CZ2008415A3 - Non-fluorescent phthalocyanine and azaphthalocyanine derivatives as fluorescence extinguisher - Google Patents

Abstract

The use of phthalocyanine, naphthalocyanine, combined phthalo / naphthalocyanine and their analogue of formula I, wherein the individual substituents are of particular interest, to quench fluorescence upon binding to a biomolecule, except in-vivo quenching where the biomolecule can be a nucleic acid or a peptide that can be conjugated to a fluorophore. Applying the above for detecting, quantifying fluorescently active molecules in a sample of a biological sample or tissue. The azaphthalocyanines, azanaphthalocyanines and combined azaphthalocyananaphthalocyanines of the general formula II, where the individual substituents are of particular importance, use them to quench fluorescence upon binding to a biomolecule, in addition to quenching in-vivo fluorescence, wherein the biomolecule can be a nucleic acid or a peptide that can be conjugated to a fluorophore, and for detecting, quantifying the fluorescently active molecules in a sample of a biological sample of material or tissue, as well as a kit wherein the compound of Formula II is bound to a specific DNA / RNA sequence.

Description

(57)

The use of phthalocyanines, naphthalocyanines, combined phthalo / naphthalocyanines and their azaanalogs of the general formula I, where the individual substituents have a specific meaning, to quench fluorescence after binding to a biomolecule. except in-vivo fluorescence quenching, wherein the biomolecule may be a nucleic acid or a peptide. which may be conjugated to a fluorophore. Use of the above for the detection, quantification of fluorescently active molecules in a sample of a biological sample of material or tissue. The azaphthalocyanines, azanaphthalocyanines and combined azaftalo / azanaflalocyanines of the general formula II, where the substituents are of particular importance, are used for quenching fluorescence after binding to a biomolecule, in addition to quenching in-vivo fluorescence. wherein the biomolecule may be a nucleic acid or peptide that can be conjugated to a fluorophore, and for detecting, quantifying fluorescently active molecules in a biological or tissue sample as well as a kit, wherein the compound of Formula II is bound to a specific DNA / RN / Y sequence .

N .... Μ N JÍN í

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Non-fluorescent derivatives of phthalocyanines and azaphthalocyanines as fluorescence quenchers

Technical field

This solution belongs to the field of organic chemistry and biochemistry, respectively. molecular genetics. In particular, it relates to the use of fluorescence quenching molecules in biochemical assays.

BACKGROUND OF THE INVENTION

The use of fluorescent detection methods is increasingly used in biochemical and biophysical research, but also in clinical diagnostics, genetic analyzes, environmental monitoring and other areas where it replaces radionuclide methods. It is a progressive detection method with minimal risks to workers' health and the environment. Indocyanine dyes, fluorescein derivatives, or 4,4-difluoro-4-boron-3a, 4a-diaza-5-indacene are most commonly used as fluorescent labeling molecules.

Fluorescence

Fluorescence occurs when radiation from the excited electron state is emitted by one or more spontaneous energy transitions. Fluorescence is therefore a spin permissible radiative transition, usually from an equilibrium vibration level of the state of Si to one of the vibration levels of the ground state S _o . Fluorescence is observable during excitation of the substance, it disappears almost immediately after its termination, post-discharge time is in the order of ¹⁰ ' ⁸ s.

Fluorescence quenching is a bimolecular process where the quantum yield of fluorescence is reduced without changing the fluorescence emission spectrum.

Some methods of detection or quantitative analysis of DNA use a combination of two dyes, the first serving as a fluorescence emitter (fluorophore) and the second for quenching the emitted fluorescence (quencher). Quenching can be static or dynamic (J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 2nd January, 1999). For static quenching forms

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PV2008-415 fluorescence donor and acceptor non-fluorescent complex. This mechanism does not require overlapping of emission and absorption spectra, it only occurs when both molecules are in close proximity (Johansson, Cook, Chemistry - A European Journal, 9 (2003) 3466-347), (Tyagi, Brat, Kramer, Nature Biotechnology) 16 (1998) 49-53. In contrast, dynamic quenching, in which energy is transferred from excited fluorophores to an acceptor, takes place over a distance (up to about 100 Å) and according to Forster's theory (Forster, Annalen der Physik. 2 (1948) 55-75) the emission spectrum of the fluorescence donor must at least partially overlap with the absorption spectrum of the acceptor. This type of quenching, also referred to as FRET (Foster Resonance Energy Transfer) is also used in hybridization methods.

Based on the use of both dynamic and static fluorescence quenching, many DNA hybridization assays in biological material or tissue are based (Marras, Kramer, Tyagi, Nucleic Acids Research 30 (2002) E122). Either pairs of probes are used, both labeled with a fluorescent dye or one with a fluorescent dye and the other with a mono-labeled probes, or with a single probe labeled with both a fluorescent dye and a quencher molecule (dual-labeled probes). ("Molecular beacon" or TaqMan probes).

Two types (generations) of molecules are currently used to extinguish fluorescence. The first type is now of only marginal importance. These include older fluorescein derivatives (eg tetramethylrhodamines) that exhibit intrinsic fluorescence. This fluorescence causes undesirable background during experiments and thus decreases their sensitivity. Substances without their own fluorescence but with absorption maxima at relatively low wavelengths belong to the first generation. Examples include 6- (N, 4'-carboxy-4- (dimethylamino)) azobenzene (Chen, Wagner, Still, Science 279 (1998), 851-853). This increases the sensitivity of the measurement, but is only applicable to a limited number of fluorophores emitting at low wavelengths.

