermediates in the synthesis of
trioxatriangulenium systems described in Bo W. Laursen et
al., J. Am. Chem. Soc., 1998, 120, 12255-12263.
Accordingly, the present invention provides in a first
aspect a reagent for use in detecting an analyte, comprising
a fluorescent energy donor and an energy acceptor, the energy
donor and the energy acceptor being such that when they are
sufficiently close to one another energy is non-radiatively
transferred from the energy donor following excitation
thereof to the energy acceptor quenching fluorescence of the
energy donor, wherein the energy acceptor is of the general
formula:
wherein:
R1, R2 and R3 are each independently H, electron donating
substituents, or electron withdrawing substituents or R3
is attached to a linker structure, provided that at
least two of R1, R2 and R3 are electron donating groups;
R4, R5, Rs, R7, R8 and R9 are each independently H,
halogen, alkyl, aryl, 0-alkyl, S-alkyl and R , R1- ,12
R13, R14 and R15 are each independently hydrogen, 0-alkyl,
S-alkyl, alkyl, or one or more pairs of groups R1 and R4
and/or R1 and R5 and/or R2 and Rfi and/or R2 and R7 and/or
R3 and R8 and/or R3 and R9 and/or R and R10 and/or R5 and
R11 and/or Rfi and R13 and/or R7 and R13 and/or Ra and R1
and/or R9 and Ris is a bridging group consisting of aryl,
alkylene, O-alkylene, S-alkylene or N-alkylene
optionally substituted with one or more of S03", P03
OH, 0-alkyl, SH, S-alkyl, COOH, COO', ester, amide,
halogen, SO-alkyl, S02-alkyl, S02NH2, S02NH-alkyl, S02Ndialkyl,
SO3-alkyl, CN, secondary amine or tertiary
araine, provided that not all of R10, R11, R12, R13, R14 and
R1S are hydrogen;
and wherein the distance between the energy donor and
the energy acceptor of the reagent is capable of
modulation by a suitable analyte to be detected.
As used herein, the term "electron donating substituent"
includes but is not limited to amino, primary amine,
secondary amine, alkyl, 0-alkyl, S-alkyl, amide (NHCOR),
ester (OCOR), OH, SH and electron rich aryl.
As used herein, the term "electron withdrawing
substituent" includes but is not limited to NO, N02, CN,
COOH, ester (COOR) , COO", amide (CONR2) , CHO, keto (COR), SOalkyl,
S02-alkyl, S02NH2, S02NH-alkyl, S02N-dialkyl, SO3-
alkyl, and electron deficient aryl.
Electron rich/deficient aryls may be defined as aryls
having electron-donating or -withdrawing substituents
respectively.
As used herein, the term "alkyl" and "alkylene" include
linear, branched and cyclic groups, saturated and unsaturated
groups (including groups containing triple bonds), groups
containing one or more substituents, and groups containing
one or more heteroatoms. Ci to Cg alky groups are
preferred.
As used herein, the term "0-alkylene", "N-alkylene" and
"S-alkylene" each include groups wherein the heteroatom is at
any position within the group. Ci to C7 O-alkylene, Nalkylene
and S-alkylene and C2 to C7 alkylene are preferred.
Where an ester substituent is referred to, the
substituent may be linked via the carbonyl portion (as
R(C=0)OR) or via the alkoxy portion, (as RO(C=0)R).
Similarly, where an amide substituent is referred to, the
substituent may be linked via the carbonyl portion (as
RCONR2) or the nitrogen portion (as R(NR)COR).
In one preferred embodiment, the energy donor and energy
acceptor are linked together by non-covalent binding. The
non-covalent binding may exist between an analybe binding
agent linked to one of the energy donor and the energy
acceptor and an analyte analogue linked to the other of the
energy donor and the energy acceptor, the non-covalent
binding being disruptable by a suitable analyte so as to
increase the distance between the energy donor and the energy
acceptor of the reagent.
Suitably, in this embodiment the analyte binding agent
is a lectin and/or the analyte analogue is a glucose
analogue. For example, the analyte analogue may be dextran.
This system can be used for glucose detection and/or
measurement as discussed above.
It is preferable for the lectin to be bound to the
energy donor and the analyte analogue to be bound to the
energy acceptor (e.g. Dextran-HMCV and Concanavalin A-Alexa
Fluor 594) . Dextran can be more heavily labelled than
Concanavalin A. If dextran is labelled with a fluorophore
there will be excess fluorescence which dilutes the signal in
a lifetime based assay.
In an alternative embodiment, the energy donor and
energy acceptor are linked together by a covalent linkage.
The covalent linkage between the energy donor and energy
acceptor may be cleavable to increase the distance between
the energy donor and the energy acceptor of the reagent.
Suitably, the energy donor and energy acceptor are linked via
a polynucleotide sequence or a polynucleotide analogue
sequence or a polypeptide sequence, the sequence having a
conformation which is capable of modulation by a suitable
analyte to be detected so as to modulate the distance between
the energy donor and the energy acceptor of the reagent. In
this case, the reagent is a "molecular beacon" as discussed
above.
In a further alternative embodimejit, the energy donor
and the energy acceptor are not linked in the absence of
analyte. This reagent may be the "dual probe" discussed
above.
Preferably, a linker structure is attached to the energy
acceptor at R3, or where a bridging group is present
optionally the linker structure is attached to the energy
donor at the bridging group.
Preferably, the electron donating substituents are
selected from amino, primary amine, secondary amine, O-alkyl,
alkyl, S-alkyl, amide, ester, OH and SH.
Preferably, one or more of R1 to R3 is dimethylamino,
diethylamino or methylethylamino, optionally substituted with
one or more of S03", P03
2", OH, O-alkyl, SH, S-alkyl, COOK,
COO", ester, amide, halogen, SO-alkyl, SO2-alkyl, S02NH2,
S02NH-alkyl, S02N-dialkyl, and S03-alkyl, CN, secondary amine
or tertiary amine.
In a preferred embodiment, R1 and R2 are each
dimethylamino. Alternatively, R1 and R2 may each be
optionally substituted methylethylamino.
Preferably, an electron withdrawing substituent is
present, and the electron withdrawing substituent is selected
from NO, N02, CN, COOH, ester, COO", amide, CHO, keto, SOalkyl,
SO2-alkyl, S02NH2l SOjNH-alkyl, S02N-dialkyl, and S03-
alkyl.
Preferably, at least one of R10, R11, R12, R13, R14 and R"
is O-alkyl.
Suitably, one or more pairs of groups R4 and R10 and/or
R5 and R11 and/or Rs and R12 and/or R7 and R13 and/or R8 and R14
and/or R9 and R15 is a bridging group consisting of alkylene ,
