CZ300906B6 - Non-fluorescent phthalocyanine and azaphthalocyanine derivatives as fluorescence extinguisher - Google Patents

Abstract

In the present invention, there is disclosed the use of phthalocyanines, naphthalocyanines, combined phthalo/naphthalocyanines and their aza analogs of the general formula I, wherein the individual substituents have specific meanings, for extinguishing fluorescence after bonding to a biomolecule except for extinguishing fluorescence in-vivo, where the biomolecule can be represented by a nucleic acid or peptide, which can be conjugated with fluorophore. There is further disclosed the use of the above indicated for the detection, quantification of fluorescent-active molecules in a sample of a biological material or tissue. There are also disclosed azaphthalocyanines, aza naphthalocyanines and combined azaphthalo / aza naphthalocyanines of the general formula II, in which the individual substituents have specific meanings, their use for extinguishing fluorescence after bonding to a biomolecule, except for extinguishing fluorescence in-vivo, where the biomolecule can be represented by a nucleic acid or peptide, which can be conjugated with fluorophore, and for the detection, quantification of fluorescent-active molecules in a sample of a biological material or tissue and also in the form of a kit, where the compound of the general formula II is bound to a specific DNA/RNA sequence.

Description

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. It relates in particular to the use of fluorescence quenching molecules in biochemical tests.

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 minimal15 risks to workers' health and the environment. Indocyanine dyes, fluororescein derivatives or 4,4-difluoro-4-boron, 4a-diaza-s-indacene are most often 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 the equilibrium vibration level of the state Si to one of the vibration levels of the ground state Sq. Fluorescence excitation is observed in the substance after stopping almost immediately disappear, decay time is in the order of 1O ^8.

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). In static quenching, the fluorescence donor and acceptor form a non-fluorescent complex. This mechanism does not require overlapping of emission and absorption spectra, only 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), on the other hand, dynamic quenching, in which energy is transferred from an excited fluorophore to an acceptor, takes place over a distance (up to about 100 Å) and according to the Förster theory (Förster, 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 hybridization determinations of DNA in biological material and tissue have been based (Marras, Kramer, Tyagi, Nucleic Acids Research 30 (2002) El 22). 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 (so-called duallabeled 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 in 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). While this increases the sensitivity of the measurement, it is only useful for a limited number of fluorophore emitting at low wavelengths,

Tzv. 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 fluorophore, but each is suitable for quenching only a limited number of fluorescent molecules. Certain, not entirely satisfactorily solved problems remain especially when extinguishing 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 5,088,044). 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 Eclips ™ Quencher (US 6,727,356), 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 ™) group consists of 4 basic structures called BHQ ™ 0 ™ 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 ^R - (4-nitroxphenyldiazo) -2-5'm -metho30 xy-methoxyazobenzen) absorbs at 579 nm may be used for Cy5 ™ fluorescence quenching, but the overlap of the emission spectrum (662 nm) 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 fluorophore Cy5.5 ™ (emission at approximately 707 nm, depending on the conjugated molecule) and minimal quenching of the Cy7 ™ dye that emits fluorescence at 770 nm are then problematic. The last of BHQ ™, BHQ-3 ™ (chemically 3-diethylamino-5-phenyl-7- (4-diethylamino) phenylazophenazinium chloride), has an absorption maximum at 672 nm, which overlaps well with the emission spectrum of the most commonly used indocyanine Cy5 fluorophore However, its disadvantage is the lack of stability under normal oligonucleotide synthesis and processing conditions 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 sufficiently cover the area above 700 nm with their absorption45. 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 the conditions of oligonucleotide synthesis and in the reagents and conditions normally used in their processing. Their further advantage is that with a suitable combination an absorption maximum in the range 620 nm to 770 nm can be achieved (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.