The so-called "dark quenchers" represent the second, significantly more important group of molecules currently used for quenching fluorescence. These substances exhibit extremely low or zero intrinsic fluorescence. Substances suitable as dark quenchers are limited because most substances have their own fluorescence. In practice, there are only a few structural types of dark quenchers, and therefore the need for completely new structural types is strongly relevant. The absorption maxima of commercially available dark quenchers cover almost the entire fluorescence emission range of the currently used fluorophores, but each is suitable for quenching

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PV2008-415 only a limited number of fluorescent molecules. Certain, not entirely satisfactorily solved problems remain above all when quenching emissions in the long wavelength range, above 650 nm. This applies to some fluorescent indocyanine dyes, eg Cy5 ™, Cy5.5 ™, Cy7 ™ (US 5,556,959 and US 5,808,044). The use of fluorescence quenching molecules in this region is limited by their lack of stability in some applications, eg, under standard conditions of chemical synthesis and processing of oligonucleotides used as labeled probes. The most commonly used dark quenchers are chemically azo dyes. This category includes, for example, the Epoch Eclipse ™ Quencher (US 6,727,356), the Black Hole Quenchers (BHQ ™) (US 7,019,129), and azopyridinium compounds (WO 2005 / 030979A2).

The Epoch Eclipse ™ Quencher, (US 6,727,356) has an absorption maximum at 522 nm, which is suitable for quenching traditionally used fluorescein, but it is too low a wavelength to quench more modern fluorophores such as indocyanine dyes (eg Cy5 ™).

The Black Hole Quenchers (BHQ ™) are comprised of 4 core structures called BHQ0 ™ to BHQ-3 ™. BHQ-0 ™ and BHQ-1 ™ have absorption maxima at 493, respectively. 534 nm and can also be effectively used only for quenching fluorescein or its close rhodamine fluorophores, emitting at lower wavelengths. BHQ-2 ™ (4 '- (4-nitro-phenyldiazo) -2'-methoxy-5'-methoxy-azobenzene) absorbs at 579 nm and can be used to quench the fluorescence of Cy5 ™, but overlapping its emission spectrum (662 nm) with the absorption spectrum of this quencher (which is one of the important conditions for efficient dynamic quenching) is far from optimal. His ability to quench the Cy5.5 ™ fluorophores (emission at approximately 707 nm, depending on the conjugated molecule) and the extrinsic quenching of the Cy7 ™ dye, which emits fluorescence at 770 nm, are then problematic. The last of the BHQ ™ family, BHQ-3 ™ (chemically 3diethylamino-5-phenyl-7- (4-diethylamino) phenylazophenazinium chloride), has an absorption maximum at 672 nm that overlaps well with the emission spectrum of the most commonly used Cy5 ™ indocyanine fluorophores, however, its disadvantage is the lack of stability under normal conditions of oligonucleotide synthesis and processing and hence problematic practical applicability. Similar to BHQ-2 ™, its absorption spectrum does not well cover Cy7 ™ emission.

Other molecules used for quenching fluorescence, QSY ™ quenchers (US 6,399,392), are chemically rhodamine derivatives whose stability under standard oligonucleotide synthesis and deprotection conditions is also not satisfactory. In addition, they also do not cover their own

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PV2008-415 absorption peaks sufficiently over 700 nm. Asymmetric cyanine dyes are also used for quenching (US 6,348,596), also not exceeding an absorption of over 700 nm.

In contrast, the compounds of the present invention are characterized by a structure that is stable both under oligonucleotide synthesis conditions and in the reagents and conditions used conventionally in their processing. Their further advantage is that a suitable combination can achieve an absorption maximum in the range of 620 nm - 770 nm (Figure 1). The region of emission of fluorescein and rhodamines at lower wavelengths then covers another significant absorption band (Figure 1). Thus, compounds optimally quenching all used fluorophores, including indocyanine emitting fluorescence high above 700 nm, can be prepared.

SUMMARY OF THE INVENTION

It is an object of the invention to use compounds of formula (I) as fluorescence quenchers.

Thus, the compounds useful according to the invention are compounds of the following formula (1):

m is 1-8

A, B, C, D are independently the following aromatic rings fused to the basic macrocycle: benzene, 2,3-pyridine, 3,4-pyridine, pyrazine, pyrimidine, pyridazine, 2,3-naphthalene, 2,3-quinoline, 2 , 3-quinoxaline, 6,7-quinoxaline, pyrazinopyrazine.

E is any substituent

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R 1, R 2 are independently H, substituted or unsubstituted saturated or partially or fully unsaturated C 1 -C ₁₈ alkyl substituted or unsubstituted C 1 -C ₁₈ cycloalkyl, which may be partially unsaturated, substituted or unsubstituted saturated or partially or fully unsaturated branched C -C 1-6 alkyl, substituted or unsubstituted 2-18 carbons, substituted or unsubstituted 2-18 carbons, substituted or unsubstituted aryl 6-18 carbons, substituted or unsubstituted heteroaryl 1-18 carbons, and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted 7-18 carbon alkyl, aryl substituted or unsubstituted 2-18 carbon heteroaryl and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted 1-acyl -18 carbons, substituted with or o 1-18 carbon unsubstituted alkoxycarbonyl, 1-18 carbon substituted or unsubstituted aminoalkyl substituent, 1-18 carbon substituted or unsubstituted alkylaminoalkyl, 1-18 carbon substituted or unsubstituted dialkylaminoalkyl, substituted or unsubstituted tri-alkylammonium alkyl containing 1- And wherein the complementary ion is F ', Cl *, B', Γ, CH 3 OSO 3 ', CH 3 CH 2 OSO 3 ·, R 1 and R 2 together form = N-Ar, where Ar is a substituted or unsubstituted aryl containing 6-18 carbons or heteroaryl containing 1 to 18 carbons and 1 to 6 heteroatoms of O, S, N, Se, R 1 and R 2 together form = N-OR 3. The above R 1 and R 2 may also be combined into a cycle which may be part of other saturated or unsaturated cycles, including a heterocycle. The foregoing R 1 and R 2 may also be linked to the ring linking two NR 1 R 2 substituents on one A to the ring. The above R 1 and R 2 may also be joined to the ring linking the two NR 1 R 2 substituents on A and B or A and C into the ring. If m> l. then the individual NR1R2 may or may not be the same. The alkyl portions of the contemplated substituents may also be unsaturated.