O-alkylene, S-alkylene or N-alkylene optionally substituted
with one or more of S03~, PO3
2~, OH, O-alkyl, SH,' S-alkyl,
COOH, COO', ester, amide, halogen, SO-alkyl, S02-alkyl,
SO2NH2, SO2NH-alkyl, S02N-dialkyl, CN, secondary amine or
tertiary amine.
Preferably, R10 to R1S are each 0-methyl or O-ethyl.
Preferably, the reagent further comprises one or more
counterions selected from halide, BF4", PF6", NOa",
carboxylate, ClO4", Li+, Na+, K, Mg2 and Zn2+ so that the
reagent is uncharged overall.
Preferably, a linker structure is present, and is formed
by reaction of a linker element selected from an active
ester, an isothiocyanate, an acid chloride, an aldehyde, an
azide, an a-halogenated ketone and an amine with a reaction
partner. Suitably, the reaction partner is selected from a
polysaccharide, polynucleotide or a protein. Suitably, the
linker element is an active ester, and is selected from
succinimidyl and pentafluorophenyl active esters.
Suitably, the energy donor is Alexa Fluor 594W.
The reagent may be used for in vivo detection of an
analyte. For example, the reagent may be contained within a
sensor which is implanted into the body. Suitably, the
sensor is implanted within the epidermis so that it will be
shed from the body over time. Suitable sensor constructions
are described in detail in W002/30275. In this case, it
should be possible to illuminate the reagent and measure
reagent fluorescence through the layers of the skin above the
implanted sensor.
Alternatively, the reagent may be contained within a
sensor which is partially implanted within the body but
extends outside the body. Such a sensor may take the form of
a needle. In this case, the reagent may be illuminated down
the bore of the needle and reagent fluorescence may be
detected in the same way without light passing through layers
of the skin.
The sensor should be permeable to analyte.
In this second aspect, the invention relates to a sensor
comprising a semi-permeable membrane enclosing a reagent for
use in detecting an analyte as described above.
In a third aspect, the present invention relates to a dye
compound having the general formula:
wherein:
R, R5, Rs, R7, Ra and R9 are each independently H,
halogen, alkyl, aryl, 0-alkyl or S-alkyl and R10, R11,
R12, R13, R14 and R15 are each independently hydrogen, 0-
alkyl, S-alkyl, or alkyl, or one or more pairs of groups
R20 and R and/or R20 and R5 and/or R4 and R10 and/or R5
and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and
R14 and/or R9 and R15 is a bridging group consisting of
aryl, alkylene, o-alkylene, S-alkylene or N-alkylene
optionally substituted with one or more of SO3", PO3
OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide,
halogen, SO-alkyl, S0z-alkyl, S02NH2, S02NH-alkyl, SO2Ndialkyl,
S03-alkyl, CN, secondary amine or tertiary
amine, provided that not all of R10, R11, R12, R13, R14 and
R15 are hydrogen;
R1S, R17, R18 and R19 are each independently H, alkyl
(preferably Ci to Cs alkyl) or aryl, or one or more of
Rls and R17 or Ria and R19 is alkylene (preferably C3 to C7
alkylene) optionally substituted with one or more of
S03", P03
2', OH, 0-alkyl, SH, S-alkyl, COOH, COO', ester,
amide, halogen, SO-alkyl, S02-alkyl, S02NH2, SO2NH-alkyl,
S02N-dialkyl, S03-alkyl, CN, secondary amine or tertiary
amine;
or one or more of pairs of groups R6 and R16, R7 and R17,
R8 and Ria and R9 and R19 is alkylene, 0-alkylene, Salkylene
or N-alkylene optionally substituted with one
or more of S03", P03
2", OH, 0-alkyl, SH, S-alkyl, COOH,
COO", ester, amide, halogen, SO-alkyl, S02-alkyl, S02NH2,
S02NH-alkyl, S02N-dialkyl, SO3-alkyl, CN, secondary amine
or tertiary amine
and
R20 is a linker element selected from an active ester, an
isothiocyanate, an acid chloride, an a-halogenated
ketone, an azide and an amine.
Suitably, at least one of R10, R11, R12, R13, R14 and R15 is
alkyl.
In a preferred embodiment, R16, R17, R16 and R19 are each
methyl or ethyl. Alternatively, R16 and R18 may each be
methyl and R17 and R19 may each be ethyl substituted at the 2-
position with S03".
Suitably, one or more pairs of groups R4 and R10 and/or
R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14
and/or R9 and R15 is a bridging group consisting of alkylene,
O-alkylene, S-alkylene or N-alkylene optionally substituted
with one or more of S03~, P03
2', OH, 0-alkyl, SH, S-alkyl,
COOH, COO", ester, amide, halogen, SO-, S02NH2, S02NH-alkyl,
S02N-dialkyl, CN, secondary amine or tertiary amine.
Preferably, R20 is a linker element having the structure:
wherein R21 is H or alkyl or aryl optionally substituted
with one or more of S03", P03
2~, OH, 0-alkyl, SH, Salkyl,
COOH, COO", ester, amide, halogen, -SO-alkyl,
SO2N-dialkyl, CN, secondary amine or tertiary amine and
R22 is alkylene, O-alkylene, S-alkylene or N-alkylene
(preferably C2 to Cs) or R21 and R22 are part of a ring
(preferably C3 to C7) , optionally substituted with one
or more of S03~, P03
2', OH, O-alkyl, SH, S-alkyl, COOH,
COO", ester, amide, halogen, SO-alkyl, S02NH2, S02NHalkyl,
S02N-dialkyl, S03-alkyl, CN, secondary amine or
tertiary amine; and
R23 is o-succinimidyl, o-pentaf luorophenyl, Cl or ahalogenated
alkyl.
Preferably, R1C to R15 are each 0-methyl or O-ethyl.
Preferably, the dye compound further comprises one or
more counterions selected from halide, BF4", PF6~, N03",
carboxylate, C1O4", Li+, Na, K, Mg2t and Zn2+.
In a fourth aspect, the present invention relates to a
method of detecting or measuring an analyte using a reagent
as described above, comprising the steps of:
contacting the reagent with a sample;
illuminating the reagent and sample with light of
wavelength within the absorption spectrum of the energy
donor;
detecting non-radiative energy transfer between the
energy donor and energy acceptor by measuring the
fluorescence of the energy donor; and
associating the fluorescence measurements with presence
or concentration of analyte.
Suitably, - the fluorescence of the energy donor is
measured by intensity based or time resolved fluorescence
measurements. Preferably, the analyte is measured by
comparing sample fluorescence measurements with fluorescence
measurements made using known concentrations of analyte.
The light used for illumination preferably has a
wavelength above 550 nm.
In a fifth aspect, the present invention relates to a
complex of an analyte and a reagent for detecting the analyte
wherein the reagent comprises a fluorescent energy donor and
an energy acceptor, the energy donor and the energy acceptor