CZ JUU ^ UO DO

SUMMARY OF THE INVENTION

It is an object of the invention to use compounds of formula I as fluorescence quenchers. Thus, the compounds useful in the present invention are those of the following formula

IN

where n is 0 to 15 10 m rode to 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

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 1 -C ₁₈ alkyl, substituted or unsubstituted polyalkyleneoxy chain of 2 to 18 carbons, substituted or unsubstituted branched polyalkylenoxy chain of 2 to 18 carbons, substituted or unsubstituted aryl of 6 to 18 carbons, substituted or unsubstituted heteroaryl of 1 to 18 carbons and 1 up to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted alkylaryl containing 7 to 18 carbons, substituted or unsubstituted alkylheteroaryl containing 2 to 18 carbons, and 1 to 6 heteroatoms O, S, N, Se, substituted or unsubstituted acyl containing 1 to 18 carbons 1 8 carbons, substituted or unsubstituted alkoxycarbonyl of 1 to 18 carbons, substituted or unsubstituted aminoalkyl of 1 to 18 carbons, substituted or unsubstituted alkylaminoalkyl of 1 to 18 carbons, substituted or unsubstituted dialkylaminoalkyl of 1 to 18 carbons, substituted or unsubstituted trialkylammonium alkyl containing 1 to 18 carbons 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 = N-Ar wherein Ar is a substituted or unsubstituted aryl of 6-18 carbons or heteroaryl containing 1 up 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 R1 and R2 may also be combined into a cycle which may be part of other cycles, both saturated and unsaturated, including the heterocycle. The above R1 and R2 may also be linked to the ring linking two NR1R2 substituents on one A to the ring. The above R1 and R2 may also be joined to the ring linking the two NR1R2 substituents on A and B or A and C into the ring. If / w> 1, then the individual NR 1 R 2 may or may not be the same. The alkyl portions of the contemplated substituents may also be unsaturated.

-3GB 300906 B6

M is 2H, 2Li, 2Na, 2K, 2Rb, 2Cs, Be, Mg, Ca, Sr, Ba, Se, Y, 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, De, 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 completely C 1 -C ₁₈ unsaturated alkyl, substituted or unsubstituted C ₃ -C ₆ alkyl; ₈ cycloalkyl which may be partially unsaturated, substituted or unsubstituted branched saturated or partially or fully unsaturated, C _t -C | ₈ 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 O, S, N, Se, substituted or unsubstituted C 7 -C 18 alkylaryl, substituted or unsubstituted C 2 -C 18 alkyl heteroaryl and 1 to 6 O, S, N, Se heteroatoms. The alkyl portions of the contemplated substituents may also be unsaturated.

E is an arbitrary substituent, preferred are the following substituents independently of each other H, substituted or unsubstituted C _{(-C 8} alkyl, substituted or unsubstituted C ₃ -C _t8 cycloalkyl, who may also be partly unsaturated, substituted or unsubstituted branched C, _C] C ₈ -C ₈ alkyl, substituted or unsubstituted partially or fully unsaturated C 1 -C ₁₈ alkyl, substituted or unsubstituted polyalkylenoxy of 2 to 18 carbons, substituted or unsubstituted branched polyalkylenoxy of 2 to 18 carbons, substituted or unsubstituted aryl of 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 alkylaryl containing 7 to 18 carbons, substituted or unsubstituted alkylheteroaryl containing 2 to 18 carbons and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 1 -C ₁₈ alkoxy, substituted or unsubstituted C 1 -C ₁₈ alkylsulfanyl, substituted or unsubstituted polyalkyleneoxy substituent of 1 to 18 carbons, substituted or unsubstituted aryloxy of 6 to 18 carbons, substituted or unsubstituted arylsulfanyl of 6 to 18 carbons substituted or unsubstituted heteroaryloxy containing from 6 to 18 carbons and 1 to 6 heteroatoms of O, S, N, Se contained in the ring, substituted or unsubstituted heteroarylsulfanyl containing from 6 to 18 carbons and lazo heteroatoms of O, S, N, Se contained in the ring, halogen , -NO ₂ , -NO, -COH, -CN, -NCS, -NCO, -C00R 3, -CONR ₁ R ₂ , -COC 1, -COBr, -SO ₂ Cl, -SO ₂ NR ₁ R ₂ , -SO ₃ R 3, -OH ( which may also be esterified), -SH (which may also be esterified), -SS- (2-pyridyl), -PO (OR 3) (OR 4), -OP (OR 3) (NR 1 R 2), -N _In Part E salts formed from both acidic and basic f The function groups are generally known. Substituent 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 to 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 a central metal (M = 2H),