M is 2H, 2Li, 2Na, 2K, 2Rb, 2Cs, Be, Mg, Ca, Sr, Ba, Sc, Y, La, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pt, Pt. Cu, Ag, Au, Zn, Cd, Hg, B, Al, Ga, In, Ti, Si, Ge, Sn, Pb, As, Sb, Bi, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tb, Yb, Lu, Th, U.

Y is halogen, OR 3, NR 1 R 2, SR 3, OSOR 3 R 4 R 5

A: is 0 to 2.

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R 3, R 4, R 5 are independently H, substituted or unsubstituted saturated or partially or fully C ₁ -C ₁₈ unsaturated alkyl, substituted or unsubstituted C 3 -C 18 cycloalkyl, which may be partially unsaturated, substituted or unsubstituted branched saturated or partially or fully unsaturated _Ci-C18 alkyl, substituted or unsubstituted polyalkyleneoxy chain containing 2-18 carbons, substituted or unsubstituted branched polyalkyleneoxy chain containing 2-18 carbons, substituted or unsubstituted aryl containing 6 to 18 carbons, substituted or unsubstituted heteroaryl containing 1 to 18 carbons, and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted alkyl of 7-18 carbon atoms, substituted or unsubstituted alkyl heteroaryl of 2-18 carbons, and 1 to 6 heteroatoms of O, S, N, Se. The alkyl portions of the contemplated substituents may also be unsaturated.

Substituent E is arbitrary, the following independent substituents are preferred: H, substituted or unsubstituted C 1 -C 18 alkyl, substituted or unsubstituted C 1 -C 18 cycloalkyl, which may also be partially unsaturated, substituted or unsubstituted branched C 1 -C 6 alkyl, substituted or unsubstituted partially or fully unsaturated C 1-6 alkyl, substituted or unsubstituted 2-18 carbon-substituted polyalkylene chain, 2-18 carbon substituted or unsubstituted branched chain polyalkyleneoxy, 6-18 carbon substituted or unsubstituted aryl, 1-18 carbonated or unsubstituted heteroaryl and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted alkyl of 7-18 carbon atoms, substituted or unsubstituted alkyl heteroaryl of 2-18 carbons, and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 1 -C 8 ₈ alkoxy, substituted or unsubstituted C 6 -C 18 alkylsulfanyl, substituted or unsubstituted 6-18 carbons, substituted or unsubstituted aryloxy containing 6-18 carbons, substituted or unsubstituted 6-18 carbonated arylsulfanyl, 6-18 substituted or unsubstituted arylsulfanyl carbon atoms and 1 to 6 heteroatoms of O, S, N, Se contained in the ring, substituted or unsubstituted heteroarylsulfanyl containing 6-18 carbons and 1 to 6 heteroatoms of O, S, N, Se contained in the ring, halogen, -NO2, -NO, - COH, -CN, -NCS, -NCO, COOR 3, -C0NR1R2, -COC1, --COBr, -SO ₂ C 1, -SO ₂ NR1R2, _-SO3 R3 -OH (which may be esterified), -SH ( which can also be esterified), -SS- (2-pyridyl), -PO (OR 3) (OR 4), 7/20

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OP (OR 3) (NR 1 R 2), -N ?. Substituent E may also include salts formed from both acidic and basic functional groups in a generally known manner. Substitute E may also include quaternary ammonium salts. The alkyl portions of the contemplated substituents may also be unsaturated.

Preferred compounds are those wherein m is 2-8. Preferred compounds are those wherein A, B, C and D independently of each other are especially pyrazine or 6,7-quinoxaline. Preferred compounds are those without central metal (M = 2H).

Particularly preferred compounds are those of formula (II),

wherein A, B, C and D are independently pyrazine or 6,7-quinoxaline,

R 6, R 7, R 8, R 9, R 10, R 11, R 12 are independently H, saturated or partially or fully unsaturated C 1 -C 6 alkyl, phenyl, heteroaryl containing 1 to 6 carbons and 1 to 6 heteroatoms of O, S, N, Se, 7-10 carbon alkyl aryl, 2 to 7 carbon alkyl heteroaryl and 1 to 6 heteroatoms of O, S, N, Se, 1-8 carbon acyl, saturated and partially unsaturated C 1 -C 10 cycloalkyl. R 6 and R 7, R 8 and R 9, R 10 and R 11, R 12 and R 13 may also be combined to form a saturated or partially unsaturated cycle. R 6 and R 8, R 10 and R 12 may also be combined to form a saturated or partially unsaturated cycle.

R 13 is suitably substituted saturated or partially or fully unsaturated C 3 -C 12 alkyl, suitably substituted phenyl, suitably substituted heteroaryl containing 1 to 6 carbons and 1 to 6 heteroatoms O, S, N, Se, suitably substituted 7-12 carbons, suitably substituted alkyl heteroaryl containing 2 to 7 carbons and 1 to 6 heteroatoms of O, S, N, Se.

A suitable substituent is -OH, -SH, -SS- (2-pyridyl), -NHR 3, -NCS, -NCO, -COH, -N ₃ ,

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-COCl, -SO2Cl, -OP (OR3) (NR4R5), both substituted and unsubstituted -COOH, -SO3H, -PO3H2.

Further suitable substituents are active esters, in particular pentafluorophenyl ester, p-nitrophenyl ester, and active esters of the -COOX type, wherein X belongs to the following structures:

M is 2H, Zn, Cu, Ni, Cd, Mg;

R 3, R 4, R 5 are as defined for formula (I).

The preferred compounds are not fully exhausted or restricted.