being such that when they are sufficiently close to one
another energy is non-radiatively transferred from the energy
donor following excitation thereof to the energy acceptor
quenching fluorescence of the energy donor, wherein the
energy acceptor is of the general formula:
wherein:
R1, R2 and R3 are each independently H, electron donating
substltuents, or electron withdrawing substltuents or R3
is attached to a linker structure, provided that at
least two of R1, R2 and R3 are electron donating groups;
R*, R5, R6, R7, R8 and R9 are each independently H,
halogen, alkyl, aryl, 0-alkyl, S-alkyl and R10, R11, R12,
R13, R14 and R1S are each independently hydrogen, O-alkyl,
S-alkyl, alkyl, or one or more pairs of groups R1 and R4
and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or
R3 and R8 and/or R3 and R9 and/or R and R10 and/or R5 and
R11 and/or Rs and R12 and/or R7 and R13 and/or R8 and R14
and/or R9 and R15 is a bridging group consisting of aryl,
alkylene, 0-alkylene, S-alkylene or N-alkylene
optionally substituted with one or more of S03", P03
OH, 0-alkyl, SH, S-alkyl, COOH, COO", ester, amide,
17
halogen, SO-alkyl, S02-alkyl, S02NH2, SOjNH-alkyl, S02Ndialkyl,
and S03-alkyl, CN, secondary amine or tertiary
amine, provided that not all of Rxo, R11, R12, R13, R14 and
R15 are hydrogen; and
wherein the presence of the analyte modulates the
distance between the energy donor and the energy
acceptor.
The invention will be further described with reference
to preferred embodiments illustrated by the Examples, and as
illustrated in Figure 1, which shows a glucose dose-response
curve, and Figure 2, which shows normalised absorption and
emission spectra of Alexa Fluor 594 and HMCV-1-dextran in
aqueous PBS buffer 50 tnM, pH=7.4 (Exitation of AF594 at 570
nm in a 0.7 uM solution).
Example
Preparation of Dye Compound 4
Synthetic Methods and Materials
All reagents used were standard grade unless otherwise
mentioned. Ether and THF were dried by distillation from
sodium/benzophenone under nitrogen. Benzene was dried by standing
over sodium. NMR spectra were on a 250 or 400 MHz spectrometer.
FABMS spectra were obtained using 1,4-dicyanobutane as matrix.
Elemental analysis was done at the University of
Copenhagen, Department of Chemistry, Elemental Analysis
Laboratory, Universitetsparken 5, 2100 Copenhagen, Denmark.
Tris (2,4, 6-trimethoxyphenyl) carbenitun Tetrafluoroborate (1-
BP4) . A solution of phenyllithium was prepared by addition of
bromobenzene (24.5 g, 156 mmol) in dry ether (50 raL) to
lithium wire (2.30 g, 328 mmol) in dry ether (100 ml).
1,3,5-Trimethoxy-benzene (25.1 g, 149 mmol) in dry benzene
(100 mL) was added, and the reaction mixture was stirred at
room temperature under argon for 70 h. Diethyl carbonate
(5.30 g, 45 mmol) in dry benzene (150 mL) was added , and the
reaction mixture was refluxed for 3 days. The cooled reaction
mixture was poured into NaOH solution (300 mL, 1 M) . The
phases were separated, and the water phase was extracted with
ether. The combined organic phases were dried over MgS04 and
filtered yielding a clear yellow solution. Addition of
aqueous HBF4 solution (12 mL, 50% approximately 100 mmol)
resulted in immediate precipitation of the deep blue
carbenium salt. The dark purple precipitate was filtered off,
washed thoroughly with dry ether, and dried over solid KOH to
yield 26.0 'g (96%) of the crude product. Reprecipitation by
addition of water to an acetonitrile solution followed by
recrystallisation from methanol gave the pure compound in 70%
overall yield. 1H NMR (250 MHz, CDC13) ; 8 6.05 (6H,s), 3.99
(9H,s), 3.59 (18H,s). 13C NMR (400 MHz, CDC13) : 5 169.70,
166.47, 163.93, 118.62, 91.53, 56.52, 56.36. MS (FAB*) : m/z
513 (M+) . UV-vis Xnax (nm (log e)) (CH2C12) : 584 (4.55), 467
(3.98), 322 (3.68), 287 (4.02). Anal. Cacd for CaaHasOgBF^: C,
56.01; H, 5.50. Found: C, 55.69; H, 5.64.
Tris(2,4,6-trimethoxyphenyl)carbenium Chloride (1-C1).
This compound was prepared analogously to 1-BF4 (starting
with 15.0 g of 1,3,5-trimethoxybenzene). Instead of HBF4,
gaseous HC1 was bubbled through the hydrolysed and dried
reaction, mixture. The dark purple crystalline precipitate was
filtered off, washed thoroughly with dry ether, and dried
over aolid KOH to yield 10.1 g (66%) of the crude product.
The MS (FAB) , H NMR, and 13C NMR spectra were identical with
those of the BF4 salt.
Synthesis of HMCV-1:
'Schema 1. I) 4-(M-methylamino)-bvitanic acid hydrochloride (1 eq.),
Diisopropylethylaroine, in acetonitri'leT 20 "C, 20 hours. II) Dimethylamine
'(excess). Ill) TSTU, Diisopropylethylamine, in acetonitrile, 20 °C, 2 hours.
4a (BF«~) : 4-(methylamino)butyric acid hydrochloride (1.36 g;
8.8 mmol), 1 (5.0 g; 8.3 mraol) , and diisopropylethylamine (5
tnL) was dissolved in acetonitrile (120 mL) . The reaction
mixture was stirred at 30-35 °C in a dry nitrogen atmosphere
for 22 h. Aqueous dimethylamine (40 mL of a 40% solution) was
added and the reaction mixture was stirred for four more
days. Solvent and excess dimethylamine were removed in vacuo
and the remaining material dissolved in chloroform. The
chloroform solution was washed twice with brine and dried
over MgSOj before evaporation of the solvent and
reprecipitation of the product from CH2Cl2/ether. Yield: 4.4
g (70%) of a dark blue powder.
MS (FAB+) : m/z 624 (M+)
(400 MHz, DMSO-ds) : 8 8.34. (1H, bs) , 6.03 (2H, s) ,
5.83 (4H, s) , 3.49 (2H, m) , 3.46 (6H, s) , 3.44 (12H, s) , 3.12
(3H, s (masked)), 3.08 (12H, a), 1.94 (2H, t) , 1.70 (2H, m) .
HMCV-1 (Cl~) : TSTU (2-succinimido-l , 1 , 3 , 3 -tetraraethyluronium
tetraf luoroborate; 0.8 g, 2.6 mmol) was added to a solution
of 4a (0.9 g, 1.26 mmol) and diisopropylethylamine (0.55 g,
4.5 mmol) in acetonitrile (15 mL) . The reaction mixture was
stirred in a closed flask for 2 h, before it was poured into
an ice-cold nearly sat. NaCl, solution (approx. 150 mL)
acidified with HCl-aq (4 mL, 2 M) . The water phase was
extracted with chloroform (2 x 150 mL) . The combined
chloroform phases was washed with brine (2 x 50 mL) -and dried
over MgSO4 . Evaporation of the solvent and reprecipitation
from CH2Cl2/ether gave a dark blue powder (0.80 g, 84%) .
MS (FAB+) : m/z 721 (M+)
'•H-NMR '•H-NMR br.(400 MHz, DMSO-d6) : 8 5.88 (2H, s) , 5.85
(4H,s), 3.60 (2H, s) , 3.46 (12H, s) , 3.45 (6H, s) , 3.15 (12H,
s) , 3.12 (3H, s) , 2.85 (4H, s) , 2.80 (2H, t) , 1.95 (2H, m) .
Synthesis of HMCV-2:
Scheana 2. I) Piperidine-4-carboxylic acid (1 eq.), Diisopropylethylamine, in