-4CZj JUU7UO DO

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 8 alkyl, phenyl, heteroaryl containing 1 to 6 carbons and 1 to 6 heteroatoms of O, S, N, Se, (C 7 -C 10) alkyl aryl, (C 2 -C 7) alkylheteroaryl (C ₁ -C 6) heteroatom, C 1 -C 8 acyl, saturated and partially unsaturated C ₃ -C 10 cycloalkyl. R6 and R7, R8 and R9, R10 and R11, R12 and R13 can 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 C 1 -C 6 heteroaryl and 1 to 6 hetero15 O, S, N, Se atoms, suitably substituted C 7 -C 12 alkylaryl, 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 , -COCl, -SO ₂ Cl, -OP (OR 3) (NR 4 R ₅ ) ), substituted and unsubstituted -COOH, -SO ₃ H, - PO ₃ H> Further suitable substituents are active esters, especially pentafluorophenyl ester, p-nitrophenyl ester, and active esters of the -COOX type, where 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 1 594 376), optical data carriers (CZ 2003-832 A3), mineral oil labeling (CZ 296 339 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, vol. 15-20. New York: Academic Press, 2003). In contrast to the above examples, the present compounds exhibit very low to zero fluorescence and, depending on the size of the conjugate system, their absorption spectrum overlaps with a sufficient amount of 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 range of approximately 620 nm to 800 nm, corresponding to the overlap with emission spectra commonly used by fluorophore, emitting at high wavelengths above 700 nm (Fig. 1).

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 in the form of 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 w, may be 1 to 8 so that the subject compounds do not have intrinsic fluorescence and thus be useful for quenching. The compounds prepared according to Examples 1 to 6 have different. Compound (Vil) (/ w = 0) 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 fluorescence yield in pyridine is 0.005). Compounds (III-VI) (m = 8) and (VIII) (w-2) no longer have detectable fluorescence and are therefore ideal for quenching fluorescence.

The invention also relates to the use of the present compounds to label solid phase (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 with either fluorophore or the subject compounds by 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 conjugates of biomolecules with the subject compounds for quenching fluorescence, both by dynamic and static quenching, for standard fluorophores emitting in the range of 400 nm to 820 nm. Example 9 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 by fluorophore or quencher). Example

10 shows the use for dual-labeled probes i.e. for conjugates that contain both fluorophore and quencher at the same time on one biomolecule.

Description of the Drawings 45

Giant. 1. Comparison of absorption spectra of compounds III, IV and V with emission maxima (top row) of commonly used fluorophore.

Giant. Synthetic route for the production of conjugates of compound (III) with nucleic acids.

Giant. 3. Directly recording 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 comparison probe labeled with the quenching molecule BHQ-2 ™, a pair of higher intensity lines represents a probe, oligonucleotide S5. The drawing shows a higher increase in fluorescence in the sample with the oligonucleotide

-6YE JUU7UU nu

S5 compared to a BHQ-2 ™ quencher-labeled probe, i.e. significantly better quencher parameters of the quencher molecule of the invention used in oligonucleotide S5 over the BHQ-2 ™ quencher 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.

Examples Examples of compounds suitable for use in the invention

Example 1: Preparation of the compound of formula III

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 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% 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 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. 150 mg of compound III is obtained, i.e. 10% of the theoretical yield. UV-vis (tetrahydrofuran): (nm) 680,654,518,370.

Example 2: Preparation of Compound of Formula IV:

7GB 300906 B6

966 mg of 5,6-bis (diethylamino) quinoxaline-6,7-dicarbonitrile and 330 mg of 4 - [(5,6-Dicyan-3-diethylaminopyrazin-2-yl) methylamino] benzoic 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% 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-visio (tetrahydrofuran): (nm) 752, 713, 685, 644, 477, 372.