The subject compounds belong to the group of phthalocyanine and azaphthalocyanine dyes and their extended homologues. To date, these dyes have been used in many different applications, such as: tobacco smoke filtration (EP 1594376), optical data carriers (CZ 2003-832 A3), mineral oil labeling (CZ 296339 B6), photodynamic therapy (Zimcik, Miletin) , Musil, Kopecky, Kubza, Brault, Journal of Photochemistry and Photobiology A: Chemistry 183 (2006) 59-69), as fluorescent probes (US 5,438,135). The method for preparing the subject compounds is based on general knowledge of the synthesis of these macrocycles (Kadish, Smith, Guilard: The Porphyrin Handbook, vols. 15-20. New York: Academic Press, 2003). In contrast to the above examples, the subject compounds exhibit very low to zero fluorescence and, depending on the size of the conjugate system, their absorption spectrum overlaps in a sufficient amount the fluorescence emission spectra of the fluorophores used (Fig. 1). They are therefore suitable for both dynamic and static quenching of fluorescence.

A common feature of the present compounds is the basic macrocyclic nucleus responsible for the absorption of radiation emitted by fluorophores. The nature of the basic skeleton (or A, B, C and D) affects the shift (bathochromic or hypsochromic) of the main absorption maximum, which is in the region of approximately 620 nm - 800 nm, corresponding to overlapping emission spectra of commonly used fluorophores, even those emitting at high wavelengths above 700 nm (Fig. I). In addition, the present compounds exhibit an additional absorption maximum in the region of about 500 nm which is suitable for fluorophores emitting at low wavelengths. The basic prerequisite for the quenching of fluorescence is the presence of at least one peripheral substituent NR1R2, which is responsible for the low and / or low carbon content. zero emission of absorbed energy

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PV2008-415 in the form of a photon and therefore also for quenching the fluorescence emitted by the fluorophore. The energy received by the quencher (the subject compound) from the fluorophore is released by electron hopping from the free electron pair of nitrogen of this group into the conjugated system (the so-called photoinduced electron transfer) and therefore no photon emission occurs. The other E substituents no longer play a significant role in the actual mechanism of action.

The number of NR 1 R 2 substituents, or m, may be 1 to 8 so that the subject compounds do not have intrinsic fluorescence and thus be useful for quenching. Compounds prepared according to Examples Ιό have different m. Compound (VII) (m-ϋ) does not quench fluorescence and even emits strong fluorescence itself (quantum yield of fluorescence in pyridine is 0.13). Compound (IX) (m = 1) already has a very low intrinsic fluorescence (quantum yield of fluorescence in pyridine is 0.005). Compounds (III-VI) (m = 8) and (VIII) (m = 2) no longer have detectable fluorescence and are therefore ideal for quenching fluorescence.

The invention also relates to the use of the present compounds for solid phase labeling (Example 7) followed by synthesis of nucleic acid-type biomolecules (Example 8), peptides, proteins, or preparation of conjugates. Biomolecules can also be labeled post-synthetically either fluorophore or subject compounds generally known methods (Haugland, Handbook of Fluorescent Probes and Research Products, 9th edition, Molecular Probes Inc.:Eugene, 2002). Preferred biomolecules are nucleic acids.

The invention furthermore relates to the use of biomolecule conjugates with the present compounds for quenching fluorescence, both by dynamic and static quenching, in standard fluorophores emitting in the range of 400 nm to 820 nm. Example 9 also demonstrates the functionality of both types of quenching. Example 9 also demonstrates the quenching functionality of the so-called mono-labeled probes (i.e., biomolecule conjugates bound with only one dye either fluorophore or quencher). Example 10 shows the use for dual-labeled probes. for conjugates that contain both dye fluorophore and quencher simultaneously on one biomolecule.

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Description of the picture

Giant. 1, Comparison of absorption spectra of compounds ΙΠ, IV and V with emission maxima (upper series) of commonly used fluorophores.

Giant. 2. A synthetic route for producing a conjugate of compound (III) with nucleic acids,

Giant. 3. Direct recording of the detected fluorescence generated by the reaction of the probe, oligonucleotide S5, prepared according to Example 8, in the reaction described in Example 10. Two adjacent lines each represent a duplicate of the reaction. A pair of lower fluorescence intensity lines represents a comparative probe labeled with the BHQ-2 ^1M quencher molecule, a pair of higher intensity lines represents a probe, oligonucleotide S5. The drawing shows a higher fluorescence increase in the S5 oligonucleotide sample compared to the BHQ-2 ™ quenching molecule probe, ie significantly better quenching parameters of the quenching molecule of the invention and used in the S5 oligonucleotide over the BHQ-2 quenching molecule. ™.

Giant. 4. Fluorescence Recording FIG. 3 on a logarithmic scale. It is evident from the figure that the Ct value is not changed, ie the value at which the detected fluorescence crosses the set threshold and therefore the essential parameters of the actual reaction are not changed.

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Examples

Examples of compounds suitable for use in the invention

Example 1: Preparation of compound of formula (III):

(1Π)

816 mg of 5,6-bis (diethylamino) pyrazine-2,3-dicarbonitrile and 330 mg of 4 - [(5,6-dicyano-3-diethylaminopyrazin-2-yl) -methylamino] benzoic acid are dissolved in 15 ml of anhydrous butanol. and 200 mg of lithium metal are added at boiling point. The mixture is kept under boiling for 3 hours, the solvent is distilled off under reduced pressure and 50 ml of a 50% acetic acid solution are added to the residue. After 30 minutes, the precipitate is filtered off and the filtered residue is washed thoroughly on the filter with water. After drying, the reaction mixture was purified by column chromatography on silica gel with dichloromethane / acetone / methanol 10/1/1. The second intense violet fraction from the column was collected. Purification of this fraction was carried out by column chromatography with hexane / ethyl acetate / acetic acid 6/6/1. 150 mg of compound (III) is obtained, i.e. 10% of the theoretical yield. UV-vis (tetrahydrofuran): λ _max (nm) 680,654, 518, 370.