NMP, 80 °C. II) Dimethylamine (excess), 20 °C. Ill) TSTU, Diiaopropylethylamine,
in acetonitrile, 20 °C, 2 hours.
4b (PF6")s A suspension of piperidine-4-carboxylic acid
(Isonipecotic acid) (0.215 g,- 1.7 mmol) and
diisopropylethylamine (1 mL) in NMP {N-methyl-2-pyrrolidone,
20 mL) was added to a solution of 1 (1.0 g; 1.7 nvmol) in 30
mL of NMP. The reaction mixture was heated in an 80-100 °C
warm oil bath for 2 h (until the reaction mixture was bright
and left at room temperature overnight. Dimethylarrdne
(10 mL of a 33% solution in absolute ethanol) was added and
the reaction mixture was stirred for another 24 h at room
temperature. The reaction mixture was poured into an aqueous
KPF6 solution (400 mL, 0.2 M) and KCl-aq (2M) was added in
small portions until precipitation. The blue precipitate was
filtered off and washed with pure water, dried, dissolved in
DCM, filtered and reprecipitated by addition of ether. Yield:
0.85 g of a dark blue powder (64%) .
MS (FAB+): m/z 636 (M+)
XH-NMR (CD3CN, int. solvent ref. 1.94 ppm) : 6 6.03 (2H, s) ,
5.81 (4H, s), 3.90 (2H, m), 3.48 (12H, s) , 3.47 (6H, s), 3.14
(12H, s) , 3.04 (2H, m) , 2.61 (1H, m) , 2.15 (1H, br) , 1.97
(2H, m), 1.71 (2H, m).
HMCV-2 (PFS~) : TSTU (2-succinimido-l, 1, 3,3-tetramethyluronium
tetrafluoroborate; 0.72 g, 2.4 mtnol) was added to a solution
of 4b (0.8 g, 1 mmol) and diisopropylethylaraine (0.47 g) in
acetonitrile (15 mL) . The reaction mixture was stirred in a
closed flask for 2 h, before it was poured into an cold NaCl
solution (ca 100 mL) acidified with HCl-aq (4 mL, 2 M
solution, ca 4 mmol}. The water phase was extracted with
chloroform (2 x 100 mL) . The combined chloroform phases were
washed with brine (2 x 50 mL) and dried over MgS04. Filter
aid (10 g) was added to the filtered chloroform phase .and the
solvent removed. Workup by column chromatography (silica,
CHCl3/MeCN 5:1) gave, after evaporation of the solvent (the
first blue fraction was collected) and re-precipitation from
CH2Cl2/ether a dark blue powder (0.55 g, 62%) .
MS (FAB+): m/Z 732 (M+)
XH-NMR (DMSO, int. solvent ref 2.50 ppm): S 6.08 (2H, s) ,
5.84 (4H, s), 4.93 (2H, m), 3.45 (12H, s ) , 3.44 (6H, s), 3.14
(12H, s) , 3.14 (2H, m (masked)), 2.82 (4H) , 2.03 (2H, m) ,
1.74 (2H, m) . (CDC13/ int. solvent ref 7.76 ppm ) 8 5.94 (2H,
s) , 5.70 (4H, S) , 3.80 (2H, m) , 3.53 (12H, s) , 3.18 (6H) ,
3.16 (2H, m (masked)), 2.97 (1H, m) , 2.85 (4H, s), 2.19 (2H,
m) , 2.04 (2H, m) .
Synthesis of HMCV-3:
Synthesis of the HMCV-3 precursor 4c has been accomplished by
two synthetic routes. A: by a "one-pot" procedure analogous
to the one used in the synthesis of HMCV-1 and HMCV-2, where
the linker carrying atnino group (4-(N-methylamino)-butanic
acid) is introduced first, followed by the two sulfonic acid
substituted amino functions (N-methyl-taurine) (Scheme 3). In
method B the sequence is reversed and performed in two steps
with isolation of the double substituted product 3c (Scheme
4).
Scheme 3 (Method A).- I) 4-(N-methylamino)-butanic acid hydrochloride (1 eg.),
Diisopropylethylamine, in DMSO, 20 °C, 24 hours. II) sodium N-methyltaurine (excess), 70
Schema 4 (Mathod B) : I) sodium N-methyltaurine (excess), 20 "C, 3 days. II)
4-(N-methylamino)-bXicanic acid hydrochloride (excess), NajC03, in DMSO, 70 "C, •
3 days .
4c (Na*) (method A): A solution of 4-(methylamino)butyric
acid hydrochloride (0.275 g; 1.8 mmol) , 1 (1.0 g; 1.7 mmol) ,
and diisopropylethylamine (I mL) in DMSO (25 mL) was stirred
for 20 h. at room temperature before sodium N-methyltaurine
(2.0 g, 12 mmol) , diisopropylethylamine- (1 mL) and DMSO (20
mL) was added. The reaction mixture was then stirred for two
days at about 70 °C and two days at room temperature. The
reaction mixture was filtered through silica followed by
thoroughly washing with methanol. The blue filtrate was
concentrated and the crude product precipitated, as a sticky
blue wax, by addition of ethyl acetate. Column chromatography
(silica, MeCN/MeOH 5:2)' gave a blue solid (first blue
fraction/band), which was further purified by dissolution in
hot ethanol followed by filtration (hot). After cooling the
ethanol solution was filtered again and the precipitate was
washed with ethanol (leaving a white material) . The blue
ethanol solution was evaporated yielding 0.3 g of 4c (18%) .
MS (FAB+, Glycerol): m/z 812 (MH2
+) , 834 (MNaH*) , 856 (MNa/)
XH-NMR (CD3OD, int. solvent ref 3.35 ppm) : 8 5.98 (6H, s) ,
3.95 (4H, t ) , 3.62 (2H, t), 3.57 (6H, s) , 3.56 (12H, s), 3.21
(3H, s), 3.20 (6H, s) , 3.15 (4H, t) , 2.41 (2H, t ) , 1.99 (2H,
m ) .
4c by Method B:
3o (Na*) : A solution of sodium N-methyltaurine (1.3 g, 8
mmol) , and 1 (1.0 g; 1.7 tnraol) in DMSO (25 mL) was stirred
for three days at room temperature. Addition of ethyl
acetate (approx. 500 mL) gave a blue precipitate (and a red
mother liquor). The solid material was heated to reflux in
absolute ethanol (approx. 150 mL) and left overnight at room
temperature. The precipitate was filtered off, dissolved in
methanol and filtered through silica (5 cm layer), which was
washed with methanol. Evaporation of the solvent and reprecipitation
from methanol/ethyl acetate yielded a blue
powder (1.1 g, 88%).
MS (FAB+, Glycerol) : m/z 727 (MH2
+) , 749 (MNaH+) , 765 (MNaK+) ,
771 (MNa2
'•H-NMR (CD3OD, int. solvent ref 3.35 ppm): 8 6.20 (2H, s) ,
5.98 (4H, s), 4.00 (4H, t), 3.87 (3H, s) , 3.58 (12H, s), 3.56
(6H, S), 3.28 (6H, s), 3.17 (2H, t).
4c (Na+) : A solution of 3c (0.5 g; 0.67 mmol), 4-(Nmethylamino)-
butanic acid hydrochloride (0.5 g, 3.26 mmol),
and dry Na2CO3 (0.5 g) in DMSO (7 mL) was stirred for 3 days
at 70 °C. The crude product was precipitated from the cooled
reaction mixture by addition of ethyl acetate, giving a
sticky blue material (ca 1.1 g.). Column, chromatography
26
(silica, MeCN/MeOH 5:2) gave the product 4c as a blue solid
after evaporation of the solvents (0.25 g, 44%).
MS (FAE+, Glycerol) : m/z 812 (MH2+)
(CD3OD, int. solvent ref 3.35 pptn) : 5 5.98 (6H, s) ,
3.95 (4H, t) , 3.62 (2H, t) , 3.57 (6H, s) , 3.56 (12H, s) , 3.21
(3H, a), 3.20 (6H, s) , 3.15 (4H, t) , 2.41 (2H, t) , 1.99 (2H,
m) .
Scheme 5. Ill) TSTU, Diisopropylethylamine, DMSO.
HMCV-3 (Na+) : TSTU (2-succinimido-1,1,3,3-tetramethyluronium
tetrafluoroborate; 0.20 g, 0.6 mmol) was added to a solution
of 4c (0.25 g, 0.3 mmol) and diisopropylethylamine (0.06 g)
in dry DMSO (10 mL) . The reaction mixture was stirred in a
closed flask for 2 h, before the crude product was
precipitated by addition of ethyl acetate. The sticky blue