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) 453 5-4542.

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] are dissolved in 15 ml of anhydrous butanol | butane 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 mixture resulting from the reaction was purified using column chromatography on silica gel

-8CL JUU7VU υυ silica gel with dichloromethane / acetone / methanol 10/1/1. The second intense violet 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. 120 mg of VI is obtained. UV-vis (tetrahydrofuran):? 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. 612 mg of 5,6-bis (tert-butylsulfanyl) pyrazine-2,3-dicarbonitrile and 544 mg of 5,6-bis15 (diethylamino) pyrazine-2,3-dicarbonitrile are then 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 was purified by column chromatography on silica gel with chloroform / toluene / ethyl acetate 7/7/1. The first intense green fraction corresponds to compound VII, the second (intense green-blue) fraction flowing from the column corresponds to compound VIII. Purification of both fractions was performed 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): (nm) 671, 640, 615, 588, 477, 366.

Compound VIII UV-vis (tetrahydrofuran): (nm) 670, 649, 569, 470, 367.

Example 6: Preparation of the compound of formula IX

9GB 300906 B6

Compound IX was prepared in a similar manner to that described in Example 5, except that 5-diethylamino-6- N -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 ΙΙΪ (Fig. 2)

Compound ΙΙΪ 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. It is allowed to stir for 24 hours at room temperature. To the succinimidyl ester formed 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 reaction with dimethoxytrityl chloride (41 mg) in anhydrous pyridine (5 ml) at room temperature for 24 h and subsequently the secondary hydroxy group is esterified with succinic anhydride (13 mg) in anhydrous pyridine. (5 mL) at 60 ° C for 12 h.

The modified compound III (9 mg) was dissolved in anhydrous dimethylformamide, to which was added O- (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (7 mg).

Anhydrous triethylamine (20 μΐ) was added over 10 minutes and this mixture was added 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. In order 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 ™.

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

An oligonucleotide was synthesized on a commercially available BHQ®-2 quenched solid phase 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.

- in.

CL · ouuyuo DO

Sl: 5-GAA GTC GGG TGG TCA AAA AGC AGA-Q-3 '

S2: 5'- GAA GTC GGG TGG TCA

S3: 5'-GAAGTCGGGTGGTCAAAAAGCAGATTTTTTTTTT-Q-3 '

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

S5: 5 - Cy5 ™ - GAA GTC GGG TGG TCA

S6: 5'- Cy5 ™ - GCA GTC GGG TGG TCA AAA AGC AGA - BHQ * -2 - 3 '

S7: 5CCC TGA ACA CGG ACT

S8: 5 - CTA TGC AAC ATT CGA GTT TCC T-3

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

Example 9: Examples of fluorescence quenching

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

Oligonucleotide solution S4, respectively, was added to the solution of oligonucleotide S4. 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 relationship F _x / Fo, where F _o 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 + S1), 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 to or better than commonly used fluorescence quenching molecules (Marras, Kramer, Tyagi, NucleicAcids Research 30 (2002) E122).

Example 10: Use of a fluorescence-quenching modified oligonucleotide in real25 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 uses so-called.

hydrolysis or hybridization probes. In the presence of the detected DNA sequence, it is amplified / multiplied and decomposes with the amount of the detected sequence in the reaction (using a hydrolysis probe) (nuclease activity of the polymerase enzyme), thereby 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 TAB 1. Real-time PCR or PCR knowledge is a general knowledge for the person skilled 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 an IH compound. Oligonucleotide S6 of the same sequence was on

-11GB 300906 B6 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 real-time PCR as hydrolysis probes. System using hydrolysis probes sequences S5 and S6 together with primers S7 and S8 are used for detection and quantification of specific Herpes virus sequence

Simplex 1 (HSV 1). The composition of the reaction mixture is shown in Table 1 and HSV1 DNA at 20 ng / μΙ was used as template DNA. And PCR was carried out in program 95 ^D C initial denaturation of 3 minutes followed by 55 cycles of 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. On the Y-axis, the fluorescence intensity of the individual samples is plotted, on the X-axis, the reaction time in terms of the number of cycles performed. Based on the so-called baseline or background fluorescence, the software determines a specific fluorescence intensity value, the so-called threshold value. At the point where the detected fluorescence of the sample crosses this imaginary line, the cycle is read on the X-axis. 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 (Table 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 the 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 shows that, compared to the commercially available quencher BHQ®-2, the subject compounds are completely comparable and show an even higher fluorescence increase (Table 2, "Avg, Increase" column).