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Example 2: Preparation of Compound of Formula (IV):

966 mg of 5,6-bis (diethylamino) quinoxaline-6,7-dicarbonitrile and 330 mg of 4 - [(5,6-Dicyano-3-diethylaminopyrazin-2-yl) -methylamino] benzoic acid are dissolved in 15 ml of anhydrous butanol, and 200 mg of lithium metal are added to the boil. The mixture is kept under boiling for 3 hours, the solvent is distilled off under reduced pressure and 50 ml of a 50% acetic acid solution are added to the residue. After 30 minutes, the precipitate is filtered off and the filtered residue is washed thoroughly on the filter with water. After drying, the reaction mixture was purified by column chromatography on silica gel with dichloromethane / acetone / methanol 10/1/1. A second intensely colored fraction emerging from the column was collected. Purification of this fraction was carried out by column chromatography with hexane / ethyl acetate / acetic acid 6/6/1. 180 mg of compound (IV) is obtained. UV-vis (tetrahydrofuran): λ max (nm) 752,713,685,644,477, 372.

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Example 3: Preparation of Compound of Formula (V)

Compound (V) is prepared as described in Musil, Zimcik, Miletin, Kopecky, Lenco, European Journal of Organic Chemistry 27 (2007) 4535-4542.

Example 4: Preparation of Compound of Formula (VI)

816 mg of 5,6-bis (diethylamino) pyrazine-2,3-dicarbonitrile and 330 mg of 4 - [(5,6-dicyano-3-diethylaminopyrazin-2-yl) methylamino] butanoic acid are dissolved in 15 ml of anhydrous butanol and 200 mg of lithium metal are added while boiling. The mixture is kept under boiling for 3 hours, the solvent is distilled off under reduced pressure and 50 ml of a 50% acid solution are added to the residue.

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PV2008-415 acetic. After 30 minutes, the precipitate is filtered off and the filtered residue is washed thoroughly on the filter with water. After drying, the reaction mixture was purified by column chromatography on silica gel with dichloromethane / acetone / methanol 10/1/1. The second intense purple fraction flowing from the column was collected. Purification of this fraction was carried out by column chromatography with hexane / ethyl acetate / acetic acid 6/6/1. The obtained product was dissolved in anhydrous dimethylformamide (10 mL) and anhydrous copper acetate (180 mg) was added and heated at 150 ° C for 4 hours. The solution was concentrated on a vacuum evaporator and distilled water (40 mL) was added. The precipitate was filtered off, washed with water and, after drying, purified by column chromatography on silica gel with chloroform / acetone / methanol 20/1/1. There were obtained 120 mg (Vij. UV-vis (tetrahydrofuran): _{λ! ΜΧ} 654, 596, 500, 362.

Example 5: Preparation of Compounds of Formula (VII) and Formula (VIII)

Magnesium (672 mg) was heated in anhydrous butanol (15 mL) for 5 hours. Then 612 mg of 5,6-bis (tert-butylsulfanyl) pyrazine-2,3-dicarbonitrile and 544 mg of 5,6bis (diethylamino) pyrazine-2,3-dicarbonitrile are added to the reaction mixture. The mixture was heated at 130 ° C for 12 hours. The butanol was distilled off using a vacuum evaporator and 50 ml of a 50% acetic acid solution were added to the mixture. After 30 minutes, the precipitate is filtered off and the filtered residue is washed thoroughly on the filter with water. After drying, the mixture was dissolved in tetrahydrofuran and 1 ml of trifluoroacetic acid was added and stirred at room temperature for 90 minutes. Then the solvents were evaporated under reduced pressure and the mixture was washed with water. After drying, the reaction mixture is purified by column chromatography on silica gel with chloroform / toluene / ethyl acetate 7/7/1. First intensively

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PV2008-415 the green fraction corresponds to compound (VII), the second fraction (intense green-blue) emerging from the column corresponds to compound (VIII). Purification of both fractions was carried out by column chromatography with chloroform / ethyl acetate 30/1 for compound (VII); and chloroform / toluene / ethyl acetate

7/7/1 for compounds (VIII); Compound (VII) UV-vis (tetrahydrofuran): λ _max (nmr) 671,640,615,

588,477, 366. Compound (VIII) UV-vis (tetrahydrofuran): λ _max (nm) 670,649,569,470,367.

Example 6: Preparation of Compound of Formula (IX)

N

N

Compound (IX) was prepared in a manner similar to Compound (VIII) described in Example 5, except that 5-diethylamino-6-tert-butylsulfanylpyrazine-2,3-dicarbonitrile was used in place of 5,6-bis (diethylamino) pyrazine-2,3-dicarbonitrile. UV-vis (tetrahydrofuran): (nm) 672,

646.619, 593, 477.366.

Examples of use according to the invention

Example 7: Preparation of solid phase for synthesis of oligonucleotide modified with fluorescence quenching compound of formula (III) (Fig. 2)

Compound (III) prepared according to Example 1 (117 mg) was dissolved in 10 ml of anhydrous tetrahydrofuran and N, N-dicyclohexylcarbodiimide (25 mg) and N-hydroxysuccinimide (14 mg) were added.

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PV2008-41S mg). It is allowed to stir for 24 hours at room temperature. To the resulting succinimidyl ester in the reaction mixture was added 3-aminopropane-1,2-diol (45 mg). The resulting amide was isolated from the reaction mixture by column chromatography using dichloromethane / acetone / methanol 30/1/1 as the mobile phase. The primary hydroxy group of 3-amino-1,2-propanediol in the prepared amide is protected by treatment with dimethoxytrites (chloride (41 mg) in anhydrous pyridine (5 mL) at room temperature for 24 h) followed by esterification of the secondary hydroxy group with succinic anhydride (13 mg). pyridine (5 mL) at 60 ° C for 12 h.

The thus-modified compound (III) (9 mg) was dissolved in anhydrous dimethylformamide, to which O- (benzotriazol-1-yl) -1,1,3,3-texramethyluronium hexafluorophosphate (HBTU) (7 mg) was added, after 10 min. add anhydrous triethylamine (20 μΐ) and add this mixture to the aminomodified solid phase (amino-lcaa-CPG) (150 mg). It is allowed to stir at room temperature for 24 hours. Then the solid phase was washed with dimethylformamide, acetonitrile and diethyl ether. To avoid synthesis of the oligonucleotide chain on the optionally unreacted free amine groups of the solid phase, these groups are acetylated with a mixture of acetic anhydride and 1-methylimidazole in pyridine and tetrahydrofuran at room temperature for 15 minutes.