material was washed from the filter with methanol and dried
at high vacuum. Workup by column chromatography (silica,
H2O/MeCN 1:10) gave, after evaporation of the solvent (high
vacuum at room temperature) and re-precipitation from
methanol/ethyl acetate a dark blue powder (0.25 g, 90%).
MS (FAB+, Glycerol) : m/z 909 (MH2
+) , 931 (MNaH+) , 947 (MKH4) ,
D, int. solvent ref 3,35 ppm) : 5 5.99 (4H, s) ,
5.94 (2H, s) , 3.95 (4H, t) , 3.68 (2H, t) , 3.57 (12H, s) , 3.58
(2H, masked), 3.55 (6H, s) , 3.21 (9H, s) , 3.16 (4H, t) , 2.88
(4H, s) , 2.80 (2H,s), 2.13 (2H, m) .
Attachment of KMCV-1 to Aminodextran
Aminodextran (130 mg, Mw 110.000, 0.5 mmol NH2/g dextran) was
dissolved in 6.5 mL aqueous sodium carbonate (15 mM, pH = 8.5
to give a solution with dextran concentration 182 uM, 20
mg/mL) . To 3.0 mL of this solution under stirring was added
HMCV-1 (5.65 mg in 200 uL DMSO) in 10 uL portions over 10
minutes. The solution was stirred at room temperature for 1
hour and dialysed.(3 mL dialysis slide, 12-14.000 MwCO
membrane) extensively against an aqueous phosphate buffer (10
mM H2P04"/HPO4
2', 4 mM K+, 145 mM Na+, 0 . 1 mM Ca2*, 0 . 1 mM Mr.2*,
pH =7.4) . The concentration of labeled dextran was determined
from the dilution during dialysis. The degree of labelling
(the DOL- value) was determined from a series of UV-Vis
spectra of the resulting solution (3.2 mL, concentration of
dextran 168 uM, DOL = 10) .
Attachment of Alexa Fluor 594™ to Concanavalin A (ConA)
A solution of Con A (Type III, Sigma) was made as follows.
Methyl -a-D-mannopyranoside (0.97 g) , 30 uL aqueous CaCl2 (0.1
M) and 30 p,L aqueous MnCl2 (0.1 M) were mixed in 20 mL 0.5 M
Na2CO3 buffer (pH = 8.5). Then ConA (1.00 g ~ 150 mg protein)
was added and the suspension stirred vigorously for 1 h. The
solution was centrifuged and the supernatant collected.
To 13.0 mL of this solution was added Alexa Fluor 594™ (10.0
mg in 800 mL dry DMSO) in 20 \iL portions over 10 min. The
resulting blue solution was stirred for 1 h and then succinic
anhydride (18.2 mg in 867 uL dry DMSO) was added in 20 [iL
portions over 10 min. The solution was stirred for another
1.5 h after which it was transferred to a 15 mL dialysis
slide and dialysed against the same buffer as described for
the HMCV-1-Dextran synthesis. To the buffer was added 2 mM
NaN3 to prevent growth. (15.4 mL, concentration of protein
dimer 59 ^M, DOL =4.0)
Use of analyte binding reagent labelled with Dye Compound 4
in FRET assay
The glucose measurement assay chemistry of the preferred
embodiment is based on the competition between binding of
dextran and glucose to Concanavalin A (Con A) as discussed
above.
Con A is labelled with Alexa Fluor 594 (AF594) (donor)
and dextran is labelled with a non-fluorescent dye, HMCV-1
(acceptor), absorbing within the emission band of AF594.
The fluorescence lifetime is measured by frequencydomain
fluoritnetry. The intensity of the excitation light is
modulated causing the excited donor to emit light modulated
with the same frequency and delayed by the lifetime of the
excited state. This results in a phase shift between the
excitation light and the emitted fluorescence.
We chose AF594-labelled (ConA) as the sugar binding
lectin and the HMCV-1 labelled dextran as a glucose analogue.
The bulk concentrations of the donor and acceptor dyes used,
[AF594] and [HMCV-1] were constant during the whole
experiment and their ratio was 1: 6. This is consistent with
the Forster theory condition that the donor concentration is
much smaller than the acceptor concentration. [Ref. : C.J,
Rolinski, D.J.S. Birch, L.J. McCartney, J.C. Pickup, J.
Photochem. Photobiol. B: Biol. 54, 26-34, 2000] It is our
finding that the Forster theory is best fulfilled when ConA
is labelled with the donor and the poly-sugar dextran is
labelled with the acceptor.
Measurements
The donor gpnjugate, the acceptor conjugate and the PBSbuffer
(50 mK PBS-buffer, pH = 7.4, ionic strength adjusted
to 150 mM with NaCl) were mixed so that the final
concentration in the assay chemistry of ConA was 20 /xM and
the concentration of dextran was 50 fiM. The molar ratio
([ConA]/[Dex] ) was 0.40 and the concentration ratio
([AF594]/[HMCV-1]) was 0.16. Two hollow cellulose fibres
(Spectrum Laboratories, Inc., regenerated cellulose, fibre
outer diameter 216 /J.mf fibre inner diameter 200 ^m, MWCO 13
kDa Reorder No. 132294) were filled with assay chemistry, and
were then mounted in a custom-made holder placed in a
fluorescence cell containing PBS-buffer.
The phase excitation shift was measured using a phase
and modulation fluorimeter (Koala from ISS, Inc., Champaign,
Illinois). The light source was a yellow LED. A XF1067 band
pass filter, which transmits light from 540 to 590 rim (Glenn
Spectra Ltd) was placed in the excitation channel as well as
in the reference channel. The emission channel was fitted
with a XF3061 band pass filter, which transmits light from
610 to 690 nm (Glenn Spectra Ltd) .
The glucose dose-response was measured by successive
replacement of the PBS buffer with PBS buffer containing
increasing glucose concentration from 0 mM to 50 mM glucose.
The results are shown in Table 1 and in Fig. 1.
(Table Removed)The above assay system was tested in vivo in a clamped pig
experiment. The assay was loaded in a hollow fiber which by a
needle was placed in the subcutus of the skin. The results
from the phase measurements showed a slight phase shift
compared with the in vitro experiment but could be correlated
very well with reference values for whole blood glucose.
The acceptors of the preferred embodiment of the present
invention have a number of advantages over known acceptors.
First, the HMCV acceptors have a broad and intense
absorption spectrum (550 to 700 nm) . This means that they
can be used with a variety of donors (See Fig. 2) .
Second, the acceptors absorb at high wavelengths. This
means that they are suitable for in vivo use at wavelengths
where autofluorescence does not interfere with the
measurements.
Third, the center carbon atom of the dyes is shielded by
ortho substituents on the aromatic moities prohibiting
degradation processes that initiates by either reductive,
oxidative or nucleophilic attack at this position.
Fourth, the absorption spectrum of the acceptors is not
significantly affected by conjugation to analyte or analyte
binding reagent. This means that the performance of the
acceptors is predictable.
Fifth, the acceptors do not fluoresce and will not
contribute to background fluorescence signal.