Table 1: Composition of the reaction mixture for 1 reaction of 20 μΐ

Total volume of mixture: [stock concentration] [reaction mixture] ddH ₂ O 7.85 PCR buffer (10x) 2.00 MgCl ₂ / [mM] $ 0 4.00 dNTP / dUTP mix i [μΜ] ÍOW 0.40 Primer F / [nM] w Primer R / [nM] 250.00 ýg; Proba / [nM] 150,00 ' te Taq polymerase / [U / μΙ] resp. [AT] 5.00 ¹ WO 0.25 Template DNA

-12 knows. juu7uu υυ

Table 2: Analysis of fluorescence data.

By name Ct diairCt SDCt increase Avg. increase SD increase Fbasrljne prihn. Fbaseline SD Fbaseline Sírep.1 31.67 31, «5 0,065 1.89 137 0,0255966 4.15 408 0,04544434 Sírep.2 31.54 1.84 404 SSrcpJ 31.4: P 31,425 0.005 2.15 203 0,0754184 409 <0 · 0,03143429 SSrep.2 31.43 2.30 403

Claims (11)

PATENT CLAIMS 10 1. Use of phthalocyanines, naphthalocyanines, combined phthalo / naphthalocyanines and their azaanalo- 15 n is 0-15; m is 1-8; A, B, C, D are independently the following aromatic rings fused to the parent 20 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 be partially unsaturated, substituted Or unsubstituted branched C 1 -C 18 alkyl, substituted or unsubstituted partially or fully unsaturated C 1 -C 18 alkyl, substituted or unsubstituted C 2 -C 18 polyalkyleneoxy chain, substituted or unsubstituted C 2 -C 18 branched polyalkyleneoxy chain, substituted or unsubstituted aryl of 6 to 18 carbon atoms, substituted or unsubstituted heteroaryl of 1 to 18 carbon atoms 30 and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted C 7 -C 18 alkylaryl, substituted or unsubstituted C 2 -C 18 alkyl heteroaryl and 1 to 6 O, S, N, Se heteroatoms substituted or unsubstituted C _t8 alkoxy, substituted or unsubstituted Ci-Cis alkylthio, substituted or unsubstituted aryloxy having 6 to 18 carbon atoms, a substituted or unsubstituted phenyl arylsulfa35 containing 6 to 18 carbon atoms, substituted or unsubstituted heteroaryloxy obsahu- 13GB JOUVUft B6 6 to .mu.Ci 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se contained in the ring, substituted or unsubstituted heteroarylsulfanyl containing 6 to 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se contained in the ring, halogen, -NO ₂ , -NO, -COH, -CN, -NCS, -NCO, -COOR 3, -C0NR1R2, -C0C1, --COBr, _-SO2 C1 -SCLNR1R2, -SO ₃ R 3, OH (which may be a weighing esterifiko5 na), -SH (which may also be esterified), -S-S- (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 portions of the contemplated substituents may also be unsaturated; io Rl, R2 are independently H, substituted or unsubstituted saturated or partially or fully unsaturated, C _t -C | C ₈ -C ₈ alkyl, substituted or unsubstituted C ₁ -C ₁₈ cycloalkyl, which may be a partially unsaturated, substituted or unsubstituted saturated or partially or fully unsaturated branched C ₁ -C ₁₈ alkyl, substituted or unsubstituted C 2 -C 18 polyalkyleneoxy chain, substituted or unsubstituted unsubstituted branched poly15 alkyleneoxy chain of 2 to 18 carbon atoms, substituted or unsubstituted aryl of 6 to 18 carbon atoms, substituted or unsubstituted heteroaryl of 1 to 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted alkylaryl C 7 -C 18 substituted or unsubstituted C 2 -C 18 alkylheteroaryl and 1 to 6 O, S, N, Se heteroatoms, substituted or unsubstituted acyl C 1 -C 18 substituted, unsubstituted C 1 -C 18 alkoxycarbonyl, C 1 -C 18 substituted or unsubstituted C 1 -C 18 substituted or unsubstituted C 1 -C 18 alkylaminoalkyl, C 1 -C 18 substituted or unsubstituted