Example 8: Synthesis of an oligonucleotide modified with a fluorescence-quenching molecule

Oligonucleotide synthesis is performed on a standard DNA / RNA synthesizer (eg, ABI 392/394 RNA / DNA Synthesizer) using a solid phase prepared according to Example 7 by a standard synthesis program after cleavage of the dimethoxytrityl protecting group in an acidic medium, with 2% (w / w) solution. trichloroacetic acid in dichloromethane. Likewise, cleavage of the oligonucleotide from the solid phase and its unblocking is carried out by a standard procedure (32% ammonia solution / 40% methylamine solution, 1/1.25 ° C, 90 minutes).

Oligonucleotides with sequences S1, S2, S3 and S5 were synthesized on a solid-phase modified fluorescence quenching molecule prepared according to Example 7. Oligonucleotide S5 is labeled at its 5 'end with a commercially available fluorescent dye Cy5 ™.

In the standard oligonucleotide synthesis phase, an oligonucleotide with a S4 sequence complementary to the S1 and 24 sequences of S2 and S3, labeled with the commercially available fluorescent dye Cy5 ™, was synthesized.

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An oligonucleotide was synthesized on a commercially available quencher labeled BHQ®-2 quencher and subsequently labeled at its 5'-end with a commercially available fluorescent dye Cy5 ™. Oligonucleotides with sequence and S8 serving as primers for PCR in Example 10 were synthesized on the standard oligonucleotide synthesis phase.

S l: 5 GAA GTC GGG TGG TCA

S2: 5'- GAA GTC GGG TGG TCA

S3: 5GAA GTC GGG TCA AAA AGC AGA TTT TTT TTT T - Q - 3 '

S4; 5Cy5 ™ - TCT GCT TTT TGA CCA CCC GAC TTC - 3 '

S5: 5'- Cy5 ™ - GAA GTC GGG TGG TCA

S6: 5'- Cy5 ™ - GAA GTC GGG TGG TCA AAA AGC - BHQ®-2 - 3 '

S7: 5 'CCC TGA ACA CGG

S8; 5'- CTA TGC AAC ATT CGA GTGA TCT T-3 '

Q is the preferred compound (III) modified with an aminopropanediol spacer to bind to the oligonucleotide (Figure 2).

Example 9: Examples of fluorescence quenching

Oligonucleotide S4 was dissolved in buffer hybridization to a concentration of 0.05 μΜ and its fluorescence was measured at 690 nm after excitation at the absorption maximum of the fluorophore (651 nm).

Oligonucleotide solution S4 and oligonucleotide solution S4 were then added. S2 or S3 in the hybridization buffer so that its concentration in the final mixture is 0,25 μΜ. After hybridization, the fluorescence of the S4 + S1, respectively, solutions was measured. S4 + S2 and S4 + S3. The measured decrease in fluorescence value (X) was calculated according to the relation F _x / Fo, where Fo is the fluorescence of the S4 solution itself and F _x is the fluorescence of the S4 solution when mixed with one of the solutions S1, S2 or S3. The X value was 89.5% (for S4 + S3), 94.0% (for S4 + S2) and 89% (for S4 + S3). Oligonucleotide S1 is suitable for the determination of static quenching, oligonucleotide S3 is suitable for the determination of dynamic quenching. Oligonucleotide S2 exhibits both quenching principles. These values are quite sufficient for efficient fluorescence quenching in DNA hybridization assays and are comparable or better than commonly used fluorescence quenching molecules (Marras, Kramer, Tyagi, NucleicAcids Research 30 (2002) El22).

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PV2008-415

Example 10: Use of a fluorescence-quenching modified oligonucleotide in real-time

PCR (polymerase chain reaction) using hydrolysis probes

Real-time polymerase chain reaction (qPCR) is a commonly used molecular genetic technique used to detect and quantify a specific DNA / RNA sequence, which is determined by the sequence of the primers and probes / probes. In one of the widely used variants it uses so called hydrolysis or hybridization probes. In the presence of the detected DNA sequence, it is amplified / multiplied, and with increasing amount of the detected sequence in the reaction, it decomposes (by using the hydrolysis probe) (nuclease activity of the polymerase enzyme), releasing the fluorescent dye from the quencher molecule and exponentially increasing fluorescence. The presence of the detected DNA / RNA sequence is determined in DNA / RNA samples isolated by conventional nucleic acid isolation techniques from biological material or tissue, or can be detected directly in tissue in-situ.

PCR or qPCR are generally used in the form of kits for the detection of specific DNA / RNA sequences of the general composition according to TAB1. Real-time PCR or real-time PCR is a general knowledge for those of ordinary skill in molecular biology.

The S5 oligonucleotide synthesized according to Example 8 is labeled at its 5 'end with a Cy5 ™ fluorescent molecule, at its 3' end with compound (III). Oligonucleotide S6 of the same sequence was labeled with the commercial quencher BHQ®-2 at its 3 'end and labeled with a Cy5 ™ fluorescent molecule at its 5' end. Oligonucleotides S5 and S6 were further used in quantitative realtime PCR as hydrolysis probes. A system using hydrolysis probes of S5 and S6 sequences together with primers S7 and S8 are used to detect and quantify a specific Herpes Simplex 1 (HSV1) sequence. The composition of the reaction mixture is shown in Table 1 (TAB.1) and HSV1 DNA of 20 ng / μΐ was used as template DNA. The actual PCR was performed in the program: 95 ° C initial denaturation for 3 minutes followed by 55 cycles: 95 ° C / 15sec., 65 ° C / 60sec. Fluorescence measurements were performed at EX625nm / EM660nm at the end of each 65 ° C step. Fluorescence is recorded continuously during qPCR. The Y-axis shows the fluorescence intensity of individual samples, the X-axis shows the reaction time expressed by the number of cycles. Based on the so-called baseline or background fluorescence, the software determines a specific fluorescence intensity value, the so-called threshold value. In place,