We Claim;
1. A reagent for detecting an analyte, comprising a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula:
(Formula Removed)
wherein:
R1, R2 and R3 are each independently H, electron donating substituents, or electron withdrawing substituents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups;
R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl, S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more -of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, S03-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen;
and wherein the distance between the energy donor and the energy acceptor of the reagent is capable of modulation by a suitable analyte to be detected.
2. A reagent as claimed in Claim 1, wherein the energy-donor and energy acceptor are linked together by a covalent linkage.
3. A reagent as claimed in Claim 2, wherein the covalent linkage between the energy donor and energy acceptor is cleavable to increase the distance between the energy donor and the energy acceptor of the reagent.
4. A reagent as claimed in Claim 2, wherein the energy donor and energy acceptor are linked via a
polynucleotide sequence or a polynucleotide analogue sequence or a polypeptide sequence, the sequence having a conformation which is capable of modulation by a suitable analyte to be detected so as to modulate the distance between the energy donor and the energy acceptor of the reagent.
5. A reagent as claimed in Claim 1, wherein the energy donor and energy acceptor are linked together by non-covalent binding.
6. A reagent as claimed in Claim 5 wherein the non-covalent binding exists between an analyte binding agent linked to one of the energy donor and the energy acceptor and an analyte analogue linked to the other of the energy donor and the energy acceptor, the non-covalent binding being disruptable by a suitable analyte so as to increase the distance between the energy donor and the energy acceptor of the reagent.
7. A reagent as claimed in Claim 6, wherein the analyte binding agent is a lectin.
8. A reagent as claimed in Claim 6 or Claim 7, wherein the analyte analogue is a glucose analogue.
9. A reagent as claimed in Claim 8, wherein the analyte
analogue is dextran.
10. A reagent, as claimed in Claim 1, wherein the energy donor and the energy acceptor are not linked in the absence of analyte.
11. A reagent as claimed in any one of the preceding claims, wherein a linker structure is attached to the energy acceptor at R3, or where a bridging group is present optionally the linker structure is attached to the energy acceptor at the bridging group.
12. A reagent as claimed in any one of the preceding claims,
' wherein the electron donating substituents are selected from amino, primary amine, secondary amine, O-alkyl, alkyl, S-alkyl, amide, ester, OH and SH.
13. A reagent as claimed in Claim 12, wherein one or more of
R1 to R3 is dimethylamino, diethylamino or
methylethylamino, optionally substituted with one or
more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-,
ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2,
SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine
or tertiary amine.
14. A reagent as claimed in any one of the preceding claims,
wherein an electron withdrawing substituent is present,
and the electron withdrawing substituent is selected
from NO, NO2, CN, COOH, ester, COO", amide, CHO, keto,
SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl,
and SO3-alkyl.
15. A reagent as claimed in any one of the preceding claims, wherein at least one of R10, R11, R12, R13, R14 ANd R15 is O-alkyl.
16. A reagent as claimed in any one of the preceding claims, wherein one or more pairs of groups R4 and R10 and/or R5 and R11 and/or R6 and Rx2 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, .SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine.
17. A reagent as claimed in any one of Claims 1 to 14, wherein R10 to R15 are each O-methyl or O-ethyl.