dialkylaminoalkyl carbon atoms, substituted or unsubstituted trialkylammonium alkyl having 1 to 18 carbon atoms, and Wherein the complementary ion is F -, Cl ^- , BY, Γ, CH 2 OSOf, ClLC 1 LOSOf, R 1 and R 2 together form ^ N-Ar, wherein Ar is a substituted or unsubstituted aryl of 6 to 18 carbon atoms or heteroaryl of 1 to 18 the carbon atoms and the 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 combined into a cycle which may be part of other cycles, both saturated and unsaturated, the aforementioned R1 and R2 may be 30 are also linked to a ring linking two NR 1 R 2 substituents on one A to the ring; the aforementioned R1 and R2 may also be joined to the ring linking two NR1R2 substituents on A and B or A and C into the ring; 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; 35 M is 2H, 2Li, 2Na, 2K, 2Rb, 2Cs, Be, Mg, Ca, Sr, Ba, Se, 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, Tl, 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; 40 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 45 may be a partially unsaturated, substituted or unsubstituted branched saturated or partially or fully unsaturated C 1 -C ₁₈ alkyl, substituted or unsubstituted polyalkyleneoxy chain of 2 to 18 carbon atoms, substituted or unsubstituted branched polyalkyleneoxy chain of 2 to 18 carbon atoms, substituted or unsubstituted aryl of 6 to 18 carbon atoms, substituted or unsubstituted heteroaryl of 1 to 18 carbon atoms 50 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se, substituted or unsubstituted alkylaryl containing 7 to 18 carbon atoms, substituted or unsubstituted alkylheteroaryl of 2 to 18 carbon atoms and 1 to 6 heteroatoms of O, S, N, Se ; the alkyl portions of the contemplated substituents may also be unsaturated, 55 to extinguish fluorescence upon binding to a biomolecule, in addition to extinguishing fluorescence in vivo. -14CL JUV7VU UU 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 a 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. ío 5. Azaphthalocyanines, azanaphthalocyanines and combined azaphthalocyanaphthalocyanines of 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 containing 1 to 6 carbon atoms and 1 to 6 heteroatoms O, S, N C 7 -C 10 alkylaryl, C 7 -C 10 alkylheteroaryl and 1 to 6 heteroatoms of O, S, N, Se, C 1 -C 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; R6 and R8, R10 and R12 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 having 1 to 6 carbon atoms and 1 to 6 carbon atoms 30 6 heteroatoms of O, S, N, Se, suitably substituted C 7 -C 12 alkylaryl, suitably substituted C 7 -C 7 alkylheteroaryl and 1 to 6 O, S, N, Se heteroatoms; 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 β-nitrophenyl ester, and the active esters of the -COOX type wherein X is among the following structures: M is 2H, Zn, Cu, Ni, Cd, Mg; R3, R4, R5 are as defined in claim 1, except for compounds wherein A = B = OD = pyrazino and M = 2H and R6 = R7 = R8 = R9 = R10 = R11 = ethyl and R12 = H and simultaneously R 13 is 2-hydroxyethyl or 2- (2-hydroxyethoxy) 10 ethyl. Use according to claim 1 of compounds of formula II according to claim 5 associated with a biomolecule for quenching fluorescence beyond quenching in vivo. 15 Dec 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 bio-molecule is conjugated to the 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 as defined in 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.