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PV2008-415 where the detected fluorescence of the sample crosses this imaginary line is subtracted on the X-axis cycle. The cycle in which the threshold line intersects is called the Ct (threshold cycle) value. The read Ct value then directly corresponds to the amount of DNA detected at the start of the reaction (ie in the sample), the more detected DNA in the sample the lower the Ct value (ie, the fluorescence increases earlier). A record of the measured fluorescence during PCR is shown in Figure 3 and a mathematically adjusted logarithmic scale record to determine the Ct value based on the specified threshold is shown in Figure 4. Subsequently, the obtained fluorescence data was analyzed in individual cycles for samples with BHQ® labeled probes. -2 or AzaPc in duplicates (TAB.2). Data analysis included determining the Ct value based on exceeding the threshold, calculating the baseline fluorescence and calculating the fluorescence increase, all including mean and standard deviations. Baseline fluorescence expresses the quencher's ability to quench a fluorophore in an intact oligonucleotide sequence at the start of a reaction, the lower the value, the more efficient quenching occurs. In contrast, the increase in fluorescence expresses the efficiency with which the probe decomposes during PCR, the higher the value, the more effectively the probe decomposes (the better). The data show that compared to the commercially available BHQ -2 quencher, the subject compounds are totally comparable and show an even higher fluorescence increase (Table 2, "average increase" column).

TAB.l: Composition of reaction mixture for 1 reaction of 20μ1.

Total volume of mixture: [stock concentration] [reaction mixture] 20,00 ddH ₂ O 7.85 PCR Buffer (10x) 2.00 MgCl ₂ / [mM] 25 5.0 4.00 dNTP / dUTP mix / [μΜ] 10 000 200 0.40 Primer F / [nM] 250.00 1.00 Primer R / [nM] 250.00 1.00 Proba / [nM] 150.00 1.00 Taq polymerase / [U / μΙ] resp. [AT] 5.00 1,25 0.25 Template DNA 2.50

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TAB.2 Analysis of fluorescence data.

Name / designation Ct diameter Ct SDCt increase Avg increase SD increase F baseline Avg F baseline SD F baseline S6 rep. 1 31.67 31,605 0,065 1.89 1.87 0,0255966 4.15 4.20 0,04544434 S6 rep.2 31.54 1.84 4.24 S5 beet 1 31.42 31,425 0.005 2.15 2.23 0,0754184 4.29 446 0,03143429 S5 rep.2 31.43 2.30 4.23

PV2008-415

Claims (11)