18. A reagent as claimed in any one of the preceding claims, further comprising one or more counterions selected from halide, BF4-, PF6-, NO3-, carboxylate, C104-, Li+, Na+, K+, Mg2+ and Zn2+.
19. A reagent as claimed in any one of the preceding claims, wherein a linker structure is present, and is formed by reaction of a linker element selected from an active ester, an isothiocyanate, an acid chloride, an aldehyde, an azide, an α-halogenated ketone and an amine with a reaction partner.

20. A reagent as claimed in Claim 19, wherein the reaction partner is selected from a polysaccharide, a polynucleotide and a protein.
21. A reagent as claimed in Claim 19 or Claim 20, wherein the linker element is an active ester, and is selected from succinimidyl and pentafluorophenyl active esters.

22. A reagent as claimed in any one of the preceding claims, wherein the energy donor is Alexa Fluor-594™.
23. A dye compound having the general formula:
(Formula Removed)
wherein:
R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl or S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O-
alkyl, S-alkyl, or alkyl, or one or more pairs of groups R20 and R4 and/or R20 and R5 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkyiene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen;
R15, R17, R18 and R19 are each independently H, alkyl or aryl, or one or more of R16 and R17 or R18 and R19 is alkylene, optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine;
or one or more of pairs of groups R6 and R16, R7 and R17, R8 and R18 and R9 and R19 is alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine
and
R20 is a linker element selected from an active ester, an isothiocyanate, an acid chloride, an α-halogenated ketone, an azide and an amine.
24. A dye compound as claimed in Claim 23, wherein at least one of R10, R11, R12, R13, R14 and R15 is alkyl.
25. A dye compound as claimed in Claim 24, wherein one or more pairs of groups R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3, PO32-, OH, o-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine.
26. A dye compound as claimed in any one of Claims 23 to 25, wherein R20 is a linker element having the structure:
(Structure Removed)
R21 is H or alkyl or aryl optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2N-dialkyl, CN, secondary amine or tertiary amine and R22 is alkylene, O-alkylene, S-alkylene or N-alkylene or R21 and R22 are part of a ring, optionally substituted with