Patent claims Use of phthalocyanines, naphthalocyanines, combined phthalocyanines and their azaanalogs of the general formula I wherein n is 0-15 m is 1-8 A, B, C, D are independently the following aromatic rings fused to the basic macrocycle: benzo, 2,3-pyrido, 3,4-pyrido, pyrazino, pyrimido, pyridazino, 2,3-naphthaleno, 2,3-quinolino, 2 3-quinoxalino, 6,7-quinoxalino, pyrazinopyrazino; E is independently H, substituted or unsubstituted C 1 -C 18 alkyl, substituted or unsubstituted C 3 -C 18 cycloalkyl, which may also be partially unsaturated, substituted or unsubstituted branched C 1 -C 18 alkyl, substituted or unsubstituted partially or fully unsaturated C 1 -C 18 alkyl; C _i8 alkyl, substituted or unsubstituted polyalkyleneoxy chain containing 2-18 carbon atoms, a substituted or unsubstituted branched polyalkyleneoxy chain containing 2-18 carbon atoms, a substituted or unsubstituted aryl containing 6 to 18 carbon atoms, substituted or unsubstituted heteroaryl containing 1 to 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 7-18 alkylaryl, substituted or unsubstituted C 2-18 alkylheteroaryl and 1 to 6 O, S, N, Se heteroatoms, substituted or unsubstituted C 1 -C 18 alkoxy, substituted or unsubstituted C 6 -C 18 alkylsulfanyl, substituted or unsubstituted aryloxy containing 6-18 carbon atoms, substituted or unsubstituted arylsulfanyl containing 6-18 atoms PV2008-41S 2 / S carbon, substituted or unsubstituted heteroaryloxy containing 6-18 carbon atoms and 1 to 6 heteroatoms O, S, N, Se contained in the ring, substituted or unsubstituted heteroarylsulfanyl containing 6-18 carbon atoms and 1 to 6 heteroatoms O, S, N, Se contained in the ring, halogen, -NO ₂ , -NO, -COH, -CN, -NCS, -NCO, -COOR ₃ , -COR ₁ R ₂ , -COC 1, COBr, -SO ₂ Cl, -SO ₂ NR ₁ R ₂ , - SO ₃ R 3, -OH (which can also be esterified), -SH (which can also be esterified), -SS- (2-pyridyl), -PO (OR 3) (OR 4), -OP (OR 3) (NR 1 R 2) -N _3; substituents E may also comprise salts formed from both acidic and basic functional groups in a generally known manner; substituent E may also include quaternary ammonium salts; the alkyl moieties of the contemplated substituents may also be unsaturated, R 1, R 2 are independently H, substituted or unsubstituted saturated or partially or fully unsaturated C 1 -C 18 alkyl, substituted or unsubstituted C 1 -C 18 cycloalkyl, which may be both partially unsaturated, substituted or unsubstituted saturated or partially or fully unsaturated branched C 1 -C 18 cycloalkyl C 18 alkyl, substituted or unsubstituted 2-18 carbon-substituted polyalkyleneoxy, substituted or unsubstituted branched 2-18 carbon-containing polyalkylenoxy, substituted or unsubstituted aryl of 6-18 carbon atoms, substituted or unsubstituted heteroaryl containing 1-18 carbon atoms, and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 7-18 alkylaryl, substituted or unsubstituted C 2-18 alkyl heteroaryl and 1 to 6 O, S, N, Se heteroatoms, substituted or unsubstituted acyl containing 1-18 carbon atoms, substituted or unsubstituted 1-18 carbon alkoxycarbonyl, 1-18 carbon substituted or unsubstituted aminoalkyl substituent, 1-18 carbon substituted or unsubstituted alkylaminoalkyl, 1-18 substituted or unsubstituted dialkylaminoalkyl carbon, substituted or unsubstituted trialkylammonium alkyl containing 1-18 carbon atoms and wherein the complementary ion is F ', Cl', Br, Γ, CH ₃ OSO ₃ ·, CH ₃ CH ₂ OSO ₃ ', R 1 and R 2 together form = NAr, where Ar is substituted or unsubstituted aryl of 6-18 carbon atoms or heteroaryl of 1 to 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se, or R1 and R2 taken together form = N-OR3; the aforementioned R1 and R2 may also be linked to a cycle that may be part of other saturated and unsaturated cycles, the aforementioned R1 and R2 may also be linked to a cycle linking two NR1R2 substituents on one A to the cycle; the above R1 and R2 may also be linked to the ring linking the two NR1R2 substituents on A PV2008-415 O 2, -3 / A and B or A and C into a cycle; if m> 1, the individual NR 1 R 2 may or may not be the same; the alkyl portions of the contemplated substituents may also be unsaturated; M is 2H, 2Li, 2Na, 2K, 2Rb, 2Cs, Be, Mg, Ca, Sr, Ba, Sc, La, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Hg, B, Al, Ga, In, Si, Ge, Sn, Pb , As, Sb, Bi, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Th, U; Y is halogen, OR 3, NR 1 R 2, SR 3, OSOR 3 R 4 R 5; k is 0 to 2; R 3, R 4, R 5 are independently H, substituted or unsubstituted saturated or partially or fully unsaturated C 1 -C 18 alkyl, substituted or unsubstituted C 3 -C 18 cycloalkyl, which may be partially unsaturated, substituted or unsubstituted branched saturated or partially or fully unsaturated C 1 -C 18 alkyl, substituted or unsubstituted polyalkylenoxy chain containing 2-18 carbon atoms, substituted or unsubstituted branched polyalkylenoxy chain containing 2-18 carbon atoms, substituted or unsubstituted aryl containing 6-18 carbon atoms, substituted or unsubstituted heteroaryl containing 1-18 atoms carbon and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 7-18 alkylaryl, substituted or unsubstituted C 2-18 alkylheteroaryl and 1 to 6 O, S, N, Se heteroatoms; the alkyl portions of the contemplated substituents may also be unsaturated to quench fluorescence upon binding to the biomolecule, in addition to quenching fluorescence in vivo. Use according to claim 1, wherein the biomolecule is: a. a nucleic acid molecule or a synthetic analogue thereof b. a protein or peptide molecule. Use according to claim 2, wherein the biomolecule is conjugated to the fluorophore beforehand or later. Use according to claims 2 or 3 for detecting and quantifying fluorescently active molecules in a sample of biological material or tissue. PV2Q08-415 5. Azaphthalocyanines, azanaphthalocyanines and combined azaphthalocyanines The general formula (II) wherein A, B, C and D are independently the following aromatic rings fused to a basic macrocycle: pyrazino or 2,3-disubstituted 6,7-quinoxalino; R 6, R 7, R 8, R 9, R 10, R 11, R 12 are independently H, saturated or partially or fully unsaturated C 1 -C 6 alkyl, phenyl, heteroaryl having 1 to 6 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se, (C ₇ -C 10) alkylaryl, (C 2 -C 7) alkylheteroaryl and (C 1 -C 6) heteroatoms of O, S, N, Se, C 1-8 acyl, saturated or partially unsaturated C ₃ -C 10 cycloalkyl; R 6 and R 7, R 8 and R 9, R 10 and R 11, R 12 and R 13 may also be combined to form a saturated or partially unsaturated cycle; R 6 and R 8, R 10 and R 12 may also be combined to form a saturated or partially unsaturated cycle; R 13 is suitably substituted saturated or partially or fully unsaturated C ₃ -C ₁₂ alkyl, suitably substituted phenyl, suitably substituted heteroaryl containing 1 to 6 carbon atoms and 1 to 6 heteroatoms O, S, N, Se, suitably substituted alkylaryl containing Ί12 carbons , suitably substituted alkyl heteroaryl containing 2 to 7 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se; a suitable substituent is -OH, -SH, -SS- (2-pyridyl), NHR ₃ , -NCS, -NCO, -COH, -N ₃ , -COC 1, -SO ₂ Cl, -OP (OR 3) (NR 4 R 5) substituted and unsubstituted -COOH, -SO ₃ H, -PO ₃ H ₂ ; further suitable substituents are the active esters of the pentafluorophenyl ester type, the pnitrophenyl ester, and the active esters of the -COOX type, wherein X belongs to the following structures: PV2008-415 M is 2Η, Ζη, Cu, Ni, Cd, Mg; R3, R4, R5 are as defined in claim 1. except compounds where A = B = C = D = pyrazino and M = 2H and R6 = R7 = R8 = R9 = R10-R11 = ethyl and R12 = H and R13 is 2-hydroxyethyl or 2- ( 2-hydroxyethoxy) ethyl. Use according to claim 1 of the compounds of formula II specified in claim 5 associated with a biomolecule for quenching fluorescence beyond quenching in vivo. Use according to claim 6, wherein the biomolecule is: a. a nucleic acid molecule or a synthetic analogue thereof b. a protein or peptide molecule. Use according to claim 7, wherein the compounds are bound to a solid phase. Use according to claims 6 to 8, wherein the biomolecule is conjugated to a fluorophore beforehand or later. Use according to claims 6 to 9 for detecting and quantifying fluorescently active molecules in a sample of biological material or tissue. A kit for use according to claim 10 comprising the compounds of claim 5 bound to a specific DNA / RNA sequence and other reagents needed to detect and quantify fluorescently active molecules in a sample of biological material or tissue.