one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2NH2, SO2NH-alkyl, SO2N-dialkyl, SO3-alkyl, CN, secondary amine or tertiary amine; and
R23 is o-succinimidyl, o-pentafluorophenyl, C1 or α-halogenated alkyl.
27. A.dye compound as claimed in any one of Claims 23 to 26, wherein R10 to R15 are each O-methyl or O-ethyl.
28. A dye compound as claimed in any one of Claims 23 to 27, further comprising one or more counterions selected from halide, BF4-, PF6-, NO3-, carboxylate, C1O4-, Li+, Na+, K+, Mg2+ and Zn2+.
29. A method of detecting or measuring an analyte using a reagent as claimed in any one of Claims 1 to 22, comprising the steps of:
contacting the reagent with a sample;
illuminating the reagent and sample with light of
wavelength within the absorption spectrum of the energy
donor;
detecting non-radiative energy transfer between the
energy donor and energy acceptor by measuring the
fluorescence of the energy donor; and
associating the fluorescence measurements with presence
or concentration of analyte.
30. A method as claimed in Claim 29, wherein the fluorescence of the energy donor is measured by measuring making intensity based or time resolved fluorescence measurements.
31. A method as claimed in Claim 29 or 30, wherein the analyte is measured by comparing sample fluorescence measurements with fluorescence measurements made using known concentrations of analyte.
32 . A complex of an analyte and a reagent for detecting the analyte wherein the reagent comprises a fluorescent energy donor and an energy acceptor, the energy donor and the energy acceptor being such that when they are sufficiently close to one another energy is non-radiatively transferred from the energy donor following excitation thereof to the energy acceptor quenching fluorescence of the energy donor, wherein the energy acceptor is of the general formula:
(Formula Removed)
wherein:
R1, R2 and R3 are each independently H, electron donating substituent.s, or electron withdrawing substituents or R3 is attached to a linker structure, provided that at least two of R1, R2 and R3 are electron donating groups;
R4, R5, R6, R7, R8 and R9 are each independently H, halogen, alkyl, aryl, O-alkyl, S-alkyl and R10, R11, R12, R13, R14 and R15 are each independently hydrogen, O-alkyl, S-alkyl, alkyl, or one or more pairs of groups R1 and R4 and/or R1 and R5 and/or R2 and R6 and/or R2 and R7 and/or R3 and R8 and/or R3 and R9 and/or R4 and R10 and/or R5 and R11 and/or R6 and R12 and/or R7 and R13 and/or R8 and R14 and/or R9 and R15 is a bridging group consisting of aryl, alkylene, O-alkylene, S-alkylene or N-alkylene optionally substituted with one or more of SO3-, PO32-, OH, O-alkyl, SH, S-alkyl, COOH, COO-, ester, amide, halogen, SO-alkyl, SO2-alkyl, SO2NH2, SO2NH-alkyl, SO2N-
dialkyl, and SO3-alkyl, CN, secondary amine or tertiary amine, provided that not all of R10, R11, R12, R13, R14 and R15 are hydrogen; and
wherein the presence of the analyte modulates the distance between the energy donor and the energy acceptor.

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Patent Number

278323

Indian Patent Application Number

3562/DELNP/2006

PG Journal Number

53/2016

Publication Date

23-Dec-2016

Grant Date

20-Dec-2016

Date of Filing

20-Jun-2006

Name of Patentee

PRECISENSE A/S

Applicant Address

DR. NEERGAARDS VEJ 3, DK-2970 HORSHOLM, DENMARK

Inventors:

#	Inventor's Name	Inventor's Address
1	NORRILD JENS CHRISTIAN	CARL PLOUGS VEJ 26, DK-3460 BIRKEOED, DENMARK
2	LAURSEN BO WEGGE	MUNKSOEGAARD 69, DK-4000 ROSKILDE, DENMARK

PCT International Classification Number

C09B 11/12

PCT International Application Number

PCT/EP2004/014199

PCT International Filing date

2004-12-14

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0329161.4	2003-12-16	U.K.